Biomarkers for therapeutic management and / or prediction of disease progression, severity and / or outcome of respiratory viral infections

Abstract

The present invention relates to a method for treatment management and / or prediction of disease progression, severity and / or outcome in a patient suffering from a respiratory viral infection. The method comprises the steps of: providing a first sample isolated from the patient and a second sample isolated from the patient at a point in time following isolation of the first sample from the patient, wherein the first and second samples are isolated within 96 hours (4 days) after consulting a medical staff; determining the level of one or more endothelial biomarkers in said first and said second sample wherein said one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal proendothelin-1 (CT-proET-1) or a fragment thereof, and / or an adrenomedullin precursor (proADM) or a fragment thereof, and wherein said one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal proendothelin-1 (CT-proET-1) or a fragment thereof, and / or an adrenomedullin precursor (proADM) or a fragment thereof; and wherein a lower or equal level of the one or more endothelial biomarkers in the second sample compared to the first sample indicates that the patient has no risk of an adverse clinical outcome within at least 28 days. The invention also relates to a kit for carrying out the method of the invention.

Description

Technical Field

[0001] This invention relates to the fields of treatment guidance, medical risk assessment, clinical diagnosis and prognosis, and corresponding methods and products.

[0002] This invention relates to a method for managing the treatment of patients with respiratory viral infections and / or predicting disease progression, severity, and / or outcomes, the method comprising the steps of: (a.) providing a first sample isolated from the patient; (b.) providing a second sample isolated from the patient at a time point following the isolation of the first sample; (c.) wherein the first and second samples are isolated within 96 hours (4 days) after admission and / or after consultation with a healthcare professional; (d.) determining the level of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and / or pro-adrenergic medullaris (proADM) or a fragment thereof; and (e.) wherein a lower or equal level of the one or more endothelial biomarkers in the second sample compared to the first sample indicates a risk of the patient having no adverse clinical outcomes for at least 28 days.

[0003] The present invention also relates to a method for the treatment management of a patient with a respiratory viral infection, the method comprising assessing whether the patient requires oxygen support, comprising the steps of: (a.) providing a first sample isolated from the patient; (b.) providing a second sample isolated from the patient at a time point after the isolation of the first sample; (c.) wherein the first and second samples are isolated within 96 hours (4 days) after admission and / or after consultation with a healthcare professional; (d.) determining the level of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and / or pro-adrenergic medullaris (proADM) or a fragment thereof; and (e.) wherein a lower or equal level of the one or more endothelial biomarkers in the second sample compared to the first sample indicates that the patient does not require oxygen support for at least 28 days.

[0004] The present invention also relates to a method for predicting disease progression, severity, and / or outcome in a patient with coronavirus infection, wherein the method comprises: (a) providing a sample from said patient; (b) determining the level of one or more biomarkers in said sample; (c) wherein said one or more biomarkers comprise one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, pro-adrenergic medullary kinase (proADM) or a fragment thereof, and / or procalcitonin (PCT) or a fragment thereof; and (d) wherein said level of said one or more biomarkers in said sample indicates the risk of adverse clinical outcome within 28 days.

[0005] The present invention also relates to a method for predicting disease progression, severity, and / or outcome in patients with coronavirus infection, wherein a low-risk level of one or more biomarkers or fragments thereof in the sample on day 1 indicates the risk of the patient having no adverse clinical outcome within 28 days, and / or wherein a low-risk ratio (day 4 / day 1 ratio) of the levels of one or more biomarkers or fragments thereof in the sample measured on day 4 indicates the risk of the patient having no adverse clinical outcome within 28 days.

[0006] The present invention also relates to a kit for implementing a method for managing and / or predicting disease progression, severity and / or outcome in patients with respiratory viral infections, the kit comprising a detection reagent for determining the level of one or more biomarkers in a sample from said patient, reference data on the risk of adverse clinical outcomes in the patient over 28 days, a detection reagent optionally for determining the presence of a respiratory viral infection, and a detection reagent optionally for determining the level of at least one additional biomarker or fragment thereof. Background Technology

[0007] Severe Acute Respiratory Syndrome (SARS) is a virus-mediated respiratory illness first discovered in 2002, typically caused by coronavirus (CoV) infection. Scientific reports suggest that all human CoVs are likely zoonotic. Once humans are infected, the virus can spread rapidly through droplets and close person-to-person contact, eventually leading to epidemic scenarios or even pandemics (Vijaykrishna et al., 2007; Yuefei et al., 2020). Typical symptoms may include fever, chills, dry cough, difficulty breathing, and diarrhea. The onset and severity of symptoms can vary among infected patients. Some patients may also appear asymptomatic (Al-Tawfiq et al., 2020). Therefore, clinical course can vary, ranging from harmless, self-limiting scenarios that do not appear in the healthcare setting to severe cases where patients cannot survive in the intensive care unit (ICU).

[0008] Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has placed enormous pressure on global healthcare systems and caused significant economic impacts. The clinical manifestations of SARS-CoV-2 encompass a wide range of symptoms, from mild flu-like symptoms to serious complications such as pneumonia, respiratory failure, and ultimately death (Ochani et al., 2021; Parasher et al., 2021).

[0009] Other coronavirus infections, either previously known or potentially arising from the development or mutation of SARS-CoV-2, may present with similar symptoms or manifestations. SARS itself is also known to co-occur with various coronavirus infections, therefore it is not limited to SARS-CoV-2. In 2002 / 2003, a previously unknown coronavirus circulated; similar to COVID-19, many infected individuals developed Severe Acute Respiratory Syndrome (SARS). At that time, this pathogen, known as SARS-CoV-1, infected more than 8,000 people in 26 countries worldwide, causing many to develop severe pneumonia; approximately 800 people died globally. SARS-CoV-1 and SARS-CoV-2 share significant similarities in their genetic basis and variants, and both, as well as future SARS-CoV variants, warrant attention.

[0010] From a pathophysiological perspective, SARS is a complex medical condition in which the virus begins to replicate in the upper respiratory tract and can further spread to the lower respiratory tract or target more non-respiratory organs and cells. Clinical studies have shown that the liver, endothelium, kidneys, heart, intestines, brain, and lymphocytes may also be affected. It has been proposed that the virus can directly promote cellular damage, thereby triggering a systemic inflammatory response, which subsequently leads to multi-organ damage. CoV also causes dysfunction of the renin-angiotensin system, which increases pulmonary vascular permeability, a key factor in the development of pulmonary edema. Both the systemic inflammatory response and the dysfunction of the renin-angiotensin system can lead to a so-called cytokine storm, thereby triggering acute respiratory distress syndrome (ARDS). Multi-organ failure and / or ARDS onset represent a severe condition in SARS patients, with a high risk of death within weeks or days (Yuefei et al., 2020; Gu et al., 2005).

