RU2782305C2 - Pro-adm as monitoring marker of therapy for critically ill patients - Google Patents

Abstract

FIELD: medicine; clinical and laboratory diagnostics.

SUBSTANCE: invention relates to medicine, namely to clinical and laboratory diagnostics; it can be used for prediction of subsequent undesirable phenomenon in a patient’s health condition. A sample from the specified patient is provided, while the patient is diagnosed as critically ill, and medical treatment has been started. The sample is isolated from the patient’s body after diagnosis and beginning of the treatment. A level of proadrenomedullin (hereinafter – proADM) or its fragment (fragments) is determined in the specified sample. A low level of severity of proADM or its fragment (fragments), determined in the sample, of less than 4 nmol/l indicates the absence of the subsequent undesirable phenomenon. A high level of severity of proADM or its fragment (fragments), determined in the sample, of more than 6.5 nmol/l indicates the subsequent undesirable phenomenon. A level of procalcitonin (hereinafter – PCT) or its fragment (fragments) is additionally determined in the sample with subsequent prediction of the undesirable phenomenon.

EFFECT: method provides for a possibility of prediction of a subsequent undesirable phenomenon in a patient’s health condition, which is: death, new infection, organ failure, and/or deterioration of clinical symptoms, due to determination in a sample of a level of proADM or its fragment (fragments) and, optionally, a level of PCT or its fragment (fragments).

19 cl, 32 tbl, 10 ex

Description

The invention relates to a method for monitoring therapy, including prognosis, risk assessment and / or risk stratification of a subsequent adverse event in a patient's health, including obtaining a sample of the specified patient, while the specified patient was diagnosed as critically ill and medical treatment was started, while the specified sample isolated from the patient's body after diagnosis and initiation of treatment; determining the level of proadrenomedullin (proADM) or its fragment(s) in the specified sample, while the specified level of proADM or its fragment(s) correlates with the likelihood of a subsequent adverse event in the state of health of the specified patient. In a preferred embodiment, the invention relates to a method further comprising determining the level of procalcitonin (PCT) or its fragment(s) in a sample isolated from a patient's body. Preferably, the method of the invention comprises determining the level of procalcitonin (PCT) or fragment(s) thereof in a first sample isolated from a patient, said first sample being isolated at or before the time point of diagnosis and initiation of treatment (time point 0) ; determination of the level of PCT or its fragment(s) in the second sample isolated from the specified patient after the isolation of the first sample and the diagnosis and initiation of treatment; and determining whether a difference in the level of PCT or its fragment(s) in the second sample is visible compared to the level of PCT or its fragment(s) in the first sample.

Background of the invention

Despite significant improvements in diagnostic and preventive measures, the incidence of sepsis continues to rise 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 the characteristic frequency countries (2, 3). Thus, an early and accurate assessment of both infection load and disease severity, in terms of overall host pathophysiological response, is especially important in the early stages of sepsis to make quick and correct decisions regarding diagnostic testing and treatment strategies, as well as in the later phase. for reliable patient management, treatment monitoring, decisions to terminate participation in the study in the presence of clinical recovery.

In this regard, it is surprising that there is currently no gold standard diagnostic test for sepsis (4). The use of procalcitonin (PCT) has partly addressed this unmet need for infectious burden assessment, with experimental and interventional data available for antibiotic use (5-7). At the same time, an exact indicator of the severity of the disease has not yet been shown.

For this reason, numerous biomarkers and clinical measures have subsequently been proposed, including the use of severity rating scales such as the Sequential Organ Failure Assessment (SOFA), Acute Physiological and Chronic Functional Change (APACHE). Health Evaluation) II and Simplified Acute Physiological Score (SAPS) II, however, these are rarely calculated on a daily basis in a standard way due to the relatively high complexity and time requirements associated with each assessment scale. The use of new biomarkers may meet this unmet clinical need, yet almost none of them have successfully entered mainstream clinical practice (8).

Of these markers, the median fragment of proadrenomedullin (SF-proADM), a peptide produced by several tissues to stabilize microcirculation and protect against endothelial permeability and subsequent organ failure (9-16), has shown significant promise, especially in areas associated with sepsis (17) , lower respiratory tract infections (18-21), lung transplantation (22) and thoracic surgery (23). In this regard, it can be noted that it is generally accepted that the endothelium and microcirculation play a significant role in the pathophysiological response of the host to sepsis (24, 25), with the regulation and distribution of blood flow in each important organ (25), and thus, they may provide an alternative measure of the severity of the overall host response compared to individual organ dysfunction scales.

Thus, the present invention uses a range of biomarkers (PCT, lactate, C-reactive protein, SF-proADM) and clinical scoring scales (SOFA, APACHE II and SAPS II) to (i) obtain an accurate assessment of disease severity within a short time frame. after diagnosis, such as within 24 hours of diagnosis, as well as within the first ten days of intensive care unit (ICU) therapy, (ii) identifying low-risk patients who may be suitable for early discharge from the ICU in the postoperative setting, and (iii) identifying patients who, despite clinical improvement (eg, reduced PCT levels), remain at high or increasing risk of mortality or other adverse events and may require urgent additional diagnostic and therapeutic activities.

Accordingly, SF-proADM can be used as a means to identify patients with high disease severity who may need alternative diagnostic and therapeutic interventions, and patients with low disease severity who could potentially be subject to early discharge from the ICU in combination with the absence of specific for ICU types of therapy.

There is a need in the field of treatment of critically ill patients, such as patients with sepsis, for additional means of monitoring therapy within a short time interval after the start of treatment, as well as tools for predicting, risk assessment and / or risk stratification of the subsequent adverse event in the health status of a critically ill patient. .

Brief description of the essence of the invention

In light of the difficulties in the prior art, the technical problem underlying the present invention is to provide a means of monitoring therapy and assessing the risk of a critically ill patient after the start of treatment, preferably within a short time interval, up to and including days or weeks after the start of treatment. An additional technical problem underlying the present invention is to provide a means of predicting, assessing risk and/or stratifying the risk of a subsequent adverse event in a critically ill patient's health, particularly within a short time interval after the start of treatment.

Thus, in this invention, attempts have been made to obtain a method, set and additional means for monitoring therapy in critically ill patients, as well as means for predicting, risk assessment and / or risk stratification of a subsequent adverse event in the health status of a critically ill patient based on the levels of proadrenomedullin (proADM), determined in a sample obtained from a patient. Thus, one object of the present invention is the use of a biomarker or combination of biomarkers to distinguish critically ill patients who have been or are being treated at high risk of an adverse event from critically ill patients who are at low risk of a subsequent adverse event.

The solution to the technical problem of the present invention is proposed in the independent claims. Preferred embodiments of the present invention are contemplated in the dependent claims.

Thus, the present invention relates to a method for monitoring therapy, including prognosis, risk assessment and/or risk stratification of a subsequent adverse event in a patient's health, including

- obtaining a sample from said patient, where the patient was diagnosed as critically ill and medical treatment was initiated, whereby the sample was isolated from the patient's body after the diagnosis was made and treatment started,

- 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 event in the health status of said patient.

In one embodiment, patients in accordance with the method of this invention have already been diagnosed as critically ill and have already received treatment. Thus, the method of the present invention can be used to monitor the effectiveness of a treatment or therapy that has been initiated based on a determination of the likelihood of a subsequent adverse event. Therapy monitoring preferably includes prediction of an adverse event and/or risk stratification or risk assessment of the patient in relation to a subsequent adverse event, said risk assessment and determination of said risk being considered as a means of monitoring the initiated therapy.

Physicians or medical personnel who treat patients who have been diagnosed as critically ill may use the method of this invention in a variety of clinical settings, such as primary care settings, or preferably inpatient settings, or in an intensive care unit (ICU). . This method is highly useful for monitoring the effect of a therapy that has been initiated on a critically ill patient, and can be used to assess whether the patient being treated is a high-risk patient who should be under close medical supervision and potentially should undergo additional medical interventions, or a low-risk patient with an improvement in health, which may not require close monitoring and additional interventions, possibly due to the fact that the initiated treatment leads to a successful improvement in the patient's condition. The initial treatments for critically ill patients can have a direct impact on the likelihood of adverse events in the patient's health status. In this regard, risk assessment / prediction of a subsequent adverse event provides feedback or monitoring of initiated therapy.

The likelihood of a subsequent adverse event may be assessed by comparing the level of proADM or its fragments in the sample with respect to a reference level (such as a threshold or cut-off value and/or population mean), while the reference level may correspond to proADM or its fragments in healthy patients or in patients who have been diagnosed as critically ill.

Accordingly, the method of the present invention can facilitate the prediction of the likelihood of a subsequent adverse event in a patient's health status. This means that said method of the present invention can differentiate high-risk patients who are more likely to suffer complications, or patients whose condition will become more critical in the future, from low-risk patients whose health is stable or even improving. therefore, they are not expected to suffer from an adverse event, such as death of the patient or worsening of the patient's clinical symptoms or signs, which may require certain therapeutic measures.

A definite advantage of the method of this invention is that a patient who has been determined to be at low risk using the method of this invention can be discharged from the ICU more quickly. In addition, in the case of patients with low risk, the intensity and/or frequency of observation of the patient's health status can be reduced. Accordingly, the hospital or other medical institution responsible for the patient can more effectively decide which patients will require intensive medical care and observation. In this way, the respective hospital or institution, for example, can more efficiently occupy ICU beds in the case of high-risk patients. This will lead to improved care for high-risk patients, as medical staff can focus on such patients, while low-risk patients can be discharged from the ICU. This will also result in significant benefits by avoiding the cost of unnecessary interventions that might otherwise be applied to low-risk patients.

The time point when patients are diagnosed as critically ill and the first treatment measures are initiated is defined as "time point 0", which may represent a failure of the sample isolation time point reference used to determine proADM or fragments thereof. If the diagnosis of a patient and the initiation of treatment do not occur at the same time, time point 0 is the time point when the last of these two events of diagnosis and initiation of medical treatment occurs. Typically, the diagnosis of critically ill patients immediately precedes or accompanies the initiation of therapy.

It was completely unexpected that the level of proADM or its fragments in a sample obtained from a patient can provide definitive information about the likelihood of a subsequent adverse event in the health status of these critically ill patients. There was no indication that a single measurement of proADM or its fragments after diagnosis and initiation of treatment in a critically ill patient could provide such important information in relation to the effectiveness of current treatment and the prognosis of the patient's health.

The use of proADM or fragments thereof as a single parameter in embodiments of the present invention is preferred over the use of other single parameters such as biomarkers or clinical rating scales because proADM is more accurate in predicting an adverse event than other markers such as PCT. , CRP, lactate, or clinical rating scales such as SOFA, SAPS II, or APACHE II.

According to a preferred embodiment, the sample is isolated from said patient within 30 minutes of said diagnosis and treatment, or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after the specified diagnosis and start of treatment. In other embodiments, the sample is isolated from the body of the specified patient after 12-36 hours and/or 3-5 days after the start of treatment.

The fact that the level of proADM or its fragments at a time point corresponding to no more than about 30 minutes after diagnosis and treatment can provide such information was completely unexpected.

In preferred embodiments of the method of this invention, said sample is isolated from said patient at approximately 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 the specified diagnosis and start of treatment.

In other embodiments, the sample is isolated at time points after said diagnosis and initiation of antibiotic therapy, corresponding to 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 the time point of obtaining the sample.

In another preferred embodiment of the present invention, the patient has been diagnosed using at least one additional biomarker or clinical score. It is particularly preferred in the context of the present invention if the patient's initial critical illness diagnosis at time point 0 was based in part on the level of at least one biomarker or a defined clinical score.

In certain embodiments, this invention includes the determination of additional parameters such as markers, biomarkers, clinical rating scales, or the like.

In another preferred embodiment of the present invention, the patient has been diagnosed with at least one of the procalcitonin (PCT), lactate and C-reactive protein biomarkers and/or at least one of the SOFA, APACHE II and SAPS II clinical scores. The detection of proADM or its fragments in samples of patients who have been diagnosed as critically ill and are on treatment has proven to be particularly useful for monitoring therapy if the patient's diagnosis was based on these markers, as the prediction of an adverse event in such patient populations may be more accurate by compared with critically ill patients who were diagnosed by other means.

In one embodiment of the present invention, a critically ill patient is a patient with an infectious disease, a patient diagnosed with an infectious disease and one or more existing organ failures, a patient diagnosed with sepsis, severe sepsis, or septic shock, and/or a patient after trauma or after surgery. intervention. In light of the data presented herein, the predictive value 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 this invention, the adverse event in the state of health of the specified patient is death, preferably death within 28-90 days after diagnosis and initiation of treatment, a new infection, organ failure and / or worsening of clinical symptoms requiring a targeted cleaning procedure, transfusion blood products, colloid infusions, emergency surgery, invasive mechanical ventilation, and/or renal or liver replacement therapy.

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

In certain embodiments of the present invention, the treatment received by the patient includes one or more types of antibiotic therapy, invasive mechanical ventilation, non-invasive mechanical ventilation, renal replacement therapy, the use of vasopressors, fluid therapy, extracorporeal blood purification and/or organ protection.

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

Preferably, in some embodiments, the method is carried out by determining 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 one or the other embodiment and may be considered in the context of each embodiment, respectively. In preferred embodiments, the "ADM fragment" may be considered to be SF-proADM.

In further embodiments of the present invention, said level of proADM or fragment(s) thereof is correlated with the likelihood of a subsequent adverse event in the health status of said patient. In a preferred embodiment, the level of proADM or fragment(s) thereof is positively correlated with the likelihood of a subsequent adverse event in the health status of said patient. In other words, the higher the level of proADM determined, the higher the likelihood of a subsequent adverse event.

In accordance with a preferred embodiment of the present invention

- low severity of proADM or its fragment(s) indicates no subsequent adverse event, or indicates a low risk of a subsequent adverse event, with low severity being less than 4 nmol/l, preferably less than 3 nmol/l, more preferably less than 2 .7 nmol/l, or

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

In accordance with a preferred embodiment of the present invention

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

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

In accordance with the present invention, the term "indicate" in the context of "indicate a subsequent adverse event" and "indicate the absence of a subsequent adverse event" is intended to indicate the magnitude of the risk and/or probability. Preferably, an "indication" of the presence or absence of an adverse event is for risk assessment and should typically not be construed as limitingly indicating the definitely absolute presence or absence of said event.

Thus, the term "indicates the absence of a subsequent adverse event" or "indicates a subsequent adverse event" may mean an indication of a low or high risk of an adverse event, respectively. In some embodiments, low risk is associated with lower risk relative to detectable proADM levels greater than the indicated values. In some embodiments, a high risk is associated with a higher risk relative to detectable proADM levels that are less than the indicated values.

Given the above, the determination of high and low levels of severity of proADM is at the same time very reliable in relation to the determination of the presence or absence of a subsequent adverse event using the breakpoints disclosed in this document, as a result of which the risk assessment allows for appropriate action by the healthcare professional. .