[0011] Infection caused by SARS-CoV-2 is known to affect endothelial cells. The virus attaches to angiotensin-converting enzyme 2 (ACE-2) receptors found on various cell types, including type II alveolar cells, bronchial epithelial cells, and endothelial cells. This typically triggers a cytokine storm, characterized by the release of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). These cytokines have significant effects on endothelial cells, leading to endothelial dysfunction, cell death, and increased vascular permeability (Six et al., 2022; Bonaventura et al., 2021), and subsequently the release of proteins produced by different cells within the endothelium. Therefore, assessing the host immune system's direct response to various etiologies, including infection, using genetically signature proteins is an evolving area of ​​interest with the potential to predict and guide clinical decision-making.

[0012] The following risk factors are believed to contribute to the severe course of this syndrome: age, cardiovascular disease, cancer, diabetes, immunosuppression, smoking, and obesity (Fei et al., 2020).

[0013] In pandemic scenarios, maintaining a fully functional healthcare system is clearly essential to ensure appropriate care for patients ranging from low to high risk. Emergency departments and intensive care units quickly become overcrowded with patients presenting with severe symptoms. For critically ill patients, biomarker-based assessments can help identify those who may require escalation of treatment interventions or not, and those who may qualify for early discharge or ICU exit. Patient outcome prediction is urgently needed to avoid unnecessary treatment for patients with good prognoses or low risk (e.g., in a hospital setting) and to reserve capacity for higher-risk patients. Patients requiring oxygen at low risk, or those at low risk of death, or those at low risk of ICU admission require effective management and appropriate treatment protocols. Given the increasing shortage of healthcare facilities and personnel, it is crucial to make the right decisions for patients seeking medical attention early on, preventing low-risk patients from receiving unnecessary medical treatment.

[0014] Compared to standard methods, different biomarkers have shown superior prognostic performance, such as healthcare professional experience, which may vary and is not a reliable characteristic; or clinical parameters or scores, which, as the sole characteristic for making an accurate decision, have shown poor prognostic performance. Biomarkers of interest include soluble fms-like tyrosine kinase-1 (sFlt-1), endothelin-1 (ET-1), pro-adrenergic medullary kinase (proADM), procalcitonin (PCT), and IL-6.

[0015] SFlt-1 is a naturally occurring soluble form of vascular endothelial growth factor (VEGF) receptor 1, and its concentration in the bloodstream increases when endothelial cells are activated, such as under hypoxic and inflammatory conditions. When sFLT-1 is produced and released, it binds to VEGF and placental growth factor (PlGF), preventing them from binding to receptors on endothelial cells. Therefore, sFlt-1 has anti-angiogenic effects in diseases such as preeclampsia and nephropathy (Maynard et al., 2003; Palmer et al., 2017; Wewers et al., 2021; Lecarpentier et al., 2016). While sFlt-1 has been known as a standard biomarker for prenatal diagnosis for many years, it has not been identified as an infection or risk marker in ICU or hospital settings when treating patients presenting with early symptoms of SARS or at risk of coronavirus infection or SARS. Recent studies have shown that SFlt-1 levels are elevated in COVID-19 infection (Mohebbi et al., 2023; Dupont et al., 2021). Several small-scale studies in pregnant patients have demonstrated that sFlt-1 is significant in predicting clinical outcomes in these pregnant patients with COVID-19 (Torres-Torres et al., 202; Espino-Y-Sosa et al., 2021; Mendoza et al., 2020; Nobrega et al., 2023).

[0016] ET-1 is a potent vasoconstrictor secreted by the endothelium in cases of inflammation and reduced tissue blood perfusion. C-terminal pro-endothelin-1 (CT-proET-1) is a stable precursor protein of ET-1 circulating in the bloodstream. It can be readily measured from blood samples as an indirect quantification of ET-1 (Buendgens et al., 2017; Papassotiriou et al., 2006). Previous studies assessing the association between acute COVID-19 infection and CT-proET-1 have shown elevated levels of this biomarker (Gregoriano et al., 2021; Abraham et al., 2022). However, previous studies have not shown prognostic effects within 28 days when the biomarker was measured within a short period of 1 to 4 days after initial examination. In the current art, patient “diagnosis” is emphasized and determined at a certain stage. However, prognostic ability cannot be extrapolated from diagnostic outcomes.

[0017] Another biomarker for risk assessment in SARS patients is pro-adrenergic medullaris (proADM). ProADM is a hormone precursor produced by multiple tissues to stabilize microcirculation and prevent endothelial permeability and subsequent organ failure (Temmesfeld-Wollbruck et al., 2007; Muller-Redetzky et al., 2014; Vallet et al., 2002; Gonzaes-Rey et al., 2006; Carizzo et al., 2007; Brell et al., 2016; Brell et al., 2005; Vigue et al., 2016). This biomarker has shown considerable promise, for example, in the field of sepsis (Andaluz-Ojeda et al., 2015) and lower respiratory tract infections (Hartmann et al., 2021; Albrich et al., 2011; Albrich and Ruegger et al., 2011). Endothelium and microcirculation are widely recognized as playing important roles in the host's pathophysiological response to sepsis, with the regulation and distribution of blood flow within each organ being crucial (Tyagi et al., 2009; Hernandez et al., 2013). Therefore, measurements of endothelium and microcirculation, such as those via proADM, can provide alternative indicators of the severity of the general host response compared to scores of individual organ dysfunction.

[0018] WO2021 / 204770 and US2023 / 160893A1 also disclose the use of MR-proADM for risk stratification of coronavirus patients. In addition, Atallah et al. ("Mid-regional Proadrenomedullin BiomarkerPredicts Coronavirus Disease 2019 Clinical Outcomes:A US-Based Cohort Study", Open Forum Infectious Diseases, 2022) disclosed the prediction of mechanical ventilation demand based on a single MR-proADM value. Montrucchio et al. ("Effectiveness of mid-regional pro-adrenomedullin, compared to other biomarkers (including lymphocyte subpopulations and immunoglobulins), as a prognostic biomarker in COVID-19 critically ill patients: New evidence from a 15-month observational prospective study", Frontiers in Medicine, 2023) published predictions of 28-day mortality in COVID patients based on MR-proADM measurements within 48 hours and on days 3 and 7. Montrucchio et al. ("Effectiveness of mid-regional proadrenomedullin (MR-proADM) as prognostic marker in COVID-19 critically ill patients: An observational prospective study", PLOS ONE, Vol. 16, No. 2, 2021) published predictions of MR-proADM measurements (days 0-3, 7, and 14) and mortality in COVID patients.

[0019] However, given the potentially rapid infection rate of coronaviruses or SARS viruses throughout the population, and the likelihood of mass hospitalizations in epidemic or pandemic situations, new methods are needed to assess the risk of disease progression in patients who have or are at risk of developing severe outcomes associated with coronavirus infection, such as after infection with SARS-CoV. Furthermore, new approaches are needed to manage the treatment of these patients with respiratory viral infections. To date, clinicians lack sufficiently reliable methods to assess whether patients require intensive treatment and / or disease surveillance, or whether they can be safely weaned off clinical care. Summary of the Invention

[0020] In view of the difficulties in the prior art, a potential technical problem of the present invention is to provide means for the treatment management of patients with respiratory viral infections and for predicting disease progression, severity and / or outcome.

[0021] Another problem of the present invention is to provide means for excluding the risk of adverse clinical outcomes within at least 28 days in patients with respiratory viral infections.

[0022] Another problem of the present invention is to provide a means of assessing whether a patient with a respiratory viral infection is at risk of adverse clinical outcomes for at least 28 days.

[0023] Another potential problem of the present invention is to provide means for managing the hospitalization and ICU treatment needs of patients with respiratory viral infections.

[0024] Another potential problem with this invention is to provide a means for assessing whether a patient can be discharged from the hospital or leave the ICU.

[0025] Another problem of the present invention is to provide a means with a high positive predictive value in assessing whether a patient with a respiratory viral infection can be discharged from the hospital or leave the ICU.

[0026] Given the difficulties in the prior art, another potential problem of the present invention is to provide means for assessing whether a patient with a respiratory viral infection requires oxygen support. Another potential problem of the present invention is to provide means for the treatment management (particularly the management of oxygen support) of patients with respiratory viral infections. Another object of the present invention is to provide means with a high negative predictive value in assessing whether a patient with a respiratory viral infection requires oxygen support.

[0027] Another problem of the present invention is to provide means for predicting disease progression and / or outcomes (particularly adverse outcomes within 28 days) in patients with coronavirus infection. Another object of the present invention is to provide means with a high negative predictive value in determining a low risk of adverse events or death or other adverse disease progression and / or outcomes (particularly adverse outcomes within 28 days) in patients with coronavirus infection.

[0028] These problems are addressed by the features of the independent claims. The dependent claims provide preferred embodiments of the invention.

[0029] In one aspect, the present invention relates to a method for managing the treatment of patients with respiratory viral infections and / or predicting disease progression, severity, and / or outcome, the method comprising the following steps:

[0030] a. Provide a first sample isolated from the patient.

[0031] b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient.

[0032] c. The first and second samples were separated within 96 hours (4 days) after consultation with medical personnel.

[0033] d. Determine the levels of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and / or pro-adrenergic medullaris (proADM) or a fragment thereof, and

[0034] e. Wherein, a lower or equal level of one or more endothelial biomarkers in the second sample, compared to the first sample, indicates a risk of the patient having no adverse clinical outcome for at least 28 days.

[0035] In embodiments, the present invention demonstrates surprisingly high negative predictive values ​​(for ruling out the risk of adverse clinical outcomes), enabling healthcare professionals to quickly (within the first 4 days after a patient’s visit) and effectively determine the need for intensive treatment in such patients with respiratory viral infections.

[0036] As stated above, there is an urgent need for patient treatment management and risk assessment to avoid unnecessary treatment for patients with good prognoses or low risk (e.g., in a hospital setting) and to reserve treatment capacity for higher-risk patients and those requiring intensive care. Patients with poor clinical outcomes and low risk of intensive care require effective management and appropriate treatment options. Given the increasing shortage of healthcare facilities and personnel, it is crucial to make the right decisions for patients seeking medical care early on, preventing low-risk patients from receiving unnecessary medical treatment. This invention enables direct and rapid biomarker detection without waiting for complex scoring to determine results, thereby introducing objective assessment independent of the experience level of healthcare personnel.

[0037] Previous studies have failed to demonstrate a clear impact on risk assessment and treatment management within 28 days when biomarker levels are measured shortly after initial visit (e.g., 1 to 4 days after initial visit). In the prior art, a patient's "diagnosis" is emphasized and determined at some stage. However, it is generally not possible to derive prognostic ability from diagnostic outcomes, and as this invention demonstrates, the measurement of biomarker levels cannot be assumed to have a high negative predictive value and an extremely low false negative rate from studies indicating the diagnostic potential of biomarkers. This invention enables the measurement of negative predictive value primarily and effectively, essentially ruling out the risk of clinically adverse events and the need for intensive treatment in certain patient populations. In other words, if a biomarker value is above a threshold, clinicians cannot rule out adverse events, and if the positive predictive value (PPV) of a biomarker at a threshold level is low, especially if the PPV is below 50%, adverse events cannot be automatically expected in the patient.

[0038] Therefore, in particular, measuring the levels of endothelial biomarkers in two samples obtained over 4 days (preferably on days 1 and 4) and comparing the levels of the biomarkers in said samples (the ratio of the second sample to the first sample) provides a surprising improvement and a high negative predictive value compared to, for example, a single measurement of the biomarker on day 1 or day 4. This ratio allows for the effective and safe exclusion of patients without risk of adverse clinical outcomes, and the adjustment of treatment for said patients accordingly. To the inventors' knowledge, no prior art discloses or implies this ratio improving the negative predictive value in two samples obtained over 4 days, and is therefore particularly useful and reliable in excluding adverse events in patients with respiratory viral infections.

[0039] In some embodiments, a positive determination is possible, although the primary effect demonstrated by this invention is based on biomarker measurements, effectively excluding any given patient from adverse clinical outcomes and the risk of needing intensive care within 28 days. In some embodiments, a high positive predictive value is provided regarding patient discharge from the ICU or hospital, thereby providing inclusion criteria. This corresponds to a high negative predictive value and exclusion of adverse clinical events and the risk of needing intensive care, since discharge from the ICU or hospital means that the patient no longer needs or requires intensive care provided by the ICU or hospital.

[0040] Endothelial biomarkers (such as sFlt-1, ET-1, or proADM) are associated with endothelial function and structure, particularly with angiogenic homeostasis. Adrenomedullin (ADM) is a vasodilatory peptide with multiple physiological functions, including maintaining vascular tone and endothelial barrier function. Furthermore, ADM has been shown to promote cardiac angiogenesis after myocardial infarction, demonstrating its role in tissue repair and regeneration, as well as its pro-angiogenic effect by supporting the formation of new blood vessels and lymphatic vessels under hypoxic conditions. ADM can be measured directly or by measuring the precursor molecule proADM or other fragments thereof, such as the more stable fragment MR-proADM, which is separated from proADM at an equimolar ratio to ADM.

[0041] sFlt-1 is a non-membrane-related splicing variant of VEGF receptor 1 (Flt-1) and binds to angiogenic factors such as VEGF (vascular endothelial growth factor) and PlGF (placental growth factor). By binding to these factors, sFlt-1 reduces the concentration of free VEGF and PlGF, thereby inhibiting angiogenesis and exhibiting an anti-angiogenic effect.