It was completely unexpected that the level of proADM or its fragments could correlate with the likelihood of the presence or absence of a subsequent adverse event in the context of critically ill patients who received treatment at these time points. The levels of proADM in samples obtained from critically ill patients according to this invention can preferably be classified into 3 different levels of severity of proADM. High levels of proADM indicate a high level of severity, intermediate levels indicate an intermediate level of severity, and low levels indicate low levels of severity. Appropriate concentrations, which define breakpoints for respective levels of severity, depend on numerous parameters such as the time point of sample isolation after the diagnosis and initiation of treatment of the patient with the method of this invention and the method used to determine the level of proADM or its fragments in said sample.

The cutoff values disclosed herein preferably refer to measurements of the level of proADM protein or fragments thereof in a blood plasma sample obtained from a patient using the KRYPTOR Thermo Scientific BRAHMS assay. Accordingly, the values disclosed herein may vary to some extent depending on the detection/measurement method used, and it is intended that the specific values disclosed herein also include corresponding values determined by other methods.

In one embodiment of the present invention, a low severity level of the proADM or fragment(s) indicates no subsequent adverse event, wherein the low severity level is less than a breakpoint in the range of 1.5 nmol/L to 4 nmol/L. Any value within these ranges may be considered as an appropriate cut-off value for low severity levels of proADM or fragments thereof, e.g. 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 one embodiment of the present invention, a high severity level of the proADM or fragment(s) is indicative of a subsequent adverse event, wherein the high severity level is above a breakpoint in the range of 6.5 nmol/l to 12 nmol/l. Any value within these ranges may be considered as an appropriate cut-off value for high severity levels of proADM or fragments thereof, e.g. 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.

The breakpoints disclosed herein associated with the level of a marker or biomarker such as proADM or PCT should be understood to be "equal to or greater than" a certain breakpoint, or "equal to or less than" a certain breakpoint. For example, an embodiment referring to a level of proADM or fragment(s) of less than 4 nmol/L, preferably less than 3 nmol/L, more preferably less than 2.7 nmol/L, should be understood to refer to a level of proADM or fragment(s) thereof. (fragments) equal to or less than 4 nmol/l, preferably equal to or less than 3 nmol/l, more preferably equal to or less than 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, is to be understood as referring to the level proADM or fragment(s) equal to or greater than 6.5 nmol/l, preferably equal to or greater than 6.95 nmol/l, more preferably equal to or greater than 10.9 nmol/l.

In the embodiments described herein, the levels of severity are preferably defined by cutoffs, which are boundaries between low, intermediate, or high levels of severity. Any embodiments that are thus boundary values may use the format of one boundary value as the boundary between two severity levels, or one boundary level for each severity level.

In some embodiments, the proADM cutoff between low and intermediate severity levels is:

2.7 nmol/L ± 20%, or 2.7 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%, and between intermediate and severe levels:

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

Said cut-off values are preferably relevant for assessing the level of severity of proADM at baseline, in other words, at diagnosis and/or initiation of treatment and/or hospitalization.

In some embodiments, the proADM cutoff between low and intermediate severity levels is:

2.80 nmol/L ± 20%, or 2.80 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%, and between intermediate and severe levels:

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

These cut-off values are preferably relevant for assessing the level of severity of proADM after 1 day, in other words, about 24 hours after baseline, in other words, about 1 day after diagnosis and/or treatment and/or hospitalization. For example, in embodiments where proADM is measured one day after the start of therapy, day 1 cutoffs may be used. at baseline, i.e., over time, even somewhat lower (but still relatively high) levels are associated with high risk and classified as high severity.

In some embodiments, the proADM cutoff between low and intermediate severity levels is:

2.80 nmol/L ± 20%, or 2.80 nmol/L ± 15%, or ± 12%, ± 10%, ± 8%, or ± 5%, and between intermediate and severe levels:

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

Said breakpoints are preferably relevant for assessing the level of severity of proADM at 4 days, in other words about 4 days after baseline, in other words about 4 days after diagnosis and/or treatment and/or hospitalization. For example, in embodiments where proADM is measured 4 days after the start of therapy, day 4 cutoffs may be used. at baseline or Day 1, i.e., over time, even somewhat lower (but still relatively high) levels are associated with high risk and are classified as high severity.

In some embodiments, the boundary levels to be used in the embodiments described above may be adjusted to the appropriate level depending on the day on which the measurement is taken. Each of the cutoffs is subject to some variation as a result of the overall variance, as one of skill in the art would expect. Relevant breakpoints are determined based on the exhaustive data provided below, but are not intended to be final or precise values in all possible embodiments. When using a similar limit value with respect to the mentioned values, i.e., within ± 20%, ± 15%, ± 12%, ± 10%, ± 8%, or ± 5%, as can be determined by a person skilled in this field of technology, similar results can be expected.

Any embodiment describing ±20% of a defined cut-off value may also be considered as revealing ±15%, ±12%, ±10%, ±8%, or ±5%.

Any embodiment describing a breakpoint for baseline, day 1, or day 4 may also be considered to disclose the corresponding breakpoints for other days, e.g., an embodiment describing a breakpoint for baseline may also be considered to refer to the same option. implementation that describes the boundary value of day 1 or day 4.

In accordance with a preferred embodiment of the present invention

- a low severity level of the proADM or fragment(s) indicates no subsequent adverse event, with a low severity level of less than 2.7 nmol/l, or

- a high severity level of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is greater than 10.9 nmol / l.

This embodiment of the present invention is particularly preferred if levels of proADM or fragments thereof are determined in a sample that was isolated on the day of diagnosis and treatment of the patient, especially approximately 30 minutes after diagnosis and treatment. This becomes apparent from the analysis proposed in Example 3.

In accordance with a preferred embodiment of the present invention

- a low severity level of the proADM or fragment(s) indicates no subsequent adverse event, with a low severity level of less than 2.7 nmol/l, or

- a high severity level of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is more than 10.9 nmol / l,

- while the level of proADM or its fragments is determined in the sample, which was isolated preferably on the day of diagnosis and start of treatment.

In accordance with a preferred embodiment of the present invention

- a low severity level of the proADM or fragment(s) indicates no subsequent adverse event, with a low severity level of less than 2.8 nmol/l, or

- a high severity level of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is greater than 9.5 nmol / l.

This embodiment of the present invention is particularly preferred if the levels of proADM or fragments thereof are determined in a sample that was isolated on day 1 after said diagnosis and initiation of treatment, as evident from Table 12.

In accordance with a preferred embodiment of the present invention

- a low severity level of the proADM or fragment(s) indicates no subsequent adverse event, with a low severity level of less than 2.8 nmol/l, or

- a high level of severity of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is more than 9.5 nmol / l,

- while the level of proADM or its fragments is determined in the sample, which was isolated preferably 1 day after the diagnosis and start of treatment.

In accordance with a preferred embodiment of the present invention

- a low severity level of the proADM or fragment (fragment) indicates the absence of a subsequent adverse event, while the low severity level is less than 2.25 nmol / l.

This low severity cutoff is particularly preferred because, in some embodiments, it has been determined to be the optimal cutoff between low and intermediate severity of proADM or fragments thereof when the sample is cleared from the patient at 4 days, 7 days, or 10 days after the specified diagnosis and start of treatment. This becomes apparent from the analysis proposed in Table 12.

In accordance with a preferred embodiment of the present invention

- a low severity level of proADM or a fragment (fragment) indicates the absence of a subsequent adverse event, while the low severity level is less than 2.25 nmol / l,

- while the level of proADM or its fragments is determined in the sample, which was isolated preferably 4, 7 or 10 days after the diagnosis and the start of treatment.

In accordance with a preferred embodiment of the present invention

- a high severity level of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is greater than 7.7 nmol / l.

This high severity cutoff is particularly preferred because, in some embodiments, it has been determined to be the optimal cutoff between intermediate and high severity of proADM or fragments thereof, when the sample is shed from the patient 4 days after said diagnosis and initiation of treatment. This becomes apparent from the analysis proposed in Table 12.

In accordance with a preferred embodiment of the present invention

- a high level of severity of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is more than 7.7 nmol / l,

- in this case, the level of proADM or its fragments is determined in the sample, which was isolated 4 days after the diagnosis and the start of treatment.

In accordance with a preferred embodiment of the present invention

- a high severity level of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is greater than 6.95 nmol / l.

This high severity cutoff is particularly preferred because, in some embodiments, it has been determined to be the optimal cutoff between intermediate and high severity of proADM or fragments thereof when the sample is shed from the patient 7 days after said diagnosis and initiation of treatment. This becomes apparent from the analysis proposed in Table 12.

In accordance with a preferred embodiment of the present invention

- a high level of severity of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is more than 6.95 nmol / l,

- while the level of proADM or its fragments is determined in the sample, which was isolated 7 days after diagnosis and treatment.

In accordance with a preferred embodiment of the present invention

- a high severity level of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high severity level is greater than 7.45 nmol / l.

This high severity cutoff is particularly preferred because it has been determined to be the optimal cutoff between intermediate and high severity of proADM or fragments thereof when the sample is shed from the patient 10 days after said diagnosis and treatment. This becomes apparent from the analysis proposed in Table 12.

In accordance with a preferred embodiment of the present invention

- a high level of severity of proADM or a fragment (fragment) indicates a subsequent adverse event, while a high level of severity is more than 7.45 nmol / l,

- in this case, the level of proADM or its fragments is determined in the sample, which was isolated 10 days after the diagnosis and the start of treatment.

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

- a low severity level of proADM or its fragment(s) indicates that the patient has been discharged from the ICU, or

- a high level of severity of proADM or fragment(s) indicates a modification of the patient's treatment in the ICU.

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

For example, if the level of proADM or its fragments falls into the category of low severity of proADM, the attending physician can make a decision with greater certainty about the discharge of the specified patient from the ICU, since it is unlikely that an adverse event in the state of health of the specified patient will occur, preferably within the next 28 days, more preferably within the next 90 days. Accordingly, it may not be necessary to keep the patient in the ICU. It may also be possible to make decisions that the current treatment leads to a successful improvement in the health status of the patient, assessed by measuring the risk of an adverse event.

In contrast, if the determination of the level of proADM or its fragments of the specified ICU patient indicates a high level of severity of proADM or its fragments, the attending physician should keep the patient in the ICU. As an adjunct, consideration should be given to adjusting the patient's treatment, since it is likely that the current treatment does not result in an improvement in the patient's health, 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, but is not limited to, adjusting the dose or mode of administration of the current drug, changing the current treatment to another treatment, adding an additional treatment option to an existing treatment, or stopping the current treatment. Various treatment options 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 less than 2.25 nmol/L, wherein said sample is isolated from an ICU patient 1 day or more after said diagnosis and treatment, and the low severity level of proADM or its fragment(s) indicates the discharge of the specified patient from the ICU.

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

- obtaining a sample from a specified patient, where the patient is an intensive care unit (ICU) patient and medical treatment has been initiated, and the sample has been isolated from the patient's body after admission to the ICU and initiation of treatment,

- determination of the level of adrenomedullin (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 event in the health status of said patient.

In the context of the method according to the invention relating to ICU patients, the reference value for the time point corresponding to the release of the sample used to determine proADM or its fragments is the time point when patients are admitted to the ICU and the first treatment measures begin (time point 0) . The specified time point corresponds to the diagnosis and initiation of treatment in the method according to this invention, referring to patients who were diagnosed as critically ill.

All embodiments of the method of this invention relating to patients who have been diagnosed as critically ill are also considered to correspond to embodiments of the method of this invention relating to ICU patients.

The invention further relates to the medically indicated methods of treatment described herein, wherein the patient population to be treated is identified, stratified, monitored, predicted, diagnosed, or otherwise assessed using the methods described herein. Appropriate treatments for the methods are disclosed herein. Thus, the present invention is particularly advantageous in identifying patients at increased risk of having an adverse event and initiating preventive or risk-reducing treatments, or initiating treatments directed at the presence of a medical condition, preferably conditions perceived as a critical illness.

Embodiments of the present 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 point of extraction of the first and second sample)

A preferred embodiment of the present invention further comprises determining the level of PCT or fragment(s) thereof in a sample isolated from a patient's body. In a preferred embodiment, a sample for determining the level of PCT or its fragment(s) is isolated before, at or after the time point of diagnosis and initiation of treatment.

It is particularly preferred 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 fragments thereof, either 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 also exist for the combined assessment of proADM or fragments thereof with other markers or clinical assessment scales such as lactate, CRP, SOFA, SAPS II, APACHE II or other clinical assessments.

In accordance with a further preferred embodiment of the present invention, the method described herein further comprises

- determining the level of PCT or fragment(s) in the first sample isolated from the patient's body, while said first sample is isolated before, at the time of or after diagnosis and treatment,

- determination of the level of PCT or its fragment(s) in the second sample isolated from the body of the specified patient, while the second sample was isolated after the first sample, preferably within 30 minutes after the isolation of 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 is isolated, and

- determination of the difference in 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 especially preferred to combine the determination of proADM or its fragments in a sample isolated from the patient's body with the determination of PCT or its fragments in the first sample and the determination of the level of PCT or its fragments in the second sample isolated after the first sample, while the sample used to determine proADM or fragments thereof, may be the same or different from the first sample or the second sample used to determine the PCT or fragments thereof.

In a preferred embodiment, the method described herein further comprises

- determination of the level of PCT or fragment(s) in the first sample isolated from the patient's body, while said first sample is isolated at or before the time point of diagnosis and start of treatment (time point 0),

- determination of the level of PCT or its fragment(s) in the second sample (sample according to claim 1) isolated from the body of the specified patient after the diagnosis and start of treatment, preferably within 30 minutes after the diagnosis and start 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 treatment, and

- determination of the difference in 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 preferred to combine the detection of proADM or fragments thereof (in the second sample) with the determination of PCT or fragments thereof in an earlier sample (first sample) that is isolated from the body of said patient and which can be used to diagnose said patient as critically ill in time. point 0, and determining the level of PCT or its fragments in the second sample isolated at a certain time point after diagnosis and start of treatment, which is preferably the same time point at which proADM or its fragments are determined. As indicated by the data presented below, the determination of the difference in the level of PCT or its fragments in the second sample compared to the first sample adds additional information to the information obtained from the levels of proADM or its fragments in the second sample. Based on said combined information, it may be possible with a higher probability to predict whether an adverse event will occur in the health state of said 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 an unexpected result since biomarkers for sepsis are typically not synergistic or complementary, but only alternative diagnostic markers.

In a preferred embodiment, the method described herein further comprises

- determining the level of lactate in the first sample isolated from the body of the patient, while the specified first sample is isolated at or before the time point of diagnosis and treatment (time point 0),

- determination of the level of lactate in the second sample (sample according to claim 1) isolated from the body of the specified patient within 30 minutes after the statement of the specified diagnosis and the start of treatment or after at least 30 minutes, preferably 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after said diagnosis was made and treatment started, and

- determination of the difference in 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 isolated from the patient's body. In a preferred embodiment, the sample for determining the level of CRP or its fragment(s) is isolated before, at or after the time point of diagnosis and initiation of treatment.

It is particularly preferred to combine detection of proADM or fragments thereof with detection 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.

In accordance with a further preferred embodiment of the present invention, the method described herein further comprises

- determining the level of CRP or fragment(s) in the first sample isolated from the patient's body, while said first sample is isolated before, at the time of or after diagnosis and treatment,

- determination of the level of CRP or its fragment(s) in the second sample isolated from the body of the specified patient, while the second sample was isolated after the first sample, preferably within 30 minutes after the isolation of 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 is isolated, and

- determination of the difference in 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, the method described herein further comprises

- determining the level of CRP or fragment(s) in the first sample isolated from the patient's body, wherein said first sample is isolated at or before the time point of diagnosis and start of treatment (time point 0),

- determination of the level of CRP or its fragment(s) in the second sample (sample according to claim 1) isolated from the body of the specified patient within 30 minutes after the establishment of the specified diagnosis and the start of treatment, or after at least 30 minutes, preferably 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 in 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.

The preferred embodiment of the present invention further includes the definition of SOFA. In a preferred embodiment, SOFA is determined before, at, or after the time point of diagnosis and initiation of treatment.

It is particularly preferred to combine the determination of proADM or fragments thereof with the determination of SOFA, wherein the time point of isolation of the sample for determination of proADM may be the same or different from the time point of SOFA determination.

In accordance with a further preferred embodiment of the present invention, the method described herein further comprises

- determination of the first SOFA value before, at the time of or after the time point of diagnosis and initiation of treatment,

- determination of the second SOFA value within 30 minutes after the determination of the first SOFA value, 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 value, and

- determining the difference between two defined SOFA values.

In a preferred embodiment, the method described herein further comprises

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

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

- determination of the difference in SOFA determined after the specified diagnosis and start of treatment, and SOFA determined at time point 0.

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

It is particularly preferred to combine the determination of proADM or fragments thereof with the determination of SAPS II, wherein the time point of extraction of the sample for the determination of proADM may be the same or different from the time point of the SAPS II determination.

In accordance with a further preferred embodiment of the present invention, the method described herein further comprises

- determining the first SAPS II value before, at or after the time point of diagnosis and treatment,

- determination of the second SAPS II value within 30 minutes after the determination of the first SOFA value, 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 value, and

- determination of the difference between two defined SAPS II values.

In a preferred embodiment, the method described herein further comprises

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

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

- determination of the difference in SAPS II, determined after the specified diagnosis and start of treatment, and SAPS II, determined at time point 0.

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

It is particularly preferred to combine the determination of proADM or fragments thereof with the determination of APACHE II, wherein the time point of isolation of the sample for the determination of proADM may be the same or different from the time point of the APACHE II determination.

In accordance with a further preferred embodiment of the present invention, the method described herein further comprises

- determination of the first APACHE II value before, at or after the time point of diagnosis and initiation of treatment,

- determination of the second APACHE II value within 30 minutes after the determination of the first APACHE II value 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 value, and

- determining the difference between two defined APACHE II values.

In a preferred embodiment, the method described herein further comprises

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

- determination of APACHE II within 30 minutes after the stated diagnosis and start of treatment, or at least 30 minutes, preferably 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

- determination of the difference in APACHE II, determined after the specified diagnosis and start of treatment, and APACHE II, determined at time point 0.

In a preferred embodiment of the present invention

- lower level of PCT or its fragment(s) in the second sample compared to the first sample, and low severity of proADM or its fragment(s), while the low severity is less than 4 nmol/l, preferably less than 3 nmol/l , more preferably less than 2.7 nmol/l, indicate the absence of a subsequent adverse event, or

- a lower level of PCT or its fragment(s) in the second sample compared to the first sample, and a high level of severity of proADM or its fragment(s), while the high severity level is more than 6.5 nmol/l, preferably more than 6, 95 nmol/l, more preferably greater than 10.9 nmol/l, indicates 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 initiated treatment. Conversely, elevated levels in the second sample compared to the first sample may indicate elevated levels of the marker during treatment.

It was a complete surprise that a patient with a decrease in PCT levels from the day of diagnosis and start of treatment to a later time point of at least 30 minutes after the start of treatment may still show an increased risk of a subsequent adverse event. In particular, this increased risk is evident if a high level of severity of proADM or its fragments has been determined.

It is believed that PCT is a marker of a patient's critical illness, in particular in the case of a patient with sepsis. Accordingly, it is believed that a decrease in PCT values during treatment indicates an improvement in the patient's health. At the same time, as disclosed herein, it has become apparent that despite a decrease in PCT value, the patient may be at risk of having a subsequent adverse event if the level of proADM or its fragments at a later time point is a high level of severity. Accordingly, the attending physician may adjust the treatment of such a patient who would not be identified as a high-risk patient without determining proADM or fragments thereof in the second sample.

On the other hand, the clinician can be sure that an adverse event is unlikely to occur if the PCT level decreases during treatment, while the level of proADM or its fragments in the second sample represents a low level of severity of proADM or its fragments. Accordingly, such patients can be defined as low risk patients. It was a complete surprise that 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 time point, leads to improved monitoring, treatment prognosis and assessment of the risk of a subsequent adverse event in the patient's health status.

According to another 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 a low level of severity of proADM or its fragment(s), while the low level of severity is less than 4 nmol/l, preferably less than 3 nmol/l, more preferably less than 2.7 nmol/l, indicate no subsequent adverse event, or

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

A great advantage of this invention is that it is possible to identify low-risk patients in a critically ill patient group based on the low severity of proADM in the second sample, even if the PCT level increases during the course of the patient's treatment. This was a complete surprise as elevated PCT levels were thought to indicate that the critically ill patient was deteriorating and thus an adverse event would be expected. Accordingly, a successful treatment may be stopped and replaced with a modified or different treatment. The additional determination of proADM at a later time point allows identification of low-risk patients in the group of patients with increasing PCT levels, which is a significant advantage of the present invention.

On the other hand, it is possible to define high-risk patients with increasing PCT levels and high severity of proADM or its fragments, which is a more precise definition of such patients who are likely to have an adverse event in the future. Accordingly, the treatment of said patients can be adjusted while minimizing the risk that said patient may be a low-risk patient.

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

A preferred embodiment of the method of this invention further includes

- determining the level of proADM or fragment(s) in the first sample isolated from the body of the patient, while the specified first sample is isolated before, at the time of or after diagnosis and treatment, and

- determination of the level of proADM or its fragment(s) in a second sample isolated from the body of the specified patient, while the specified second sample was isolated after the first sample and after the time point of diagnosis and treatment, preferably within 30 minutes after the isolation of the first sample, or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 or 10 days after the first sample is isolated, and

- determining whether the difference in 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 is obvious.

The first and second sample used to determine the level of proADM or its fragment(s) may be the same or different from the first and second sample used to determine the level of PCT or its fragment(s).

A preferred embodiment of the method of this invention further includes

- determining the level of proADM or fragment(s) in the first sample isolated from the patient's body, said first sample being isolated at or before the time point of diagnosis and initiation of treatment (time point 0), and

- determination of the level of proADM or its fragment(s) in the second sample after diagnosis and initiation of 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 was made and treatment started, and

- determining whether the difference in 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 is obvious.

A preferred embodiment of the method of this invention further includes

- determining the level of proADM or fragment(s) in the first sample isolated from the patient, wherein said first sample is used to diagnose said patient as critically ill (time point 0), and

- determination of the level of proADM or its fragment(s) in the second sample after diagnosis and initiation of 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 was made and treatment started, and

- determining whether the difference in 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 is obvious.

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

- determination of the level of proADM or fragment(s) and optionally PCT or its fragment(s) in the first sample isolated from the patient's body, while said first sample is isolated at or before the time point of diagnosis and start of treatment (time point 0), and

- determination of the level of proADM or its fragment(s) and optionally PCT or its fragment(s) in the second sample isolated from the body of the specified patient, after the statement of the specified diagnosis and the start of treatment, preferably within 30 minutes or at least 30 minutes after making said diagnosis and initiating treatment, preferably 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 in the level of proADM or its fragment(s) and/or the difference in the level of PCT or its fragments 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 of this invention further comprises

- determining the level of proADM or fragment(s) and optionally PCT or fragment(s) thereof in the first sample isolated from the patient's body, said first sample being used to diagnose said patient as critically ill (time point 0), and

- determination of the level of proADM or its fragment(s) and optionally PCT or its fragment(s) in the second sample isolated from the body of the specified patient, after the statement of the specified diagnosis and the start of treatment, preferably within 30 minutes or at least 30 minutes after making said diagnosis and initiating treatment, preferably 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 in the level of proADM or its fragment(s) and/or the difference in the level of PCT or its fragments in the second sample compared to the level of proADM or its fragment(s) in the first sample.

Surprisingly, determining the change in levels of proADM or its fragments from the time point of diagnosis and initiation of treatment to a later time point may provide additional information regarding the occurrence of a subsequent adverse event in the health status of a patient who has been diagnosed as critically ill. A significant advantage of this embodiment of the present invention is that the same sample that is used to determine the diagnostic marker at time point 0 can also be used to determine the baseline level of proADM or its fragments, which can be compared with the level of proADM or its fragments in more late time point after diagnosis and initiation of treatment. By determining the change in the level of proADM or its fragments during the treatment of patients, the accuracy of predicting the occurrence of an adverse event in the patient's health status can be further improved.

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.

It was unexpected that based on the change in the level of proADM or its fragments, it is possible to reliably predict the likelihood of an adverse event in the patient's state of health without determining additional markers. An increase in the level or severity of proADM or fragments thereof from the time point of diagnosis and initiation of treatment indicates that an adverse event is likely to occur. Accordingly, based on the change in proADM or fragments thereof during treatment, the clinician may decide whether to change or modify the patient's treatment or to adhere to the original treatment.

In a preferred embodiment of the method of this 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 compared to the first sample is associated with an increased level of severity 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 is associated with a lower level of severity of proADM or fragments thereof in the second sample compared to the first sample.

A significant advantage based on a change in the level of proADM or fragments thereof in combination with a certain change in PCT or fragments thereof during the treatment of a critically ill patient is that the likelihood of an adverse event in the patient's state of health can be determined. Accordingly, it is possible to reliably identify high-risk patients and low-risk patients based on changes in these two markers. It was particularly surprising that a decrease in PCT levels may be associated with an increased likelihood of a subsequent adverse event if they coincide with increasing levels of proADM or its fragments.

Additional Embodiments of the Invention

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

- lower level of PCT or its fragment(s) in the second sample compared to the first sample, and low severity of proADM or its fragment(s), while the low severity is less than 4 nmol/l, preferably less than 3 nmol/l , more preferably less than 2.7 nmol/l, indicate the discharge of the specified patient from the ICU;

- a lower level of PCT or its fragment(s) in the second sample compared to the first sample, and a high level of severity of proADM or its fragment(s), while the high severity level is more than 6.5 nmol/l, preferably more than 6, 95 nmol/l, more preferably greater than 10.9 nmol/l, indicates the need to modify the patient's treatment in the ICU;

- an increased level of PCT or its fragment(s) in the second sample compared to the first sample, and a low level of severity of proADM or its fragment(s), while the low level of severity is less than 4 nmol/l, preferably less than 3 nmol/l, more preferably less than 2.7 nmol/l, indicate the discharge of the specified patient from the ICU; or

- an increased level of PCT or its fragment(s) in the second sample compared to the first sample, and a high level of severity of proADM or its fragment(s), while the high level of severity is more than 6.5 nmol/l, preferably more than 6.95 nmol/l, more preferably greater than 10.9 nmol/l, indicate the need to modify the patient's treatment in the ICU.

It is preferred that by combined analysis of the change in PCT level or fragments thereof and the severity level of proADM or fragments thereof at the second sample isolation time point (later time point) in an ICU patient, it can be decided whether the patient is a patient with is a low-risk patient who can be discharged from the ICU while maintaining current treatment, or is the patient a high-risk patient who requires modification or adjustment of current ICU therapy to prevent the occurrence of an adverse event indicated by an appropriate combination of changes in PCT levels and the current level of severity of proADM.

In a further embodiment, this invention relates to a kit for carrying out the method of this invention, wherein the kit contains

- detection reagents for determining the level of proADM or its fragment(s) and optionally for determining the level of PCT, lactate and/or C-reactive protein or their fragment(s) in the sample obtained from the subject, and

- reference data, such as a reference level corresponding to high and/or low levels of severity of proADM, with low severity being less than 4 nmol/l, preferably less than 3 nmol/l, more preferably less than 2.7 nmol/l, and high the severity level 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 reference data being preferred stored on a computer-readable medium and/or used in the form of a computer executable code configured to compare certain levels of proADM or fragment(s) thereof and optionally certain levels of PCT, lactate and/or C-reactive protein or fragment(s) thereof with said reference data.

In a further embodiment, this invention relates to a kit for carrying out the method of this invention, wherein the kit contains

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

- reference data, such as a reference level corresponding to high and/or low levels of severity of proADM, with low severity being less than 4 nmol/l, preferably less than 3 nmol/l, more preferably less than 2.7 nmol/l, and high the severity level 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 levels, said reference data being preferably stored on a computer-readable medium and/or used in in the form of a computer executable code configured to compare certain levels of proADM or fragment(s) thereof and optionally certain levels of PCT or fragment(s) thereof with said reference data.

Detection reagents for determining the level of proADM or its fragment(s) and optionally for determining the level of PCT, lactate and/or C-reactive protein or fragment(s) thereof are preferably selected from the reagents necessary for carrying out the method, for example, antibodies directed to 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 its fragment(s) and optionally additionally other biomarkers such as, for example, PCT or its fragment(s) is determined using a method selected from the group consisting of mass spectrometry (MS), immunoluminescent assay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescence immunoassays, enzyme immunoassay (EIA), enzyme-linked immunosorbent assays (ELISA), luminescent chips using beads, chips using magnetic beads, assays for based on protein microarrays, rapid test formats such as, for example, immunochromatographic test strips, rare earth cryptate assays, and automated systems/analyzers.

The method of the invention may further be carried out as a homogeneous method, wherein 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 to use two antibodies, both antibodies being labeled with parts of the detection system, resulting in signal generation or signal activation if both antibodies are integrated into one sandwich.

Such techniques are carried out, in particular, in the form of detection methods based on fluorescence enhancement or fluorescence quenching. A particularly preferred aspect relates to the use of detection reagents that must be used in pairs, such as, for example, those described in US 4,882,733 A, EP-B1 0,180,492 or EP-B1 0,539,477 and the prior art cited herein. In this regard, measurements become possible in which only reaction products containing both labeling components in the same immune complex in the reaction mixture are detected.

For example, such technologies are offered under the trade names TRACE® (Time Resolved Amplified Cryptate Emission) or KRYPTOR®, embodying the ideas of the applications mentioned above. Thus, in certain 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 additional marker of the method proposed herein is determined. In certain preferred aspects, the diagnostic device is a KRYPTOR®.