[0042] ET-1 is also an endothelial regulator and biomarker, and as a vasoconstrictor secreted by endothelial cells, it participates in arteriosclerosis and vascular remodeling. Pre-endothelinogenin is a precursor to the peptide ET-1. Endothelial cells convert pre-endothelinogenin into pro-endothelinogen, which is subsequently converted into mature endothelin released by the cell. ET-1 also has angiogenic effects on endothelial and perivascular cells, and has been shown to have indirect effects through increased release of the potent pro-angiogenic substance vascular endothelial growth factor (VEGF) via hypoxia-inducible factor-1.

[0043] sFlt-1, proADM, and ET-1 all play a crucial role in maintaining angiogenesis homeostasis in subjects, thereby maintaining the function of the vascular system. According to the invention, adverse clinical outcomes in patients with respiratory viral infections are often associated with injury, inflammation, and hypoxia (lack of oxygen) in diseased tissues, leading to activation of angiogenesis, which in turn forms new blood vessels to improve oxygen supply to said tissues. During angiogenesis, various biomarkers with angiogenic or anti-angiogenic effects are activated to maintain homeostasis. Therefore, according to the invention, such biomarkers involved in angiogenesis, particularly SFlt-1, ET-1, and proADM or fragments thereof (proportionally representing the level and activity of ADM), are associated with adverse clinical outcomes, and their measurement advantageously allows for the assessment of the risk of such adverse clinical outcomes in patients with respiratory viral infections. Although different endothelial biomarkers are covered, these different embodiments of the invention are therefore preferably unified by their role in angiogenesis (endothelial biomarkers) and their ability to effectively and accurately assess the risk of adverse clinical outcomes in patients with respiratory viral infections based on these endothelial biomarkers.

[0044] In this embodiment, a higher level of one or more endothelial biomarkers in the second sample compared to the first sample does not necessarily indicate that the patient will require oxygen support. In this embodiment, patients with higher levels in the second sample are those at risk of adverse clinical outcomes that cannot be “excluded” by biomarker measurement, and do not necessarily indicate “inclusion.” In a preferred embodiment, patients with lower or equal levels in the second sample are effectively excluded from the said risk of adverse clinical outcomes indicated by a high negative predictive value (NPV), as described in more detail below.

[0045] In one embodiment, a lower or equal level of one or more endothelial biomarkers in a second sample compared to a first sample indicates that the patient is not at risk of adverse clinical outcomes requiring intensive treatment and / or monitoring for at least 28 days.

[0046] In one embodiment, a lower or equal level of one or more endothelial biomarkers in a second sample compared to a first sample indicates that the patient is not at risk of requiring de-escalation of treatment and / or reduction of intensive medical monitoring and / or discharge from the ICU or hospital and / or that the patient will show improved adverse clinical outcomes on the Covid-19 Sequential Scale.

[0047] In one embodiment, a lower or equal level of one or more endothelial biomarkers in a second sample compared to a first sample indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

[0048] In one embodiment, a lower or equal level of one or more endothelial biomarkers in a second sample compared to a first sample indicates that the patient does not require supplemental oxygen for at least 28 days.

[0049] In one embodiment, the first and second samples are obtained by healthcare professionals who first see the patient after the onset of respiratory viral infection symptoms. This could be at primary care, a general practitioner's office, a hospital ward, or in the emergency department or ICU. In another embodiment, the first sample may also be obtained upon admission or in any setting within the hospital (ED, ICU, or other).

[0050] In one embodiment, a lower or equal level of one or more endothelial biomarkers in the second sample compared to the first sample indicates a risk of no adverse clinical outcome for the patient at least 28 days after initial consultation with a healthcare professional and / or receipt of the first sample. In one embodiment, a lower or equal level of one or more endothelial biomarkers in the second sample compared to the first sample indicates a risk of no adverse clinical outcome related to the need for supplemental oxygen for the patient at least 28 days after initial consultation with a healthcare professional and / or receipt of the first sample. In one embodiment, a lower or equal level of one or more endothelial biomarkers in the second sample compared to the first sample indicates that the patient does not require supplemental oxygen for at least 28 days after initial consultation with a healthcare professional and / or receipt of the first sample.

[0051] In one embodiment, the patient exhibits at least two symptoms selected from the group consisting of fever > 38°C and pulmonary infiltration at the time of initial contact with healthcare personnel. In another embodiment, the patient displays a COVID-19 ordinal scale score of ≤ 4 on day 1 following the initial contact with healthcare personnel.

[0052] In one embodiment, the patient is hospitalized and / or admitted to the intensive care unit (ICU) after the patient’s first contact with medical staff.

[0053] In one embodiment, the first sample is obtained within 24 hours (1 day) after consulting with a medical professional, and / or the second sample is obtained within 72 to 96 hours (4th day) after consulting with a medical professional.

[0054] In one embodiment, (a.) the first sample is obtained within 24 hours (1 day) after consulting a medical professional, preferably within 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours after consulting a medical professional, and / or (b.) the second sample is obtained within 72 to 96 hours (day 4) after consulting a medical professional, preferably within 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96 hours after the first consultation with a medical professional.

[0055] In this embodiment, the time point provided regarding "after consulting a medical professional" can also be applied to the same time point as the definition of admission, such as obtaining a sample within X hours (X days) of "admission (to the hospital) and / or after consulting a medical professional." This terminology can be applied to any embodiment regarding time points as disclosed herein.

[0056] As a non-limiting example, in some embodiments, the analysis time may be performed as follows:

[0057] Example: On Monday, Day 1, at 9:00 AM, the patient visits, and the first blood sample is collected within 24 hours until the start of Day 2, Tuesday, at 9:00 AM (24 hours). Day 2 will proceed from Tuesday at 9:00 AM to Wednesday at 9:00 AM (48 hours). Day 3 will proceed from Wednesday at 9:00 AM to Thursday at 9:00 AM (72 hours). Day 4 will proceed from Thursday at 9:00 AM to Friday at 9:00 AM (96 hours). In this embodiment, the second sample may be collected on Day 2, Day 3, or Day 4, preferably on Day 4. In this embodiment, the second sample may be collected within 60-96 hours after the first visit, preferably within 72-96 hours after the first visit.

[0058] In embodiments, the invention is not necessarily limited to obtaining and measuring two samples from a patient. In embodiments, the methods described herein can also enable diagnoses or prognostic determinations described using an analysis of a single sample (e.g., a sample from day 1 or day 4) to make relevant diagnoses or prognostic determinations. Any embodiment of a disclosed diagnosis or prognostic determination (i.e., regarding the management of treatment for patients with respiratory viral infections and / or the prediction of disease progression, severity, and / or outcome) is potentially related to both methods using two or one sample. Detailed data are presented below from which corresponding embodiments can be observed.

[0059] In one embodiment, the patient is infected with a coronavirus, preferably SARS coronavirus, more preferably SARS-CoV2 coronavirus.