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

In additional embodiments of the method described herein, the method further includes molecular analysis of the sample obtained from the specified patient, to determine the infection. The sample used for molecular analysis to determine infection is preferably a blood sample. In a preferred embodiment, the molecular analysis is a method directed to the determination of one or more biomolecules derived from 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. According to preferred embodiments, biomolecules are detected using 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, enzyme detection. activities and methods of detection based on immunological assays. Additional molecular analysis methods are known to the person skilled in the art and consist of the method of this invention.

In one embodiment of the method described herein, the first antibody and the second antibody are present dispersed in a liquid reaction mixture, and the first labeling component, which is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, is associated with the first antibody. , and the second labeling component of the specified labeling system is associated with the second antibody in such a way that after binding of both antibodies to the specified proADM or its fragments to be detected, a measurable signal is formed that ensures the detection of the obtained sandwich complexes in the measurement solution.

In one embodiment of the method described herein, the labeling system comprises a rare earth metal 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 comprises comparing the determined level of proADM, or fragment(s) thereof, with a reference level, cutoff, and/or population mean corresponding to proADM, or fragments thereof, in patients who have been diagnosed as critically ill. and receiving medical treatment, while said comparison is carried out in the computer processor using executable code.

The methods of this invention may be partly computerized. For example, the step of comparing a detectable level of a marker, eg proADM or fragments thereof, with a reference level may be performed on a computer system. In a computer system, a certain level of marker a(s) can be combined with levels of other markers and/or subject parameters to calculate a rating scale that is indicative of diagnosis, prognosis, risk assessment and/or risk stratification. For example, the determined values may be entered (either manually by a healthcare professional or automatically from the device(s) in which the level(s) of the respective marker has/were determined) into the computer system. The computer system may be located directly at the point of care (eg, primary care, ICU or EEP, emergency department) or may be a remote location connected via a computer network (eg, via the Internet or a specialized medical cloud system, optionally , combinable with other IT systems or platforms such as Hospital Information Systems (HIS)). Typically, a computer system will store values (e.g. marker levels or parameters such as age, blood pressure, body weight, sex, etc. or clinical scoring systems such as SOFA, qSOFA, BMI, etc.). ) on a computer-readable medium and calculate the rating scale based on predetermined and/or pre-stored reference levels or reference values. The resulting rating scale will be displayed and/or printed to the user (typically a healthcare professional such as a physician). Alternatively, or in addition, the associated prognosis, diagnosis, score, treatment instructions, management instructions, or stratification will be displayed and/or printed to the user (typically a healthcare professional such as a physician).

In one embodiment, a software system where the machine learning algorithm is self-explanatory can be used, preferably to identify hospitalized patients at risk for sepsis, severe sepsis, and septic shock using electronic health record (EHR) data. The machine learning approach can be tested on a random forest model classifier using EHR data (such as laboratory tests, biomarker expression, vital signs and demographics) obtained from patients. Machine learning is a type of artificial intelligence that provides computers with the ability to learn complex patterns in data without being explicitly programmed, as opposed to simpler rule-based systems. Earlier studies used electronic health record data to trigger readiness to measure clinical deterioration in general. In one embodiment of the present invention, the processing of proADM levels can be included in a suitable computer program for comparison with existing datasets, for example, proADM levels can also be processed in a machine learning computer program to facilitate diagnosis or prediction of 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 pattern may refer to the same pattern or to different patterns. The assay used to detect and determine proADM and, for example, PCT may be the same or different, for example, an immunoassay may be used to determine one of the above markers. More detailed descriptions of suitable assays are provided below.

Breakpoints and other reference levels of proADM or fragments thereof in patients who have been diagnosed as critically ill and are receiving treatment can be determined using previously described methods. For example, methods are known to the person skilled in the art for applying the coefficient of variation in assessing the variability of assays to determine reference values and/or breakpoints (George F. Reed et al., Clin Diagn Lab Immunol. 2002; 9(6): 1235 -1239).

In addition, the sensitivity of functional assays can be determined to indicate statically significant values for use as reference levels or breakpoints in accordance with conventional techniques. Laboratories are able to independently determine the sensitivity of functional assays 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 thus represents an indication of assay precision at low assay levels. Thus, CV is a standard deviation (SD) standardization that allows comparison of estimates of variability independent of analyte concentrations 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. Receiver operating characteristics (ROC) curves measure the efficiency of sorting model-fitted probabilities in order to sort response levels. ROC curves can also facilitate the establishment of criteria points in diagnostic tests. The higher the curve is located from the diagonal, the more effectively it is selected. If the logistic fit has more than two levels of response, then it leads to the formation of a generalized ROC curve. On such a graph, there is a curve for each response level, which is the ROC curve of that level relative to all other levels. Computer software is available to perform this type of analysis to determine appropriate reference levels and cutoffs, eg JMP 12, JMP 13, Statistical Discovery from SAS.

Boundary values can be defined in a similar way for PKT. Literature is available to one skilled in the art to determine the appropriate limit value, eg Philipp Schuetz et al. (BMC Medicine. 2011; 9:107) describe that at a cut-off value of 0.1 ng/mL, PCT determination had a very high sensitivity to rule out infection. Terence Chan et al. (Expert Rev.Mol.Diagn.2011; 11(5), 487.496) have described that metrics such as positive and negative likelihood ratios, which are calculated based on sensitivity and specificity, are also useful in assessing the strength of a diagnostic test. Values are typically plotted for multiple breakpoints (CV) as a receiver operating curve. The area under the curve value is used to determine the best diagnostically meaningful CV. This literature describes the variation of CV (boundary values that depend on the analysis and design of the study) and the corresponding methods for determining the boundary values. Population mean levels of proADM or fragments thereof can also be used as reference values, e.g., population mean proADM values, whereby patients who are diagnosed as critically ill can be compared with a control population, with the control group preferably containing 10, 20, 30, 40, 50 or more subjects. In one embodiment of the present invention, the breakpoint for PCT can be a value in the range of 0.01 to 100.00 ng/mL in a blood serum sample using, for example, the MAC Pix E-Bioscience Luminex assay or the PCT-Kryptor BRAHMS assay. In a preferred embodiment, the cut-off level of PCT 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 the specified ranges may be considered as the appropriate limit 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.

Embodiments of the present invention related to the implementation of antibiotic therapy, stratification and / or control of a patient suffering from an infectious disease and receiving antibiotic therapy

One embodiment of the present invention relates to a method for providing antibiotic therapy, stratification and/or control of a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents, the method comprising

isolating the first sample from said patient,

- isolation of the second sample from the body of the specified patient at a time point after the isolation of the first sample and the start of antibiotic therapy,

- determination of the levels of procalcitonin (PCT) or its fragment(s) 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.

In a preferred embodiment of the method for performing antibiotic therapy, stratification and/or monitoring of a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents described herein, the first sample is isolated before, at the time point, or after the symptoms of the infectious disease in the specified patient. A further embodiment of the present invention relates to a method for performing antibiotic therapy, stratification and/or control of a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents, the method comprising

isolating the first sample from the body of said patient in determining the symptoms of an infectious disease in said patient,

- isolation of a second sample from the body of the specified patient after the initial identification of the indicated symptoms and the start of antibiotic therapy,

- determination of the levels of procalcitonin (PCT) or its fragment(s) 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.

Shedding in detecting a symptom of an infectious disease refers to shedding shortly after detecting symptoms of an infectious disease, and may also be referred to as a time point of detecting symptoms of an infectious disease.

In one embodiment, the method is characterized in that said second sample is isolated from the body of said patient within 30 minutes after the detection of symptoms of an infectious disease and the start of antibiotic therapy, or after at least 30 minutes, 1 hour, 2 hours, 6 hours and/or 12 hours after identifying the symptoms of an infectious disease and starting antibiotic therapy.

In one embodiment, the method is characterized in that said second sample is isolated from the body of said patient 12-36 hours and/or 3-5 days after the symptoms of the infectious disease are identified and antibiotic therapy is started.

In one embodiment, the method is characterized in that the patient is diagnosed as suffering from sepsis and/or septic shock.

In one embodiment, the method includes determining the level of SP-proADM.

In one embodiment, the method is characterized in that

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

- intermediate or high severity level of proADM or its fragment(s) in the second sample indicates that a change in one or more antibiotic agents is required - while the intermediate level of severity of proADM or its fragment(s) is more than 4 nmol/l, preferably more than 3 nmol/l, more preferably more than 2.7 nmol/l, and less than 6.5 nmol/l, preferably less than 6.95 nmol/l, more preferably 10.9 nmol/l, and

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

In one embodiment, the method further comprises determining the level of proADM or fragment(s) thereof in the first sample. Preferably, an increased level of proADM or fragment(s) thereof in the second sample compared to the first sample indicates that a change in one or more antibiotic agents is required. In one embodiment, the method is characterized in that

- a lower level of PCT or its fragment(s) in the second sample compared to the first sample, and - a higher level of 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, such as an increase from low severity to intermediate or high severity, or from intermediate severity to high severity,

- indicate that a change in one or more antibiotic agents is required,

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

- the intermediate level of severity of proADM or its fragment(s) is more than 4 nmol/l, preferably more than 3 nmol/l, more preferably more than 2.7 nmol/l, and less than 6.5 nmol/l, preferably less than 6.95 nmol /l, more preferably 10.9 nmol/l, and

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

In one embodiment, the method is characterized in that

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

- an intermediate level of severity of proADM or its fragment(s) in the second sample compared to a low level of severity of proADM or its fragment(s) in the first sample indicate that a change in one or more antibiotic agents is required.

In one embodiment, the method is characterized in that

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

- a high level of severity of proADM or its fragment(s) in the second sample compared to an intermediate level of severity of proADM or its fragment(s) in the first sample indicates that a change in one or more antibiotic agents is required.

In one embodiment, the method is characterized in that the first and second sample are selected from the group consisting of a blood sample, a blood serum sample, a blood plasma sample, and/or a urine sample.

In one embodiment, this invention relates to a method of providing antibiotic therapy, therapy stratification and/or patient control; suffering from an infectious disease and receiving treatment with one or more of the antibiotic agents described herein, further comprising

- determining the level of at least one additional biomarker or fragment(s) thereof in the first and second sample, wherein 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 its fragment(s) in the second sample indicate whether a change in current antibiotic therapy is required.

In one embodiment, this invention relates to a method of providing antibiotic therapy, therapy stratification and/or control of a patient suffering from an infectious disease and receiving treatment with one or more antibiotic agents described herein, further comprising

- determining at least one clinical rating scale at the time point of the first sample release and at the time point of the second sample release, wherein at least one clinical rating scale is preferably SOFA, APACHE II and/or SAPS II,

wherein at least one clinical score at the time points of isolation of the first and second samples, and the level of proADM or its fragment(s) in the second sample indicate whether a change in current antibiotic therapy is required.

In one embodiment, this invention relates to a kit for implementing the method described herein, containing:

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

- reference data, such as a reference level corresponding to the severity levels of proADM according to claim 6 and/or claim 9, and optionally PCT levels, while said reference data is preferably stored on a computer-readable medium and/or used in the form of a computer executable code executed with the ability to compare certain levels of proADM or its fragment(s) and optionally certain levels of PCT or its fragment(s) with the specified reference data.

Detailed description of the invention

The present invention relates to a method for monitoring therapy, including prognosis, risk assessment and/or risk stratification of a subsequent adverse event in a patient's state of health, including obtaining a sample of said patient, wherein said patient has been diagnosed as critically ill and medical treatment has been initiated, wherein said the sample is isolated from the patient's body at least 30 minutes after diagnosis and initiation of treatment; determining the level of proADM or its fragment(s) in said sample, wherein said level of proADM or its fragment(s) correlates with the likelihood of a subsequent adverse event in the state of health of said patient.

As is evident from the data presented herein, the likelihood of the presence or absence of an adverse event in a patient's health condition is indicated by the level of proADM or its fragment(s).

This invention has the following advantages over standard methods: The methods and kits of this invention are fast, objective, easy to use and accurate for monitoring therapy in critically ill patients. The methods and kits of this invention relate to markers and clinical scores that are easily measurable using standard methods in hospitals, since the levels of proADM, PCT, lactate, C-reactive protein, SOFA, APACHE II, SAPS II can be determined in a standard in the manner obtained from blood samples or additional body fluids obtained from the subject.

As used herein, the term "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 this invention, the term "adverse event in the health of the patient" refers to phenomena that indicate complications or deterioration in the health of the patient. Such adverse events include, but are not limited to, death of the patient, death of the patient within 28-90 days of diagnosis and initiation of treatment, 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 in which worsening of clinical symptoms indicates a need for therapeutic interventions such as directed debridement, transfusion of blood products, colloid infusion, invasive mechanical ventilation, non-invasive mechanical ventilation, emergency surgery, organ replacement therapy, such as renal or liver replacement therapy, and vasopressor therapy.

A patient described in this document as "critically ill" may be diagnosed as an intensive care unit (ICU) patient, a patient who requires constant and/or careful monitoring of their health status, 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 after an injury such as a patient after an accident, a patient with burns, a patient with one or more injuries. The subject described herein may be in an emergency or intensive care unit, or in other medical care settings, such as in an emergency vehicle such as an ambulance, or in the presence of a general practitioner, who encountered a patient with these symptoms. Patients with suspected SIRS are not necessarily considered critically ill.

The term "ICU patient" refers to, but is not limited 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 (ITU) or intensive care unit (CCU), and is a special department of a hospital or healthcare facility that provides intensive care medication. ICU patients usually suffer from severe and life-threatening diseases and injuries that require constant close monitoring and support with special equipment and medications to ensure normal bodily functions. Common conditions treated in the ICU include, but are not limited to, adult acute 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 the clinical condition of the subject associated with an infectious disease. Also, assessment of the severity of an infectious disease may be covered by the term "diagnosis". The term "prognosis" refers to the prediction of the outcome of a particular risk for a subject based on an infectious disease. It may also include an assessment of the likelihood of recovery or the likelihood of an adverse event for said patient.

The methods of this invention can also be used for monitoring. The term "monitoring" refers to tracking an already diagnosed infectious disease, disorder, complication, or risk, for example, to analyze the progression of a disease or the effect of a particular treatment or therapy on the progression of a critically ill patient's disease or 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 grouping of subjects into different risk groups in accordance with their future prognosis. Risk assessment also refers to stratification in order to apply preventive and/or therapeutic measures. Examples of risk stratification are the low, intermediate, and high risk levels described in this document.

As used herein, the term "treatment delivery" refers to the administration of certain therapies or medical interventions based on the value of one or more biomarkers and/or clinical parameters and/or clinical scores.

It is to be understood that, in the context of the present invention, the term "determining the level of proADM or its fragment(s)" or the like. refers to any means of determining 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, provided that the fragment provides an unambiguous determination of the level of proADM or a fragment thereof. In certain preferred aspects of the present invention, the term "determination of the level of proADM" refers to the determination of the level of mid-regional proadrenomedullin (SF-proADM). SF-proADM is a fragment and/or region of a proADM. The peptide adrenomedullin (ADM) was discovered as a 52 amino acid antihypertensive 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 EDSSPDAARI RVKRYRQSMN

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. The term "proadrenomedullin" ("proADM") refers to pre-proADM without the signal sequence (amino acids 1-21), ie, amino acid residues 22-185 of pre-proADM. The term "mid-regional proadrenomedullin" ("SF-proADM") refers to amino acids 42-95 of pre-proADM.

An exemplary amino acid sequence of SF-proADM is shown in SEQ ID NO: 2.

SEQ ID NO:2: SF-proADM 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 153-185 pre-proADM) is called adrenotensin. ProADM peptide fragments SP-proADM may contain, for example, at least about 5, 10, 20, 30 or more amino acids. Accordingly, the proADM fragment, for example, can be selected from the group consisting of SF-proADM, PAMP, adrenotensin, and mature adrenomedullin, preferably herein, said fragment is SF-proADM.

The definition of these different forms of ADM or proADM and their fragments also encompasses the measurement and/or selection of specific sub-sites of these molecules, for example, using antibodies or other affinity reagents directed against a certain part of the molecules, or by determining the presence and/or number of molecules by measuring a portion of a 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 the determination of at least one additional biomarker, marker, clinical scoring and/or parameter, in addition to proADM.

As used herein, a parameter is a characteristic, property, or measurable factor that may facilitate the identification of a particular system. The parameter is an important element for assessments related to health and physiological status, such as the risk of disease/disorder/clinical pathological condition, preferably organ(s) dysfunction(s). In addition, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes or pharmacological responses to a therapeutic event. An illustrative example can be selected from the group consisting of the Acute and Chronic Functional Changes Score II (APACHE II), the Simplified Acute Functional Changes Score (SAPSII Score), the Dynamic Organ Failure Scale (SOFA Score), the Rapid Dynamic Organ Score. deficiency (qSOFA), body mass index, weight, age, sex, IGS II, fluid intake, white blood cell count, sodium content, potassium content, temperature, blood pressure, dopamine level, bilirubin level, respiratory rate, oxygen partial pressure, rating scales World Federation of Neurosurgical Societies (WFNS) and the Glasgow Coma Scale (GCS).

As used herein, terms such as "marker", "surrogate", "prognostic marker", "factor", or "biomarker" are used interchangeably and refer to biological markers to be measured and quantified (e.g., a specific protein or enzyme concentration or fragment, the concentration of a certain hormone or fragment thereof, or the presence of biological substances or a fragment thereof) that act as indicators for assessments of the state of health and physiological state, such as the risk of a disease/disorder/clinical pathological condition, preferably an adverse event. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes or pharmacological responses to a therapeutic event. Biomarkers can be measured in a sample (such as blood, plasma, urine, or tissue sample).

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 acute functional changes (SAPSII scoring scale) of the specified subject, the acute and chronic functional changes assessment scale II (APACHE II) of the specified subject and the level of soluble fins-like tyrosine kinase-1 (sFlt-1), histone H2A, histone H2B, histone H3, histone H4, calcitonin, endothelial-1 (ET-1), arginine vasopressin (AVP), atrial natriuretic peptide (ANP), neutrophil gelatinase-associated lipocalin (NGAL), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), lithostatin (PSP), trigger receptor expressed on myeloid cells 1 (TREM1), interleukin-6 (IL-6), interleukin-1, interleukin -24 (IL-24), interleukin-22 (IL-22), interleukin (IL-20), other ILs, presepsin (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 (P1GF), chromatogranin A, protein S100A, protein S100B and tumor necrosis factor α (TNFα), 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/MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7/ Ppbp, CXCL9, IL8/CXCL8, XCL1, XCL2, FAM19A1, FAM1 9A2, 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/IFNWl, BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L/CD178, EDA-A1, TNFSF14, LTA/TNFB, LTB, TNFa, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, IL18, IL18BP, ILIA, IL1B, IL1F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33 or a fragment thereof.

As used herein, the term "procalcitonin" or "PKT" refers to a peptide encompassing amino acid residues 1-116, 2-116, 3-116, or procalcitonin peptide fragments thereof. PCT is a peptide precursor of the hormone calcitonin. Thus, procalcitonin fragments are at least 12 amino acids in length, preferably more than 50 amino acids, more preferably more than 110 amino acids. PCT may contain post-translational modifications such as glycosylation, liposide and derivatization. Procalcitonin is a 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 the immunoassays 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 also known to those skilled in the art, for example using products obtained from Thermo Fisher Scientific/B-R-A-H-M-S GmbH.

Lactate, or lactic acid, is an organic compound with the formula CH ₃ CH(OH)COOH, which is found in biological fluids, including blood. Blood tests for lactate are performed to determine the status of acid-base homeostasis in the body. Lactic acid is a product of cellular metabolism that can accumulate when cells are deficient in oxygen (hypoxia) and must turn to a less efficient means of generating energy, or when a pathological condition results in excess production or impaired excretion of lactate. Lactic acidosis can be caused by insufficient oxygen to cells and tissues (hypoxia), for example, if the individual has a medical condition that can lead to a decrease in the amount of oxygen delivered to cells and tissues, such as shock, septic shock, or chronic heart failure, test for lactate can be used to facilitate the detection and assessment of the severity of hypoxia and lactic acidosis.

C-reactive protein (CRP) is a pentameric protein that can be found in biological fluids such as blood plasma. CRP levels may increase in response to inflammation. Measuring and charting CRP values can be helpful in determining the progression of a disease or the effectiveness of treatments.

As used herein, the term "dynamic organ failure score" or "SOFA score" is one score used to track a patient's status during their stay in the intensive care unit (ICU). The SOFA rating scale is a scoring system for the purpose of determining the degree of organ functioning or the degree of organ failure of an individual. This rating scale is based on six different rating scales, each for the respiratory system, cardiovascular system, liver system, blood coagulation system, kidney system, and nervous system. Both the mean and maximum SOFA scores are predictive of treatment outcome. Raising the SOFA score within the first 24-48 hours in the ICU suggests a mortality rate of at least 50% to 95%. Rating scales less than 9 suggest a mortality rate of 33%, while more than 14 may be around or greater than 95%.

The SOFA rating scale (qSOFA) as used herein is a rating scale that indicates organ dysfunction or risk of mortality in a patient. This rating scale is based on three criteria: 1) a change in mental status, 2) a decrease in systolic blood pressure to less than 100 mm Hg. Art., 3) a respiratory rate of more than 22 breaths per minute. Patients with two or more of these conditions have a higher risk of having organ dysfunction or death.

As used herein, the term "APACHE II" or "Acute and Chronic Functional Change Scale II" is a classification system for assessing disease severity (Knaus et al., 1985). It can be applied within 24 hours of admission to the intensive care unit (ICU) and can be determined based on 12 different physiological parameters: AaDO2 or PaO2 (depending on FiO2), temperature (rectal), mean arterial pressure, pH arterial blood, heart rate, respiration rate, sodium (serum), potassium (serum), creatinine, hematocrit, leukocyte count and Glasgow Coma Scale.

As used herein, the term "SAPS II" or "Simplified Acute Physiology Score II" refers to a classification system for the severity of a disease or disorder (see Le Gall JR et al., A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study JAMA 1993;270(24):2957-63). The SAPS II scoring scale consists of 12 physiological variables and 3 disease-related variables. The scoring scale is calculated on the basis of 12 standard physiological measurements, information about the previous state of health and some information obtained during admission to the ICU. The SAPS II scoring scale can be determined at any time, preferably on day 2. The term "worst" measurement is defined as the score that correlates with the highest score. The SAPS II scoring scale ranges from 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 content, potassium content, bicarbonate content , bilirubin content, leukocyte content, chronic diseases and type of administration. There is a sigmoid relationship between mortality and the overall SAPS II rating scale. The subject has a 10% mortality on a SAPSII score of 29, a 25% mortality on a SAPSII score of 40, a 50% mortality on a SAPSII score of 52, and a 75% mortality on a SAPSII score of 64 points, mortality is 90% with a SAPSII score of 77 points (Le Gall, ibid.).

Used in this document, the term "sample" is a biological sample that is obtained or isolated from the body of a patient or subject. The term "sample" as used herein, for example, may refer to a biological fluid or tissue sample obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. Herein, preferably, said sample is a biological fluid sample such as blood, blood serum, blood plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, cells, cell extract, tissue biopsy, stool sample, and the like. In particular, said sample is blood, blood plasma, blood serum or urine.

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

The term "plasma" in the context of this invention is essentially a 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 heparized blood), for example for at least 15 minutes at 2000-3000 g.

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

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

In preferred embodiments of the present invention, the patient has been diagnosed as having sepsis. More specifically, the patient could be diagnosed as having severe sepsis and/or septic shock.

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

The term "sepsis" as used herein includes, but is not limited to, sepsis, severe sepsis, septic shock. Severe sepsis refers to sepsis associated with organ dysfunction, an underperfusion disorder, or sepsis-induced hypertension. Disorders manifested by underperfusion include lactic acidosis, oliguria, and acute alteration of mental status. Sepsis-induced hypotension is defined as systolic blood pressure less than about 90 mm Hg. Art., or its decrease by approximately 40 mm. rt. Art. or more from baseline in the absence of other causes of hypotension (eg, cardiogenic shock). Septic shock is defined as severe sepsis with sepsis-induced hypotension that persists despite adequate fluid therapy, along with the presence of abnormalities manifested by underperfusion 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 a host's dysregulated response to infection. For clinical practice, organ dysfunction can be represented by an increase in the Dynamic Organ Failure Score (SOFA) of 2 points or more, which is associated with in-hospital mortality of more than 10%. Septic shock can be defined as a type of sepsis in which severe circulatory, cellular, and metabolic disturbances are associated with a higher risk of mortality than sepsis alone. Patients with septic shock may be clinically identified as a result of the need for vasopressors to maintain a mean arterial pressure of 65 mmHg. Art. or more and a serum lactate level of more than 2 mmol/L (>18 mg/dL) in the absence of hypovolemia.

The term "sepsis" as used herein 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). The term "sepsis" as used herein refers to all possible stages in the development of sepsis.

As used herein, the term "infection" within the scope of this invention means a pathological process caused by invasion of a normally sterile tissue or fluid by pathogenic or potentially pathogenic agents/pathogens, organisms and/or microorganisms, and refers preferably to infection(s) caused by (caused 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 this invention, a viral infection may be considered as an infection caused by a microorganism.

In addition, a subject having an infection may have more than one source of infection at the same time. For example, a subject having an infection may have a bacterial infection and a viral infection; viral infection and fungal infection; a bacterial and fungal infection, as well as a bacterial infection, a fungal infection and a viral infection, or have a mixed infection that includes one or more of the infections listed herein, including potentially superinfection, for example, 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 investigated 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 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, Enterocococcus, Enterecococcus, such as Enterecococcus, Enterecococcus, such as Enterecococcus , 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, Mycobacte rium 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 pneumoniae. 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 their ability to cause 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 disease caused by a pathogenic fungus. Aspergillus can cause disease in three main ways: through the production of mycotoxins; as a result of the induction of allergic reactions; and as a result of localized or systemic infections. In the case of the last two categories, the immune status of the host is of decisive importance. The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus. Aspergillus flavus produces aflatoxin, which is both a toxin and a carcinogen and has the potential to lead to food contamination. Aspergillus fumigatus and Aspergillus clavatus can cause disease. Cryptococcus neofbrmans can cause disease in humans. Cryptococcus neofbrmans is a major human and animal pathogen. Cryptococcus laurentii and Cryptococcus albidus have been known to inadvertently cause moderate to severe disease in immunocompromised human patients. Cryptococcus gattii is endemic to tropical regions of Africa and Australia and can cause disease. Histoplasma capsulatum can cause histoplasmosis in humans, dogs, and cats. Pneumocystisjirovecii (or Pneumocystis carinii) can cause a form of pneumonia in people with weakened immune systems, such as premature babies, the elderly, and patients with AIDS. Stachybotrys chartarum or "black mold" can cause respiratory damage and severe headaches.

In one embodiment, the infection to be detected or investigated 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 Bacillus anthracis albicans, Staphylococcus aureus, Candida dubliniensis, Staphylococcus epidermidis, Candida glabrata, Staphylococcus haemolyticus, Candida krusei, Staphylococcus hominis, Candida parapsilosis, Staphylococcus saccharolyticus, Candida tropicalis, Staphylococcus wameri, Capnocyt ophaga 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 pseudotuberculosis and Klebsiella oxytoca. In accordance with 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 receive medical treatment.

In the context of this invention, the term "medical treatment" or "treatment" includes various treatments and therapeutic strategies, which include, but are not limited to, anti-inflammatory strategies, administration of ADM antagonists such as therapeutic antibodies, si-RNA or DNA, extra corporeal blood purification or removal of harmful compounds by apheresis, dialysis, adsorbents to prevent cytokine storm, removal of inflammatory mediators, plasmapheresis, administration of vitamins such as vitamin C, ventilation of the lungs, such as mechanical ventilation and non-invasive ventilation of the lungs to ensure body with sufficient oxygen, such as directed debridement, transfusion of blood products, colloid infusion, renal or hepatic replacement therapy, antibiotic therapy, invasive mechanical ventilation, non-invasive mechanical ventilation, vasopressors, fluid resuscitation, apheresis, and intervention ia to protect organs.

Additional treatments of the invention include administration of cells or cell products, e.g. stem cells, blood, or blood plasma, as well as stabilization of patient blood flow and protection of the endothelial glycocalyx, e.g. through optimal fluid therapy strategies, e.g. to achieve normovolemia and prevention or treatment of hypervolemia or hypovolemia. In addition, vasopressors or, for example, catecholamine as well as albumin, or heparanase inhibition with unfractionated heparin or N-desulfated pe-N-acetylated heparin are suitable treatments for maintaining blood flow and the endothelial layer.

In addition, the medical treatments of this invention include, but are not limited to, blood coagulation, iNOS inhibitors, anti-inflammatory agents such as hydrocortisone, sedatives and analgesics, and insulin.

The term "renal replacement therapy" (RRT) refers to therapy that is used to replace the normal function of the kidneys to filter the blood. Renal replacement therapy may refer to dialysis (eg, hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration. These techniques are different ways of directing blood into the device, 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 in which 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 venovenous route (in which blood leaves a vein and returns to a vein). This leads to different types of RRTs. 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 (CVVH or CVVHF), continuous hemodiafiltration (CHDF), continuous arteriovenous hemodiafiltration (CAVHDF), continuous venovenous hemodiafiltration (CVVHDF), intermittent renal replacement therapy (IRRT), intermittent hemodialysis (IHD) ), intermittent venovenous hemodialysis (IWHD), intermittent hemofiltration (IHF), intermittent venovenous hemofiltration (IVVH or IVVHF), intermittent hemodiafiltration (IHDF), and intermittent venovenous hemodiafiltration (IVVHDF).

Artificial and mechanical ventilation are effective approaches to enhance appropriate gas exchange and ventilation and facilitate life saving during severe hypoxemia. Mechanical ventilation refers to facilitating 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 mechanical endowment or replacement of spontaneous breathing. It may include a device called a fan. Mechanical ventilation can be high frequency oscillatory ventilation or partial fluid ventilation.