[0060] In one embodiment, an adverse event is death within at least 28 days after obtaining the first sample.

[0061] In one embodiment, an adverse event is an adverse respiratory and / or infectious clinical outcome that requires oxygen support and intensive treatment for at least 28 days.

[0062] In one embodiment, adverse respiratory and / or infectious clinical outcomes are intensive care unit (ICU) treatment, mechanical ventilation, and / or additional infections.

[0063] In one embodiment, the patient is infected with coronavirus and the adverse event is a deterioration in the COVID Scale score (e.g., a deterioration in the COVID Scale score to >4) and / or continued hospitalization on or after day 4 of admission.

[0064] In one embodiment, oxygen support includes administering low-flow-rate or high-flow-rate oxygen. In one embodiment, low-flow-rate oxygen includes administering oxygen at a rate equal to or less than 6 L / min. In one embodiment, high-flow-rate oxygen includes administering oxygen at a rate >6 to ≤10 L / min. In one embodiment, mechanical ventilation includes non-invasive mechanical ventilation, invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO).

[0065] In one embodiment, adverse clinical outcomes are: admission to the intensive care unit (ICU), clinical deterioration on the COVID-19 ordinal scale, death within 28 days, a COVID-19 ordinal scale score ≥ 4 on day 4, prolonged hospital stay beyond day 4, a COVID-19 ordinal scale score equal to or worse than day 1 on day 4, mechanical ventilation within 28 days, any thrombotic event (such as deep vein thrombosis (DVT), pulmonary embolism (PE), and stroke), a composite severity endpoint including death within 28 days, at least one of ICU admission and mechanical ventilation, infection within 28 days (excluding COVID-19-related treatment), and / or infection within 28 days (>= grade 3).

[0066] In one embodiment, the patient's age is between 19 and 85 years.

[0067] In one embodiment, the sample is selected from blood samples, such as whole blood samples, serum samples, or plasma samples.

[0068] In one embodiment, the levels of the corresponding biomarkers in the first and second samples (day 1, day 4, and day 1 vs. day 4 (also known as the hazard ratio)) and clinical outcomes can be derived from the statistical results disclosed in Tables 1 to 49.

[0069] In one embodiment, the levels and ratios of the corresponding biomarkers in the first and second samples disclosed in Tables 1 to 49 (day 1, day 4, and day 1 vs. day 4 (also known as hazard ratios)) represent the cutoff values ​​and / or hazard ratios of the corresponding biomarkers and / or combinations of biomarkers, and the corresponding clinical outcomes.

[0070] In one embodiment, the method additionally includes

[0071] a. Determine the level of at least one additional biomarker in the first and / or second sample, wherein the at least one additional biomarker is C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, procalcitonin (PCT) or a fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes and / or C-reactive protein (CRP), and

[0072] b. Wherein, compared with the first sample, the lower or equal level of the at least one additional biomarker and / or the one or more endothelial biomarkers in the second sample indicates the risk that the patient has no adverse clinical events for at least 28 days.

[0073] In one embodiment, a lower or equal level of at least one additional biomarker and / or one or more endothelial biomarkers in the second sample, compared to the first sample, indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

[0074] In one embodiment, a lower or equal level of at least one additional biomarker and / or one or more endothelial biomarkers in a second sample, compared to a first sample, indicates that the patient does not require supplemental oxygen for at least 28 days.

[0075] In one embodiment, the method additionally includes

[0076] a. Determine the level of at least one additional biomarker in the first and / or second sample, wherein the at least one additional biomarker is C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, procalcitonin (PCT) or a fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes and / or C-reactive protein (CRP), and

[0077] b. Wherein, compared with the first sample, the lower or equal level of the at least one additional biomarker and the one or more endothelial biomarkers in the second sample indicates that the patient does not require supplemental oxygen for at least 28 days.

[0078] In one aspect, the present invention relates to a kit for the treatment management and / or prediction of disease progression, severity and / or outcome in patients with respiratory viral infections, comprising:

[0079] - A detection reagent for determining the level of one or more endothelial biomarkers in a sample from a patient, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof and / or pro-adrenergic medullary kinase (proADM) or a fragment thereof (preferably MR-proADM), and

[0080] - Reference data on the risk of adverse clinical outcomes in patients within 28 days, particularly reference data on risk thresholds, critical values, or hazard ratios, wherein the reference data is preferably stored on a computer-readable medium and / or in the form of computer-executable code configured to compare the measured levels of one or more endothelial biomarkers in a sample from the patient with the risk thresholds, critical values, or hazard ratios.

[0081] - Optional diagnostic reagents for determining the presence of respiratory viral infection, preferably for coronavirus infection, and more preferably for the presence of SARS-CoV2 virus infection.

[0082] - Optional assay reagents for determining the levels of at least one additional biomarker or fragment thereof in samples from patients, preferably pro-endothelin-1 (CT-proET-1) or fragment thereof, procalcitonin (PCT) or fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, and C-reactive protein (CRP), and reference data, such as reference data for the at least one additional biomarker, preferably pro-endothelin-1 (CT-proET-1) or fragment thereof, procalcitonin (PCT) or fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, and C-reactive protein (CRP), particularly risk thresholds, cutoff values, or hazard ratios, wherein the reference data is preferably stored on a computer-readable medium and / or in the form of computer-executable code configured to compare the measured levels of the at least one additional biomarker or fragment thereof with the thresholds, cutoff values, or hazard ratios.

[0083] Examples related to sFlt-1 or fragments thereof as biomarkers:

[0084] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise sFlt-1 or a fragment thereof, and

[0085] - Compared to the first sample, the second sample showed lower or equal levels of sFlt-1 or fragments thereof, and

[0086] - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates a risk of no adverse clinical outcome for the patient over at least 28 days.

[0087] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise sFlt-1 or a fragment thereof, and

[0088] - Compared to the first sample, the second sample showed lower or equal levels of sFlt-1 or fragments thereof, and

[0089] - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

[0090] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise sFlt-1 or a fragment thereof, and

[0091] - Compared to the first sample, the second sample showed lower or equal levels of sFlt-1 or fragments thereof, and

[0092] - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient will not require oxygen support for at least 28 days and is not at risk of death.

[0093] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise sFlt-1 or a fragment thereof, and

[0094] - Compared to the first sample, the second sample showed lower or equal levels of sFlt-1 or fragments thereof, and

[0095] - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient will not require oxygen support for at least 28 days and will not be at risk of death or additional infection.

[0096] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise sFlt-1 or a fragment thereof, and

[0097] - Compared to the first sample, the second sample showed lower or equal levels of sFlt-1 or fragments thereof, and

[0098] - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient will not require oxygen support and will not be at risk of death or additional infection for at least 28 days after consultation with a healthcare professional.

[0099] In one embodiment, the method includes the following steps

[0100] a. Provide a first sample isolated from the patient, wherein the patient is infected with coronavirus.