The term "fluid therapy" refers to the monitoring and control of a subject's fluid status and the administration of fluids to stabilize blood flow or maintain organ viability, for example, by oral, enteral, or intravenous fluid administration. It includes the stabilization of fluid and electrolyte balance or the prevention or correction of hyper- or hypovolemia, as well as the supply of blood products.

Emergency surgery/surgical emergency is necessary if the subject has an emergency and immediate surgery may be required to maintain survival or health status. The subject in need of emergency surgery may be selected from the group consisting of those with acute trauma, active uncontrolled infection, organ transplantation, organ prophylaxis or organ stabilization surgery, or cancer.

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

In the case of a critical illness 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 optimal therapy and control. Therapeutic approaches must be very individual and differ from case to case. Therapy monitoring is essential for best practice therapy and is influenced by the timing of treatment, the use of combination therapies, and the optimization of drug dosing. Incorrect or omitted therapy or control will lead to increased mortality at any time.

The medical treatment of the present invention may be antibiotic therapy, wherein 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.

Antibiotics or antibiotic agents in accordance with this invention also potentially cover antifungal or antiviral compounds used to treat diagnosed infection or sepsis. Antibiotic agents widely used in the treatment of a particular infection, when divided into pathogen classes, are presented below.

Action against gram-positive bacteria: penicillins (ampicillin, amoxicillin) resistant to penicillinase (dicloxacillin, oxacillin), cephalosporins (1st and 2nd generation), macrolides (erythromycin, clarithromycin, azithromycin), quinolones (gatifloxacin, moxifloxacin, levofloxacin), vancomycin, sulfonamide/trimethoprim, clindamycin, tetracyclines, chloramphenicol, linezolid, synercid.

Action against gram-negative bacteria: broad-spectrum penicillins (ticarcillin, clavulanate, piperacillin, tazobactam), cephalosporins (2nd, 3rd and 4th generation), aminoglycosides, macrolides, azithromycin, quinolones, (ciprofloxacin), monobactams ( azetreonam), sulfonamide/trimethoprim, carbapenems (imipenem), chloramphenicol.

Action against Pseudomonas: ciprofloxacin, aminoglycosides, some 3rd generation cephalosporins, 4th generation cephalosporins, broad-spectrum penicillins, carbapenems.

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

Antivirals: abacavir, acyclovir (acyclovir), activated caspase oligomerizer, adefovir, amantadine, amprenavir (agenerase), ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, double-stranded RNA, docosanol, edoxudin, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliever, famciclovir, fixed dose combination (antiretroviral), fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitor, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indavir, inosine, integrase inhibitor, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxidine, metisazon, morpholinos, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogs, novir, oseltamivir (tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor (pharmacology), ralteglavir, reverse transcriptase inhibitor, ribavir in, ribozymes, rifampicin, rimantadine, ritonavir, RNase H, protease inhibitors, pyramidin, saquinavir, sofosbuvir, stavudine, synergistic enhancer (antiretroviral), telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (valtrex ), valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza), zidovudine.

In addition, antibiotic agents include bacteriophages for treating bacterial infections, synthetic antimicrobial peptides, or iron antagonists/iron chelators. In addition, therapeutic antibodies or antagonists against 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 critically important therapies. sick patients such as patients with sepsis. Additional antibiotic agents/treatments or therapeutic strategies against infection or to prevent new infections include the use of antiseptics, decontamination products, antivirulence agents such as liposomes, debridement, wound care, surgery.

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

In accordance with the present invention, proADM and optionally PCT and/or other markers or clinical rating scales are used as markers for monitoring therapy, including diagnosis, prognosis, risk assessment, and risk stratification of the subsequent adverse event in the health status of the patient who has been diagnosed. as critically ill.

A person skilled in the art is able to obtain or develop means for detecting, measuring, detecting and/or quantifying any of the above proADM molecules or fragments or variants thereof, as well as other markers of this invention in accordance with standard molecular biology practice.

The level of proADM or fragments thereof, as well as the levels of other markers of this invention can be determined using any assay that reliably determines the concentration of the marker. In particular, mass spectrometry (MS) and/or immunological assays can be used as illustrative 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 or immunoglobulin binding fragment.

Methods for determining proADM or other markers such as PCT used in the context of this invention are provided in this invention. As an example, a method selected from the group consisting of mass spectrometry (MS), immunoluminescent assay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay (EIA), enzyme-linked immunosorbent assays (ELISA) ), luminescent bead chips, magnetic bead chips, protein microarray assays, rapid test formats such as, for example, immunochromatographic test strips, rare earth cryptate assays, and automated systems/analyzers. Determination of proADM and optionally other markers based on antibody recognition is a preferred embodiment of the present invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunologically reacts) an antigen. In accordance with this 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 the molecule of interest, such as proADM or a fragment thereof, is at least 50 times greater, preferably 100 times greater, most preferably at least 1000 times greater 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 specific specificity. In the context of this invention, monoclonal antibodies are preferred. An antibody or antibody binding fragment specifically binds to the markers or fragments thereof defined herein. In particular, an antibody or antibody binding fragment binds to ADM or proADM peptides as defined herein. Thus, the peptides defined herein may also be epitopes to which antibodies specifically bind. In addition, the antibody or antibody binding fragment is used in the methods and kits of this invention that specifically bind to ADM or proADM, in particular SF-proADM.

In addition, the 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 other markers of the invention, such as PCT. Illustrative immunoassays may be immunoluminescent assay (LIA), radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme immunoassay (EIA), enzyme-linked immunosorbent assays (ELISA), luminescent beads chips, magnetic beads chips, assays based on protein microarrays, express test formats, analysis using rare earth metal cryptates. In addition, assays suitable for point-of-care testing and rapid test formats, such as, for example, immunochromatographic test strips, may be used. Automated immunoassays such as the KRYPTOR assay are also provided.

Alternatively, instead of antibodies, other capture molecules or molecular scaffolds that specifically and/or selectively recognize proADM may be included within the scope of this invention. 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, SF-proADM, and PCT) from a sample . Thus, capture molecules must have an appropriate shape, both spatially and in terms of surface characteristics such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and/or acceptors, 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 interactions, or a combination of two or more of the above interactions, or covalent interactions between capture molecules or molecular scaffolds 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). Affimers and the like are included.

In certain aspects of this 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 the second epitope of said proADM, and

b) detecting the 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 this assay, one of the antibodies is labeled while the other is either bound to the solid phase or may be selectively bound to the solid phase. The first antibody and the second antibody may be present dispersed in a liquid reaction mixture, wherein the first labeling component, which is part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, is associated with the first antibody, and the second labeling component of said labeling system is associated with the second antibody in such a way that after the binding of both antibodies to the specified proADM or its fragments to be detected, a measurable signal is formed, which ensures the detection of the resulting sandwich complexes in the measurement solution. The labeling system may contain a rare earth metal cryptate or chelate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

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

The method of the invention may further be carried out as a homogeneous method, wherein the sandwich complexes formed by the antibody/antibodies and the marker, proADM or fragment thereof, to be detected remain suspended in the liquid phase. In this case, it is preferable to use two antibodies, both antibodies being labeled with parts of the detection system, resulting in signal generation or signal activation if both antibodies are integrated into one sandwich. Such techniques are carried out, in particular, in the form of detection methods based on fluorescence enhancement or fluorescence quenching. A particularly preferred aspect relates to the use of detection reagents that must be used in pairs, such as, for example, those described in US 4,882,733, EP 0,180,492 or EP 0,539,477 and the prior art cited herein. In this regard, measurements become possible in which only reaction products containing both labeling components in the same immune complex in the reaction mixture are detected. For example, such technologies are offered under the trade names TRACE® (Time Resolved Amplified Cryptate Emission) or KRYPTOR®, embodying the ideas of the applications mentioned above. Thus, in certain preferred aspects, a diagnostic device is used to implement the method proposed herein. For example, the level of proADM protein or fragments thereof and/or the level of any additional marker of the method proposed herein, such as PCT, is determined. In certain preferred aspects, the diagnostic device is a KRYPTOR®.

The level of the marker according to this invention, for example, proADM or fragments thereof, PCT or fragments thereof or other markers can also be determined using methods based on mass spectrometry (MS). Such a method may include detecting the presence, amount, or concentration of one or more peptides of modified or unmodified fragments, such as proADM or PCT, in said biological sample, or cleaving a protein (e.g., tryptic cleavage) from said sample, and optionally separating the sample using chromatographic techniques and processing the resulting and optionally a separated sample for MS analysis. For example, selective reaction monitoring (SRM), multiple reaction monitoring (MRM), or parallel reaction monitoring mass spectrometry (PRM) can be used in MS analysis, in particular to quantify proADM or fragments thereof.

In this document, the term "mass spectrometry" or "MS" refers to an analytical technique that allows the determination of compounds by their mass. To improve the mass resolution and mass determination capabilities of mass spectrometry, samples can be processed prior to MS analysis. Accordingly, this invention relates to MS detection methods that can be combined with immunological enrichment 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. chromatography (UHPLC). Sample preparation methods include lysis, fractionation, sample digestion to peptides, depletion, enrichment, dialysis, desalting, alkylation, and/or peptide reduction techniques. At the same time, these steps are optional. Selective detection of analyte ions can be performed using tandem mass spectrometry (MS/MS). Tandem mass spectrometry is characterized by a mass selection step (as used herein, "mass selection" refers to the isolation of ions having a certain m/z value or a narrow range of m/z), followed by fragmentation of the selected ions and analysis of the ion mass of the resulting product (fragments). One skilled in the art will know how to quantify the level of a marker in a sample using mass spectrometric techniques. For example, "rSRM" relative scoring or absolute scoring can be used as described above.

In addition, levels (including reference levels) can be determined using mass spectrometry-based methods, such as relative quantitation or absolute quantitation of the protein or fragment of interest. Relative quantification of "rSRM" can be achieved as follows.

1. Determination of increased or reduced presence of a target protein by comparing the peak area of the SRM characteristics (selective response monitoring) of a certain target fragment peptide detected in the sample with the same peak area of the SRM characteristics of the target fragment peptide in at least the second, third, fourth or more biological samples.

2. Determination of increased or decreased presence of a target protein by comparing the peak area of SRM characteristics from a given target peptide detected in a sample with the peak areas of SRM characteristics derived from peptide fragments from other proteins in other samples obtained from different and separate biological sources, wherein a comparison of the peak area of the SRM characteristics between two samples for a peptide fragment is normalized, for example, to the amount of protein analyzed in each sample.

3. Determination of increased or decreased presence of a target protein by comparing the peak area of SRM characteristics for a particular target peptide with the peak areas of SRM characteristics from other peptide fragments derived from different proteins in the same biological sample in order to normalize changing levels of histone proteins to levels of others proteins that do not change their expression levels under different cellular conditions.

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

Absolute quantification of a particular peptide can be achieved as follows.

1. Comparison of the peak area of SRM/MRM characteristics for a peptide of a certain fragment from target proteins in a separate biological sample with the peak area of SRM/MRM characteristics of an internal standard of a peptide fragment introduced into a protein lysate from a biological sample. The internal standard may be a labeled synthetic peptide version of a fragment from the target protein under study or a labeled recombinant protein. The specified standard is added to the sample in known amounts before (required for a recombinant protein) or after digestion, and the peak area of the SRM/MRM characteristics can be determined for both the internal standard of the fragment peptide and the native fragment peptide in the biological sample separately, and then comparing both peak areas. This can be applied to both unmodified fragment peptides and modified fragment peptides, modifications including, but not limited to, phosphorylation and/or glycosylation, acetylation, methylation (e.g., mono-, di-, or tri-methylation) , citrullination, ubiquitinylation, and while the absolute levels of the modified peptides can be determined in the same way as the determination of the absolute levels of unmodified peptides.

2. Peptides can also be quantified using external calibration curves. The normal curve approach uses a constant amount of heavy peptide as an internal standard and a varying amount of light synthetic peptide added to the sample. An exemplary matrix, similar to the matrix of the samples under study, should be used to construct standard curves in order to take into account the effect of the matrix. In addition, the inverse curve method overcomes the problem of endogenous analyte in the matrix, whereby a constant amount of light peptide is added on top of the endogenous analyte to create an internal standard, and varying amounts of heavy peptide are added to create a set of concentration standards. The same amount of standard peptide was added to test samples for comparison with normal or reverse curves as the internal standard added to the matrix used to create the calibration curve. This invention also relates to kits, the use of kits and methods in which such kits are used. The present invention relates to kits for carrying out the methods described herein above and below. The definitions provided herein, such as those proposed for methods, also apply to the kits of this invention. In particular, this invention relates to kits for monitoring therapy, including prognosis, risk assessment or risk stratification of a subsequent adverse event in a patient's health status, while this kit contains

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

- reference data, such as a reference level corresponding to high and/or low levels of severity of proADM, with low severity being less than 4 nmol/l, preferably less than 3 nmol/l, more preferably less than 2.7 nmol/l, and high the severity level 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 reference data being preferred stored on a computer-readable medium and/or used in the form of a computer executable code configured to compare certain levels of proADM or fragment(s) thereof and optionally certain levels of PCT, lactate and/or C-reactive protein or fragment(s) thereof with said reference data.

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

The kit may additionally contain objects suitable for obtaining a sample, such as a blood sample, for example, the kit may contain a container, while the specified container contains a device for attaching the specified container to a cannula or syringe, is a syringe suitable for bleeding, has an internal pressure below atmospheric pressure, one that is suitable for drawing a predetermined volume of sample into said container, and/or contains additional detergents, chaotropic salts, ribonuclease inhibitors, chelating agents such as guanidine isothiocyanate, guanidine hydrochloride, sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate, inhibitor proteins RNases and their mixtures and/or filter system containing nitrocellulose, silica matrix, ferromagnetic spheres, excess removal vessel, trehalose, fructose, lactose, mannose, polyethylene glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, aniline, pi prrol, citrate and mixtures thereof.

As used herein, the term "detection reagent" or the like are reagents that are suitable for detecting the marker(s) described herein, e.g., proADM, PCT, lactate, and/or C-reactive squirrel. 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 contained in the kit and are referred to herein as detection reagents. Detection reagents can also refer to reagents that are used to detect markers or fragments thereof using MS-based methods. Thus, such a detection reagent may 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 in accordance with this invention can also be a calibration solution(s), for example, which(which) can be used to determine and compare the level of the marker(s).

The sensitivity and specificity of a diagnostic and/or prognostic study depends on more than just the analytical "quality" of the study, they also depend on the definition of what constitutes an abnormal result. In practice, receiver operating characteristic curves (ROC curves) are typically calculated by plotting the value of a variable against its relative frequency in "normal" (i.e., outwardly healthy) individuals without infection and in populations of "diseased" for example, subjects having an infection. For any given marker (such as ADM), the distribution of marker levels for subjects with and without the disease/condition is likely to overlap. Under such conditions, the study does not completely distinguish the norm from the disease with 100% accuracy, and the area of overlap may indicate where the study cannot distinguish the norm from the disease. The boundary value is chosen such that below it, the study is considered abnormal, and above it, the study is considered normal, or below or above it, the study indicates a certain pathological condition, for example, an infection. The area under the ROC curve represents the probability value that the proposed measurement will correctly determine the pathological condition. ROC curves can be used even if test results do not necessarily give an accurate number. As long as you can rank the results, you can plot the ROC curve. For example, the results of the study of "pathological" samples can be ranked according to the degree (for example, 1=low, 2=normal and 3=high). This ranking can be correlated with results in a "normal" population and an ROC curve is plotted. These methods are well known in the art; see, for example, Hanley et al. 1982. Radiology 143: 29-36. Preferably, the cutoff value is chosen to provide an ROC curve area of greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.85. 0.9. The term "approximately" in this context refers to +/- 5% of a particular measurement.

The horizontal axis of the ROC curve represents (1 specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positive results. Thus, for a certain selected cutoff value, a value (1 specificity) can be determined and an appropriate sensitivity can be obtained. The area under the ROC curve represents the probability that the measured level of the marker will correctly identify the disease or pathological condition. Thus, the area under the ROC curve can be used to determine the performance of a study.

Accordingly, this invention includes the introduction of an antibiotic suitable for treatment, based on information obtained using the method described in this document.

As used herein, the terms "comprising" and "comprising" or their grammatical variants should be understood as describing the specified properties, integers, steps or components, but not excluding the addition of one or more additional properties, integers, steps, components, or groups thereof. . This term encompasses the terms "consisting of" and "consisting essentially of". Thus, the terms "comprising"/"comprising"/"having" mean that any additional component (or similar properties, integers, steps, etc.) may be present. The term "consisting of" means that an additional component (or similar properties, integers, stages, etc.) is not present. The term "consisting essentially of" or its grammatical variants when used in this document should be understood as describing the specified properties, integers, steps or components, but not excluding the addition of one or more additional properties, integers, steps, components or groups thereof, but only if the additional properties, integers, steps, components, or groups thereof do not significantly change the main and new characteristics of the claimed composition, device, or method.

Thus, the term "consisting essentially of" means that certain additional components (or similar properties, integers, steps, etc.) may be present, namely components that do not significantly affect the main characteristics of the composition, device or method. In other words, the term "consisting essentially of" (which may be used interchangeably herein with the term "comprising essentially") allows for the presence of other components in a composition, device, or method in addition to the required components (or similar properties, integers, stages, 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 techniques, tools, techniques and procedures for performing a specific task, including, but not limited to, such techniques, tools, techniques and procedures, either known or easily developed from known techniques, tools, techniques. and procedures by practitioners of the chemical, biological and biophysical fields. The present invention is further described with reference to the following non-limiting examples.

Examples

Ways of examples

Study Design and Patients

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

Biomarker measurements

Patients were enrolled within 24 hours of diagnosis of severe sepsis or septic shock, and PCT, CRP, and lactate levels were measured immediately thereafter. The PCT level was measured on instruments with a measuring range of 0.02-5000 ng/ml and functional assay sensitivity and lower detection limit of at least 0.06 ng/ml and 0.02 ng/ml, respectively. Additional blood samples from all patients were collected and stored at 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. Clinical severity scores, including the Dynamic Organ Failure Score (SOFA), the Acute and Chronic Functional Change Score (APACHE) II, and the Simplified Acute Functional Change Score (SAPS) II, were determined at study entry.

Statistical analysis

Differences in demographic and clinical characteristics with respect to 28-day mortality were assessed using χ2 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 variables were expressed as mean (standard deviation) and median [first quartile - third quartile], respectively. The association between mortality and each biomarker and clinical rating scale at all time points was assessed using the area under the receiver operating characteristics 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, intermediate, and high) based on the calculation of two population-wide AUROC thresholds for each biomarker and a clinical scoring scale at each time point with a predetermined sensitivity and specificity close to 90%. The subgroup of clinically stable patients was subsequently defined as those without any ICU-related procedures or complications (including directed cleaning procedures, emergency surgery, new infections, transfusion of blood products, colloid infusion, invasive mechanical ventilation, renal/ liver replacement therapy or vasopressor therapy and worsening of the patient's general clinical signs and symptoms), and an additional group was defined as patients with corresponding low concentrations of SF-proADM who 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 specific time point.

Ultimately, two models were created stratifying patients with changes in PCT levels of 20% (from baseline to day 1, based on the average decrease in PCT levels observed over a specified time period) and 50% (from baseline to day 4, based on the previously created model (26)). Patient subgroups were subsequently defined based on the severity levels of SF-proADM and the corresponding mortality rates were calculated. 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 guided cleaning procedures and emergency surgery within 4-7 days subsequently examined the models from baseline to day 4. All data were analyzed using statistical software R (version 3.1.2).

Example 1 Patient Characteristics

Characteristics of patients at inclusion in the study are presented 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. Patients included in the study had a mean age of 65.7 (13.7) years and a mean SOFA score of 10.0 (3.3) points. The all-cause mortality rate at 28 days (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 pulmonary (N=324; 30.1%), intraperitoneal (N=252; 23.4%), urogenital (N=57; 5.3%) and bone/soft tissue (N=50; 4.6%) origin was the most common. The corresponding mortality rates were 26.5%, 24.6%, 22.8% and 28.0%, respectively. Multiple origin of infection 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 an overall mortality rate of 8.6%. Restriction therapy was used in 3.4% of patients.

Example 2 Association of Baseline Biomarkers and Clinical Rating Scales with Mortality

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

Similar results were also obtained for 7-day, 90-day prediction of ICU mortality and hospital mortality (Table 3), with the addition of SF-proADM to all potential biomarkers and combinations of clinical rating scales (N=63), significantly increasing predictive power ( Table 4).

Example 3 Definition of High Risk Patients

The overall patient population was further stratified according to existing levels of SOFA severity as well as characteristics of biomarkers and clinical scoring in predicting 28-day mortality assessed in each subgroup. The level of SF-proADM showed the highest accuracy among all parameters in the subgroups of low (SOFA ≤7) and moderate (8 ≤ SOFA ≤13) severity of SOFA (Table 5; Table 6).

Subsequently, two corresponding breakpoints of SF-proADM were calculated to define subgroups of low (≤2.7 nmol/L) and high (>10.9 nmol/L) severity at baseline. Compared to SOFA, more accurate reclassification could be performed at both 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 breakpoints (Table 7).

A subgroup of 94 patients (9.3%) with high SF-proADM concentrations and corresponding low or intermediate SOFA values had 28- and 90-day mortality rates of 57.4% and 68.9%, respectively, compared with 19.8 % and 30.8% in the remaining patient population with low and intermediate SOFA values. Similar patterns could be found for SAPS II, APACHE II and lactate levels, respectively (Table 8-10).

Example 4 Identification of low-risk patients during ICU stay

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

The level of SP-proADM showed the strongest association with 28-day mortality at all subsequent time points (Table 11) and could provide a stable breakpoint of ≤2.25 nmol/L in defining the low-risk patient population, leading to classification more patients with lower mortality rates compared to other biomarkers and clinical rating scales (Table 12). Accordingly, 290 patients with low severity SF-proADM could be identified on day 4, 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 intermediate to low severity could be observed in 28 (35.4%) patients. The median ICU stay was 8 [7-10] days, and the 28- and 90-day mortality rates were 0.0% and 1.4%, respectively. By comparison, only 43 patients were actually discharged from the ICU on Day 4, with 28- and 90-day mortality rates of 2.3% and 10.0%. Analysis of the concentrations of SF-proADM in this group of patients showed a range of values, with 20 (52.6%), 16 (42.1%) and 2 (5.3%) patients having low, intermediate and high concentrations of severity, respectively. Similar results were obtained for patients who remained in the ICU on days 7 and 10. The level of SF-proADM with a stable breakpoint of ≤2.25 nmol/L could determine more patients at low risk with lower mortality rates compared with other biomarkers and clinical rating scales. Based on this, more patients could be discharged from the ICU compared to classifications without ADM. By discharging more patients, the hospital can allocate ICU beds more efficiently and benefit from avoidable costs.

Example 5 Additional effect of SF-proADM on procalcitonin 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 resulting in significantly stronger associations with 28- daily mortality (Table 14).

Thus, two models of algorithms using PCT were created, examining changes in PCT from baseline to either day 1 or day 4, with appropriate subgroup analyzes performed based on SF-proADM severity classifications.

Patients with a decrease in PCT concentration of ≥20% from baseline to day 1 (Table 15 and Table 16) or ≥50% from baseline to day 4 (Table 17 and Table 18) were found to have 28 -day mortality rate of 18.3% (N=458) and 17.1% (N=557), respectively. This indicator decreased to 5.6% (N=125) and 1.8% (N=111) if patients had persistently low levels of SF-proADM, but increased to 66.7% (N=27) and 52, 8% (N=39) in patients with consistently high SF-proADM (HR [95% CI]: 19.1 [8.0-45.9] and 43.1 [10.1-184.0]) .

In addition, patients with a decrease in PCT values by ≥50% (from baseline to day 4), but persistently high or intermediate concentrations of SF-proADM, had a significantly higher risk of developing subsequent nosocomial infections (HR [95% CI]): high concentrations: 3.9 [1.5-10.5]; intermediate concentrations: 2.4 [1.1-5.1] compared with patients with persistently low concentrations; intermediate concentrations: 2.9 [1.2–6.8]) or decrease from intermediate to low concentrations) or need for emergency surgery (HR [95% CI]: intermediate concentrations: 2.0 [1.1–3 ,7] compared to the reduction from intermediate to low concentrations). In contrast, patients with elevations from intermediate to high concentrations were more likely to require clearance of infection compared to patients with persistently intermediate levels (HR [95% CI]: 3.2 [1.3-7.6]) or declining values (RR [95% CI]: intermediate to low: 8.7 [3.1-24.8]); high to intermediate: 4.6 [1.4-14.5]). If PCT levels did not decrease by ≥50%, then there was a significant increase in the risk of requiring emergency surgery if SF-proADM concentrations were either at persistently high (HR [95% CI]: 5.7 [1.5-21.9] ), or intermediate (RR [95% CI]: 4.2 [1.3–13.2]) as opposed to persistently low.

Example 6 Association of Baseline Biomarkers and Clinical Rating Scales with Mortality

The level of SF-proADM showed the strongest association in patients with pulmonary and intraperitoneal infections, as well as in patients with gram-positive infections, regardless of the infectious focus (Table 19-20). When patients were grouped according to history of surgical emergency, non-operative emergency, and elective surgery leading to ICU admission, SF-proADM level 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 Value at Baseline and Day 1

SF-proADM had the highest correlation of all biomarkers with the SOFA score at baseline, which increased significantly when baseline values correlated with SOFA scores on day 1. The highest correlation could be found between SF-proADM and SOFA at baseline. day 10, with differences between the individual SOFA score subscales found throughout (Table 22-24).

Example 8 Definition of High Risk Patients

Similar results may have occurred in a subgroup of 124 patients (12.0%) with high SF-proADM levels and low or intermediate SAPS II values (high SF-proADM subgroup: [mortality 54.8% and 65.6%]); the remaining population with SAPS II [mortality 19.7% and 30.0%]), as well as in 109 (10.6%) patients with low or intermediate APACHE II values (high SF-proADM subgroup: [mortality 56, 9% and 66.7%]; remaining population with APACHE II: [mortality 19.5% and 30.3%]).

Example 9 Improved Procalcitonin (PCT) Therapy by Combining PCT and ADM

Two models of PCT algorithms were designed to examine changes in PCT from baseline to either day 1 or day 4, with appropriate subgroup analyzes performed based on SF-proADM severity classifications (Tables 25-30). The previous examples show added value for ADM in patients who have a <20% or <50% decrease in PCT levels, as well as in patients who have a PCT level decrease of ≥20% or ≥50%. However, additional analysis shows that ADM may represent an additional element regardless of the % reduction or even increase in PCT. Decreased PCT values may have reflected patients in whom antibiotic therapy appeared to be effective and where the clinician believed they were on the path to survival (i.e., elimination of the underlying cause of sepsis, bacteria, should result in improvement patient). For example, some patients had reduced PCT levels from baseline (day of admission) to day 1, with a 28-day mortality rate of 19%. When additionally measuring ADM, based on patients with low ADM, it can be concluded that there is a much higher probability of survival or a much lower probability of death (Table 25; compare 19% decrease in PCT level alone and 5% decrease in PCT level + low level ADM). As a result of the reduced risk of death, patients could be discharged from the ICU with greater certainty, or fewer diagnostic tests were required (i.e., if they were known to be on their way to recovery).

On the other hand, new interventions should be considered in patients with high ADM. They are at much higher risk in terms of mortality (compare only PCT reduction at 19% mortality rate with PCT level + high ADM at 58.8% mortality rate). According to the doctor, the patient's condition improves 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 is not effective and needs to be adapted as soon as possible. Similarly, the ADM level may facilitate the stratification of patients with elevated PCT values (Table 25).

Development of new infections

Changes in PCT and SF-proADM levels were analyzed in two models, either from baseline to day 1 or from baseline to day 4. Patients were grouped according to overall changes in PCT levels and SF-proADM severity 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 after the discharge of new infections did not develop. Patients with multiple infections during observation days were considered to have one new infection.

As a clinical outcome, 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 for targeted cleaning

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

The number of targeted clearing events 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.

The need for emergency surgery

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

The number of emergency surgery needs/events 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 for change or modification of antibiotics

When combined with an antibiotic therapy algorithm using a PCT level, the level of SF-proADM may help to stratify those patients who will require further change or modification of antibiotic therapy from patients who will not.

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

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

In patients with a decrease in PCT values by >50%

Patients with elevated levels of SF-proADM, from low to intermediate to moderate severity, were more likely to require modification of antibiotic therapy on day 4 than patients who had persistently low levels (hazard ratio [95% CI]: 1.5 [0.6 -4.1]).

In patients with a decrease in PCT values by <50%

Patients with either elevated levels of SF-proADM, from intermediate to high severity, or persistently high concentrations, were also more likely to require changes in antibiotic therapy on day 4 than those with persistently low levels of SF-proADM (hazard 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 concentrations of SP-proADM had significantly less modification to their antibiotic therapy than patients with persistently intermediate or high concentrations.

As a clinical outcome, in the presence of increased PCT concentrations, the clinician should check the patient's SP-proADM levels before deciding to change antibiotics. For patients with low concentrations of SF-proADM, either an increased dose or an increased concentration of the same antibiotic should be considered before changes are considered. For patients with higher concentrations of SF-proADM, earlier antibiotic changes should be considered (ie, on days 1 to 3 as opposed 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 insufficient treatment may lead to an overall worsening of the patient's clinical condition, causing further treatment to become less effective and increase the likelihood of a less effective 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 Score (SOFA) now being identified as the most appropriate tool, resulting in a central role in the definition of sepsis. 3 2016 (4). In this secondary analysis, the SISPCT study (26) compared for the first time sequential measures of standard biomarkers and clinical scoring scales such as lactate, procalcitonin (PCT) and SOFA with measures of a marker of microcirculatory dysfunction, SF-proADM, in a large population of patients with severe sepsis and septic shock.