[0101] b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient.

[0102] c. Wherein the first sample was obtained within 1 day after consultation with the medical personnel, and the second sample was obtained within 72 to 96 hours (day 4) after consultation with the medical personnel.

[0103] d. Determine the level of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in the sample, and

[0104] e. Wherein, a lower or equal level of sFlt-1 or a fragment thereof in the second sample, compared to the first sample, indicates that the patient does not require oxygen support for at least 28 days and is not at risk of death or additional infection.

[0105] Variations in the sFlt-1 cutoff value can be used, such as any value of 60, 65, 70, 75, 80, 81, 81.3, 83.9, 84, 85, 88, 88.4, 90, 95, 100, 105, or 110 pg / L, or any value within these values, or any value within a range formed by any two endpoints of this list. Any cutoff value can also be a variation of ±1, 2, 3, 4, 5, 10, 15, 20, 25, or 30% relative to a specific value. Measurements using these cutoff values ​​are preferably applicable to serum samples in B.R.A.H.M.S. KRYPTOR assays. Such possible variations in cutoff values ​​apply to any given embodiment of the relevant biomarker in this disclosure.

[0106] Examples related to CT-proET-1 or fragments thereof as biomarkers:

[0107] In one embodiment, determining the level of proADM or a fragment thereof includes determining the level of CT-proET-1 or a fragment thereof in the sample.

[0108] In one embodiment, one or more biomarkers include CT-proET-1 or fragments thereof, and

[0109] - A low-risk level of CT-proET-1 or its fragments measured on day 1 indicates that the patient is not at risk of ICU admission, mechanical ventilation, death, and / or composite severity endpoints, and / or the patient will be discharged before or on day 4, or

[0110] - A high-risk level of CT-proET-1 or its fragments measured on day 1 indicates that the patient is at risk of admission to the ICU, need for mechanical ventilation, death and / or composite severity endpoints, and / or the patient will be admitted to the hospital or ICU before or on day 4.

[0111] In one embodiment,

[0112] - The low-risk level of CT-proET-1 or its fragments measured on day 1 was ≤ 83.6 pmol / L ±20%, or

[0113] - The high-risk level of CT-proET-1 or its fragments measured on day 1 was > 83.6 pmol / L ± 20%, and optionally...

[0114] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 83.6 pmol / L ± 20%.

[0115] In one embodiment,

[0116] - A level of CT-proET-1 or a fragment thereof measured on day 1 ≤ 87.4 pmol / L ± 20% indicates that the patient is not at risk of death and / or need for mechanical ventilation within 28 days, or

[0117] - A level of CT-proET-1 or a fragment thereof measured on day 1 > 87.4 pmol / L ± 20% indicates a risk of death and / or need for mechanical ventilation within 28 days, and optionally

[0118] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 87.4 pmol / L ± 20%.

[0119] In one embodiment,

[0120] - A level of CT-proET-1 or a fragment thereof measured on day 1 ≤ 91.8 pmol / L ± 20% indicates that the patient is not at risk of requiring mechanical ventilation, ICU admission, and / or composite severity endpoints.

[0121] - A level of CT-proET-1 or a fragment thereof measured on day 1 > 91.8 pmol / L ± 20% indicates that the patient is at risk of requiring mechanical ventilation, ICU admission, and / or composite severity endpoints, and optionally

[0122] - The method includes comparing the assay level of one or more biomarkers in the sample with a critical value of 91.8 pmol / L ± 20%.

[0123] In one embodiment,

[0124] - A level of CT-proET-1 or a fragment thereof measured on day 1 ≤ 97.6 pmol / L ± 20%, preferably ≤ 102.7 pmol / L ± 20%, indicates that the patient has no risk of a COVID-19 ordinal scale score ≥ 4 on day 4, or

[0125] - A level of CT-proET-1 or a fragment thereof measured on day 1 > 97.6 pmol / L ± 20%, preferably > 102.7 pmol / L ± 20%, indicates a risk of a COVID-19 ordinal scale ≥ 4 on day 4, and optionally

[0126] - The method includes comparing the detection levels of one or more biomarkers in the sample with critical values ​​of 97.6 pmol / L ± 20% or 102.7 pmol / L ± 20%.

[0127] In one embodiment,

[0128] - A level of CT-proET-1 or a fragment thereof measured on day 1 ≤ 78.7 pmol / L ± 20% indicates the need to discontinue supplemental oxygen, or

[0129] - A CT-proET-1 level or fragment thereof measured on day 1 > 78.7 pmol / L ± 20% indicates continued supplemental oxygen, and optionally

[0130] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 78.7 pmol / L ± 20%.

[0131] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and

[0132] - In the second sample, the level of CT-proET-1 or its fragments was lower or equal compared to the first sample, and / or

[0133] - A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates a risk of no adverse clinical outcome for the patient over at least 28 days.

[0134] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise CT-proET-1 or a fragment thereof, and

[0135] - In the second sample, the level of CT-proET-1 or its fragments was lower or equal compared to the first sample, and / or

[0136] - A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

[0137] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise CT-proET-1 or a fragment thereof, and

[0138] - In the second sample, the level of CT-proET-1 or its fragments was lower or equal compared to the first sample, and / or

[0139] - A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates that the patient is not at risk of death for at least 28 days (and preferably does not require oxygen support).

[0140] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise CT-proET-1 or a fragment thereof, and

[0141] - In the second sample, the level of CT-proET-1 or its fragments was lower or equal compared to the first sample, and / or

[0142] A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates that the patient does not require mechanical ventilation and is not at risk of death for at least 28 days after consultation with medical personnel.

[0143] Variations in the CT-proET-1 cutoff value can be used, such as any value of 60, 65, 70, 75, 80, 83, 83.6, 84, 85, 87, 87.4, 90, 91, 91.8, 95, 100, 102, 102.7, 105, 110, 115, or 120 pmol / L, or any value within these values, or any value within a range formed by any two endpoints of this list. Any cutoff value can also be used with a variation of ±1, 2, 3, 4, 5, 10, 15, 20, 25, or 30% relative to a specific value. Measurements using these cutoff values ​​are preferably applicable to serum samples in B.R.A.H.M.S. KRYPTOR assays. Such possible variations in cutoff values ​​apply to any given embodiment of the relevant biomarker in this disclosure.

[0144] In one embodiment, the low-risk level of CT-proET-1 or a fragment thereof is ≤ 83.6 pmol / L ±20%, and the low-risk ratio indicates that the patient has no risk of death within 28 days, admission to the ICU, need for mechanical ventilation and / or composite severity endpoints and / or discontinuation of supplemental oxygen.

[0145] Examples related to proADM or fragments thereof as biomarkers:

[0146] In one embodiment, determining the level of proADM or a fragment thereof includes determining the level of mid-pro-adrenergic medullaris peptide (MR-proADM) in the sample.