Our results show that initial use of SF-proADM within the first 24 hours of sepsis diagnosis resulted in the strongest association with short-, medium- and long-term mortality compared to all other biomarkers or scores. Previous studies largely support our findings (17, 28, 29), however conflicting results (30) may be partly explained by the smaller sample size analyzed, as well as other factors noted in the study, such as microbial species, origin 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 in outcomes in small study populations. In addition, our study also significantly confirms the results of a previous study (17), indicating the highest efficacy of SF-proADM in patients with low and intermediate severity of organ dysfunction. Indeed, Andaluz-Ojeda et al. (17) attach great importance to the group of patients with low levels of organ dysfunction because "this group represents either the earliest manifestation of the clinical course of sepsis and/or a less severe form of the disease." However, acceptable rates can be maintained in all severity groups in terms of predicting mortality, which was also the case in both groups of patients defined according to the criteria for sepsis-3 and septic shock-3.

Analysis of serial measurements taken after the onset of sepsis made it possible to identify certain groups of patients based on the severity of the disease. The definition of low and high risk patients was of significant interest in our analysis. In many ICUs, the need for beds may occasionally exceed availability, which can lead to insufficient triage, rationing of resources, 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 mortality who may qualify for early discharge from the ICU to the postoperative unit may be of significant benefit. At each time point measured in our study, the level of SF-proADM could determine a higher number of patients with low severity with the shortest ICU stay, hospital and 28-day mortality rates. Further analysis of the group of patients with low severity and no additional specific ICU therapy indicated that an additional 4 days of ICU stay was observed at each time point after biomarker measurements were taken. Compared to a population of patients who were actually discharged at each time point, a biomarker approach to accurately identify patients with low severity resulted in lower 28- and 90-day mortality rates. Indeed, patients who were discharged had a range of low, intermediate, and high severity concentrations of SF-proADM, which subsequently resulted in higher mortality. At the same time, it is not known whether a few more patients in this group required further ICU treatment for non-microcirculatory, non-life-threatening problems, or whether these post-operative beds were available. However, such a biomarker approach for ICU discharge, in addition to the clinician's clinical assessment, may improve proper patient stratification, with associated clinical benefits and potential cost savings.

Alternatively, 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 algorithm in SISPCT and other studies (26, 36, 37), but relatively high mortality rates can still be observed even when PCT values appear to be constant. are declining. Our study showed that the addition of SF-proADM to the model of PCT reduction on subsequent days in the ICU allowed for the identification of groups of patients with low, medium and high risk, while the increase and decrease in the severity of SF-proADM from baseline to day 1 provided a sensitive and an early indicator of treatment efficacy. In addition, anticipating the need for future guided cleaning or emergency surgery, as well as susceptibility to developing new infections, can be a significant advantage in initiating additional therapeutic and interventional strategies, thereby attempting to prevent any subsequent clinical complications at an early stage. The potential of our study includes a thorough examination of several different low- and high-severity subgroups from a database of randomized trials, taking into account possible confounding factors, and including the largest sample size of patients with sepsis characterized by both SEPSIS 1 and 3 definitions and information about kinetics of SF-proADM.

In conclusion, SF-proADM is superior to other biomarkers and clinical scores for the ability to identify risk of mortality in patients with sepsis, both at initial diagnosis and during ICU treatment. Accordingly, SF-proADM can be used as a means to identify patients with high disease severity who may need alternative diagnostic and therapeutic interventions, and patients with low disease severity who could potentially be subject to early discharge from the ICU in combination with the absence of specific for ICU types of therapy.

tables

ICU: resuscitation and intensive care unit; COPD: chronic obstructive pulmonary disease; SF-proADM, mid-regional proadneromedullin; PCT: procalcitonin; CRP: C-reactive protein; SOFA: dynamic organ failure score; SAPS II: Simplified Acute Functional Changes Assessment Scale; APACHE II: acute and chronic functional changes score. Data are presented as absolute numbers and percentages in brackets, indicating the proportion of survivors and non-survivors at 28 days.

N: quantity; AUROC: area under the receiver operating curve; LR y2: RR: risk ratio; IQR: interquarter range. All multivariate analyzes were associated at p < 0.0001 with 28-day mortality.

OP IQR [95% CI] indicates the odds ratio for SF-proADM in each bivariate and multivariate model. 2 degrees of freedom in each 2D model compared to II in each multidimensional model.

N: quantity; AUROC: area under the receiver operating curve; LR χ2: RR: risk ratio; IQR: interquartile range.

SF-proADM: mid-regional proadrenomedullin; SOFA: dynamic organ failure score

SF-proADM: mid-regional proadrenomedullin; SAPS II: Simplified Assessment of Acute Functional Changes II

SF-proADM: mid-regional proadrenomedullin; APACHE II: Acute and Chronic Functional Change Scale II

SF-proADM: mid-regional proadrenomedullin

SF-proADM: mid-regional proadrenomedullin; DF: degrees of freedom

повышение от низких до промежуточных по сравнению с постоянно низкими значениями;

повышение от промежуточных до высоких по сравнению с постоянно промежуточными значениями;

снижение с высоких до промежуточных значения по сравнению с постоянно высокими значениями;

снижение с промежуточных до низких значений по сравнению с повышением от промежуточных до высоких значений.Hazard ratio for patients with * persistently intermediate to low values; ** consistently high compared to intermediate values***, consistently high compared to low values;

an increase from low to intermediate compared to persistently low values;

an increase from intermediate to high compared to constantly intermediate values;

decrease from high to intermediate values compared to persistently high values;

a decrease from intermediate to low values compared to an increase from intermediate to high values.

Kaplan-Meier plots either show individual subgroups of patients or group subgroups with an increase or decrease.

повышение от низких до промежуточных значений по сравнению с постоянно низкими значениями;

повышение от промежуточных до высоких значений по сравнению с постоянно низкими значениями;

снижение с высоких до промежуточных значений по сравнению с постоянно высокими значениями;

снижение с промежуточных значений до низких значения по сравнению с промежуточными значениями.Hazard ratio for patients with * persistently intermediate to low values; ** consistently high compared to intermediate values***, consistently high compared to low values;

increase from low to intermediate values compared to persistently low values;

increase from intermediate to high values compared to persistently low values;

decrease from high to intermediate values compared to persistently high values;

decrease from intermediate values to low values compared to intermediate values.

повышение от низких до промежуточных значений по сравнению с постоянно низкими значениями;

повышение от промежуточных до высоких значений по сравнению с постоянно низкими значениями;

снижение с высоких до промежуточных значений по сравнению с постоянно высокими значениями;

снижение с промежуточных значений до низких значения по сравнению с промежуточными значениями.Hazard ratio for patients with * persistently intermediate to low values; ** consistently high compared to intermediate values***, consistently high compared to low values;

increase from low to intermediate values compared to persistently low values;

increase from intermediate to high values compared to persistently low values;

decrease from high to intermediate values compared to persistently high values;

decrease from intermediate values to low values compared to intermediate values.

повышение от низких до промежуточных значений по сравнению с постоянно низкими значениями;

повышение от промежуточных до высоких значений по сравнению с постоянно низкими значениями;

снижение с высоких до промежуточных значений по сравнению с постоянно высокими значениями;

снижение с промежуточных значений до низких значения по сравнению с промежуточными значениями.Hazard ratio for patients with * persistently intermediate to low values; ** consistently high compared to intermediate values***, consistently high compared to low values;

increase from low to intermediate values compared to persistently low values;

increase from intermediate to high values compared to persistently low values;

decrease from high to intermediate values compared to persistently high values;

decrease from intermediate values to low values compared to intermediate values.

The AUROC values of SF-proADM are significantly higher than all other parameters, except for the APACHE II value in pulmonary infections.

Literary sources

1. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl 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 Syst Rev. 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. May 2002;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. Apr 2005;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 pro adrenomedullin as mortality predictor in severe sepsis. J Infect. Jul 2015;71(1): 136-139.

18. Hartmann O, 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. Int J 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, Irice C. Microcirculation in sepsis: new perspectives. Curr Vase 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 (46)

1. A method for predicting a subsequent adverse event in a patient's health, including providing a sample from said patient, whereby the patient has been diagnosed as critically ill and medical treatment has been initiated, whereby the sample has been isolated from the patient's body after the diagnosis has been made and treatment commenced, determination of the level of proadrenomedullin (proADM) or its fragment (s) in the specified sample, however, a low severity level of proADM or its fragment(s) detected in the sample, which is less than 4 nmol/l, indicates the absence of a subsequent adverse event, or however, a high level of severity of proADM or its fragment (s), determined in the sample, amounting to more than 6.5 nmol/l, indicates a subsequent adverse event. 2. The method of claim. 1, characterized in that the specified sample is isolated from the body of the specified patient within 10 days after the specified diagnosis and treatment. 3. The method according to any one of paragraphs. 1 or 2, characterized in that the critically ill patient is a patient with an infectious disease, a patient with a diagnosed infectious disease and one or more existing organ failures, a patient diagnosed with sepsis or septic shock, while the patient may have an injury and / or undergo surgical intervention. 4. The method according to any one of paragraphs. 1-3, characterized in that the adverse event in the state of health of the specified patient is death within 28 days or death within 90 days after diagnosis and initiation of treatment, a new infection, organ failure and / or worsening of clinical symptoms requiring a directed procedure cleaning, transfusion of blood products, colloid infusion, emergency surgery, invasive mechanical ventilation and/or renal or liver replacement therapy. 5. The method according to any one of paragraphs. 1-4, characterized in that the treatment received by the patient includes one or more of the types of antibiotic therapy, invasive mechanical ventilation of the lungs, non-invasive mechanical ventilation of the lungs, renal replacement therapy, the use of vasopressors, infusion therapy, extracorporeal blood purification and / or organ protection. 6. The method according to any one of paragraphs. 1-5, characterized in that said sample is selected from the group consisting of a blood sample, a blood serum sample, a blood plasma sample and/or a urine sample. 7. The method according to any one of paragraphs. 1-6, characterized in that the determination of the level of proADM or its fragment (fragments) includes the determination of the level of average regional proadrenomedullin (SF-proADM) in the sample. 8. The method according to p. 1, characterized in that the patients are patients of the intensive care unit (ICU), while a low severity level of proADM or its fragment(s) indicates that the patient has been discharged from the ICU, or a high level of severity of proADM or fragment(s) indicates a modification of the patient's treatment in the ICU. 9. The method according to claim 8, characterized in that the low severity level of proADM is less than 2.25 nmol/l and the specified sample is isolated from the patient's body 1 day or more after the specified diagnosis and treatment. 10. The method according to any one of paragraphs. 1-9, which additionally includes determining the level of PCT or its fragment (s) in the sample isolated from the patient's body. 11. The method according to any one of paragraphs. 1-10, including additionally: determining the level of PCT or fragment (s) in the first sample isolated from the patient's body, while the specified first sample is isolated at or before the time point of diagnosis and start of treatment, determination of the level of PCT or its fragment (s) in the second sample, corresponding to the sample according to paragraph 1, isolated from the body of the specified patient within 10 days after the diagnosis was made and the start of treatment, and determining whether the difference in the level of PCT or its fragment (s) in the second sample is obvious compared to the level of PCT or its fragment (s) in the first sample. 12. The method according to p. 11, characterized in that a lower level of PCT or its fragment(s) in the second sample compared to the first sample and a low level of severity of proADM or its fragment(s), with a low severity level of less than 4 nmol/L, indicating no subsequent adverse event, or a lower level of PCT or its fragment(s) in the second sample compared to the first sample and a high severity level of proADM or its fragment(s), with a high severity level of more than 6.5 nmol/l, indicate a subsequent adverse event. 13. The method according to p. 11, characterized in that an increased level of PCT or its fragment(s) in the second sample compared to the first sample and a low severity level of proADM or its fragment(s), with a low severity level of less than 4 nmol/l, indicate the absence of a subsequent adverse event, or an increased level of PCT or its fragment(s) in the second sample compared to the first sample and a high level of severity of proADM or its fragment(s), with a high severity level of more than 6.5 nmol/l, indicate a subsequent adverse event. 14. The method according to any one of paragraphs. 1-13, including additionally determining the level of proADM or fragment(s) in the first sample isolated from the patient's body, wherein said first sample is isolated at or before the time of diagnosis and initiation of treatment, and determining the level of proADM or fragment(s) thereof in a second sample isolated from said patient, said second sample being isolated after the first sample and after the time point of diagnosis and within 10 days of isolating the first sample, and determining whether a 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 is apparent. 15. The method according to any one of paragraphs. 1-14, including additionally determining the level of proADM or fragment(s) thereof and optionally PCT or fragment(s) thereof in the first sample isolated from the patient, said first sample being isolated at or before the time point of diagnosis and initiation of treatment, and determining the level of proADM or fragment(s) thereof and optionally PCT or fragment(s) thereof in a second sample corresponding to the sample of claim 1 isolated from said patient within 10 days of said diagnosis and initiation of treatment, and determining 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. 16. The method of claim 15, wherein an increased level of proADM or its fragment(s) in the second sample compared to the first sample is indicative of a subsequent adverse event. 17. The method according to p. 16, characterized in that 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 indicate 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 indicate a subsequent adverse event. 18. The method according to p. 10, characterized in that the patients are patients of the intensive care unit (ICU), and at the same time: a lower level of PCT or its fragment(s) in the second sample compared to the first sample and a low severity level of proADM or its fragment(s), while the low severity level is less than 4 nmol/l, indicate the discharge of the indicated patient from the ICU; a lower level of PCT or its fragment(s) in the second sample compared to the first sample and a high level of severity of proADM or its fragment(s), with a high severity level of more than 6.5 nmol/l, indicate the need to modify the patient's treatment in the ICU; an increased level of PCT or its fragment(s) in the second sample compared to the first sample and a low severity level of proADM or its fragment(s), while the low severity level is less than 4 nmol/l, indicate the discharge of the indicated patient from the ICU; or an increased level of PCT or its fragment(s) in the second sample compared to the first sample and a high level of severity of proADM or its fragment(s), with a high severity level of more than 6.5 nmol/l, indicate the need to modify the patient's treatment in ICU. 19. Set for implementing the method according to any one of paragraphs. 1-18 containing: detection reagents for determining the level of proADM or its fragment(s) and optionally for determining the level of PCT or its fragment(s) in the sample obtained from the subject, wherein the detection reagents are antibodies specific for the respective proADM and PCT, and reference levels corresponding to high and low severity levels of proADM, with low severity being less than 4 nmol/l and high severity being more than 6.5 nmol/l, and optionally PCT levels, with said reference levels being stored on a computer-readable medium and/or are used in the form of a computer executable code capable of comparing certain levels of proADM or its fragment(s) and optionally certain levels of PCT or its fragment(s) with said reference levels.

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