[0147] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and

[0148] - Compared to the first sample, the second sample showed lower or equal levels of proADM or its fragments, and

[0149] - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates a risk of no adverse clinical outcome for the patient over at least 28 days.

[0150] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and

[0151] Compared to the first sample, the second sample had lower or equal levels of proADM or its fragments, and

[0152] - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, as determined in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient is not at risk of adverse clinical outcomes associated with the need for supplemental oxygen for at least 28 days.

[0153] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and

[0154] Compared to the first sample, the second sample had lower or equal levels of proADM or its fragments, and

[0155] - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient does not require oxygen support and is not at risk of death for at least 28 days.

[0156] In one embodiment, one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and

[0157] - Compared to the first sample, the second sample showed lower or equal levels of proADM or its fragments, and

[0158] - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient will not require oxygen support for at least 28 days and will not be at risk of death, ICU admission, or mechanical ventilation.

[0159] In one embodiment, the method includes the following steps

[0160] a. Provide a first sample isolated from the patient, wherein the patient is infected with coronavirus.

[0161] b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient.

[0162] c. Wherein the first sample was obtained within 24 hours (1 day) after consulting with the medical personnel, and the second sample was obtained within 72 to 96 hours (4th day) after consulting with the medical personnel.

[0163] d. Determine the level of pro-adrenomedullin (proADM) or a fragment thereof in the sample, and

[0164] e. Wherein, a lower or equal level of proADM or a fragment thereof in the second sample compared to the first sample indicates that the patient does not require oxygen support for at least 28 days and is not at risk of death, ICU admission, or mechanical ventilation.

[0165] Variations in the proADM, preferably MR-proADM, threshold values ​​can be employed, such as any value of 0.5, 0.6, 0.7, 0.8, 0.87, 0.9, 1.0, 1.10, 1.20, 1.30, 1.33, 1.40, 1.50, 1.58, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.25, 2.30, 2.40, 2.50 or 3, 4, 5, 6, 7, 8, 9 or 10 nmol / L, or any value within these values, or any value within a range formed by any two endpoints of this list. Any threshold value can also be varied by ±1, 2, 3, 4, 5, 10, 15, 20, 25 or 30% relative to a specific value. Such possible variations in threshold values ​​apply to any given embodiment of the relevant biomarker in this disclosure.

[0166] In other embodiments, the MR-proADM cutoff value used is 0.87, 0.9, or 1 nmol / L, which can provide an assessment of clinical risk, and values ​​above this level may indicate the need for hospitalization. Alternatively, in some cases, a cutoff value of 2.25 nmol / L may be used, and values ​​below this level may indicate that ICU treatment is no longer required. Measurements using these cutoff values ​​are preferably applicable to serum samples in the B.R.A.H.M.S. KRYPTOR assay.

[0167] Other aspects and embodiments of the invention:

[0168] In one aspect, the present invention relates to a method for the treatment and management of a patient suffering from a respiratory viral infection, the method comprising assessing whether the patient requires oxygen support, including the following steps:

[0169] a. Provide a first sample isolated from the patient.

[0170] b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient.

[0171] c. The first and second samples were separated within 96 hours (4 days) after consultation with medical personnel.

[0172] d. Determine the levels of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are pro-adrenergic medullaris (proADM) or a fragment thereof (preferably MR-proADM) and / or soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, and

[0173] e. Wherein, a lower or equal level of one or more endothelial biomarkers in the second sample, compared to the first sample, indicates that the patient does not require supplemental oxygen for at least 28 days.

[0174] In embodiments, the present invention demonstrates a high negative predictive value (for ruling out the need for oxygen support), enabling healthcare professionals to quickly (within the first 4 days after a patient’s visit) and effectively determine the need for oxygen support and / or intensive treatment in such patients with respiratory viral infections.

[0175] As stated above, there is an urgent need for patient treatment management and risk assessment to avoid unnecessary treatment for patients with good prognoses or low risk (e.g., in a hospital setting) and to reserve treatment capacity for higher-risk patients and those requiring intensive care. Low-risk patients requiring oxygen support and intensive care need effective management and appropriate treatment options. Given the increasing shortage of medical facilities and personnel, it is crucial to make the right decisions for patients seeking medical care early on, preventing low-risk patients from receiving unnecessary medical treatment. This invention enables direct and rapid biomarker detection without complex scoring determination, thereby introducing objective assessment independent of the experience level of medical personnel.

[0176] Previous studies have failed to demonstrate a clear impact on risk assessment and treatment management within 28 days when biomarkers are measured shortly after initial visit (e.g., 1 to 4 days after initial visit). In the prior art, a patient's "diagnosis" is emphasized and determined at some stage. However, it is generally not possible to derive prognostic ability from diagnostic outcomes, and as this invention demonstrates, the measurement of biomarker levels cannot be assumed to have high negative predictive values ​​and extremely low false-negative rates from studies indicating the diagnostic potential of biomarkers. This invention is able to primarily and effectively measure negative predictive values, substantially excluding the need for oxygen support in certain patient populations. In some embodiments, a positive determination is possible, although the primary effect demonstrated by this invention is based on biomarker measurements that effectively exclude the need for oxygen support in any given patient within 28 days.

[0177] In embodiments, a higher level of one or more endothelial biomarkers in a second sample compared to a first sample does not necessarily indicate that a patient will require oxygen support. In embodiments, patients with higher levels in the second sample are those whose need for oxygen support cannot be “excluded” by biomarker measurements, and not necessarily “included.” In preferred embodiments, patients with lower or equal levels in the second sample effectively exclude the oxygen support requirement, as indicated by a strong NPV, as described in more detail below.

[0178] In one embodiment, one or more endothelial biomarkers in the second sample, compared to the first sample, additionally indicate that the patient has no risk of death for at least 28 days after receiving the first sample.

[0179] In one embodiment, a lower or equal level of one or more endothelial biomarkers in a second sample, compared to a first sample, indicates that the patient is not at risk of adverse respiratory and / or infectious clinical outcomes requiring oxygen support and intensive treatment for at least 28 days.

[0180] In one embodiment, adverse respiratory and / or infectious clinical outcomes are intensive care unit (ICU) treatment, mechanical ventilation, and / or additional infections.

[0181] In one aspect, the present invention relates to a method for managing the treatment of patients with respiratory viral infections and / or predicting disease progression, severity, and / or outcome, wherein the method comprises:

[0182] a. Provide samples from the patient.

[0183] b. Determine the levels of one or more endothelial biomarkers in the sample.

[0184] c. The one or more endothelial biomarkers mentioned above are one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, pro-adrenergic medullary hormone precursor (proADM) or a fragment thereof, and procalcitonin (PCT) or a fragment thereof.

[0185] d. The level of one or more endothelial biomarkers in the sample indicates the risk of adverse clinical outcomes within 28 days.

[0186] In one embodiment, the level of one or more endothelial biomarkers in the sample indicates the risk of the patient not having an adverse clinical outcome within 28 days.

[0187] In one embodiment, levels of one or more endothelial biomarkers in a sample below a threshold level indicate that the patient is at risk of no adverse clinical outcome within 28 days.

[0188] In one embodiment, one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and procalcitonin (PCT) or a fragment thereof, preferably soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof.

[0189] In an embodiment, the present invention relates to a method for predicting disease progression, severity, and / or outcome in patients with coronavirus infection, wherein the method comprises:

[0190] a. Provide samples from the patient.

[0191] b. Determine the levels of one or more biomarkers in the sample.

[0192] c. The one or more biomarkers mentioned above include one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, pro-adrenergic medullary hormone precursor (proADM) or a fragment thereof, and / or procalcitonin (PCT) or a fragment thereof.

[0193] d. The level of one or more of the biomarkers in the sample indicates the risk of adverse clinical outcomes within 28 days.

[0194] In one embodiment, the levels of one or more biomarkers indicate whether a patient is at risk of adverse clinical outcomes requiring intensive treatment and / or monitoring.

[0195] In one embodiment, the levels of one or more biomarkers indicate whether a patient is at low or no risk of adverse clinical outcomes, requires de-escalation of treatment and / or reduction of intensive medical monitoring and / or discharge of the patient from the ICU or hospital, and / or when the patient will show improvement on the Covid-19 Sequential Scale.

[0196] In one embodiment, the levels of one or more biomarkers are determined in samples obtained by the first healthcare workers who treat the patient after the onset of symptoms of coronavirus infection.

[0197] In one embodiment, the sample is obtained within 24 hours (day 1) and / or 96 hours, preferably 72-96 hours (day 4), after the first contact with a healthcare worker.

[0198] In one embodiment, the sample is obtained on day 4, and the level of one or more biomarkers in the sample indicates whether the patient is at low or no risk of adverse clinical outcomes, requiring de-escalation of treatment and / or reduction of intensive medical monitoring and / or discharge of the patient from the ICU or hospital. In another embodiment, the sample is obtained on day 4, and the level of one or more biomarkers in the sample indicates de-escalation of treatment and / or reduction of intensive medical monitoring and / or discharge of the patient from the ICU or hospital.

[0199] In one embodiment, the initial contact with medical personnel takes place in a hospital.

[0200] In one embodiment, the patient is admitted to the hospital after their first contact with healthcare personnel.

[0201] In one embodiment, the patient is treated with supplemental oxygen.

[0202] In one embodiment, the patient is assessed to determine whether they require supplemental oxygen, invasive mechanical ventilation, and / or ECMO.

[0203] In one embodiment, the risk of a patient developing a thrombotic event is assessed.

[0204] In one embodiment, the survival prediction of a patient is assessed for the next 28 days following the first (blood) sample collection.

[0205] In one embodiment, the patient was not admitted to the intensive care unit (ICU) at the time of their first contact with healthcare personnel.

[0206] In one embodiment, the patient showed a COVID-19 ordinal scale score of ≤ 4 on day 1.

[0207] In one embodiment, the levels of one or more biomarkers indicate whether a patient is at risk of: admission to the intensive care unit (ICU), clinical deterioration on the COVID-19 ordinal scale, death within 28 days, a COVID-19 ordinal scale score ≥ 4 on day 4, prolonged hospital stay beyond day 4, a COVID-19 ordinal scale score equal to or worse than day 1 on day 4, mechanical ventilation within 28 days, any thrombotic event (such as deep vein thrombosis (DVT), pulmonary embolism (PE), and stroke), a composite severity endpoint including death within 28 days, admission to the ICU, and mechanical ventilation, infection within 28 days (excluding COVID-19-related treatment), and / or infection within 28 days (>= grade 3).

[0208] In the embodiments, the present invention demonstrates a high negative predictive value (for ruling out risk or adverse events), enabling healthcare professionals to quickly (e.g., within the first 4 days after a patient’s visit), on the first day after a visit, or in relevant assessments approximately 3-4 days after the initial visit, effectively determine the risk of such patients.

[0209] As stated above, there is an urgent need for patient outcome prediction to avoid unnecessary treatment for patients with good prognoses or low risk (e.g., in a hospital setting) and to reserve treatment capacity for higher-risk patients. Patients with low risk of thrombotic events, low risk of needing oxygen, low risk of death, or low risk of needing ICU admission require effective management and appropriate treatment options. Given the increasing shortage of medical facilities and personnel, it is important to make the right decisions for patients seeking medical care early on, preventing low-risk patients from receiving unnecessary medical treatment. This invention enables direct and rapid biomarker detection without complex scoring determination, thereby introducing objective assessment independent of the experience level of medical personnel.

[0210] However, previous studies have failed to demonstrate a clear prognostic effect within 28 days when biomarkers are measured within a short period of 1 to 4 days after the initial examination. In the prior art, a patient's "diagnosis" is emphasized and determined at a certain stage. However, it is generally not possible to derive prognostic ability from diagnostic results, and as this invention demonstrates, biomarker levels with high negative predictive value cannot be assumed to have a very low false negative rate from studies indicating the diagnostic potential of a biomarker or even an association between high risk and high biomarker levels. This invention is able to measure negative predictive value primarily and effectively, substantially excluding risk in certain patient populations.

[0211] In some embodiments, a positive determination is possible, although the primary effect demonstrated by this invention is based on biomarker measurements that effectively exclude any given patient from risk within 28 days. In some embodiments, a high positive predictive value is provided regarding a patient's discharge from the ICU or hospital, thereby providing inclusion criteria. This corresponds to a high negative predictive value and exclusion of adverse clinical events and the need for intensive care, since leaving the ICU or hospital means that the patient no longer needs or requires intensive care provided by the ICU or hospital.

[0212] In one embodiment, the method includes:

[0213] - Provide samples from the patient, and

[0214] - To determine the levels of one or more biomarkers in the sample.

[0215] - The one or more biomarkers mentioned above include one or more of CT-proET-1 or a fragment thereof, sFlt-1 or a fragment thereof, proADM or a fragment thereof, and PCT or a fragment thereof.

[0216] a. Where a low-risk level of one or more biomarkers or fragments thereof in the sample on day 1 indicates a risk of the patient having no adverse clinical outcome within 28 days, and / or

[0217] b. The low risk ratio (day 4 / day 1 ratio) of the levels of one or more biomarkers or fragments thereof in the samples measured on day 4 and day 1 indicates the risk of the patient having no adverse clinical outcome within 28 days.

[0218] In one embodiment, the method includes:

[0219] - Provide the first sample from the patient.

[0220] - Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient.

[0221] - The first and second samples were separated within 96 hours (4 days) after consultation with medical personnel.

[0222] - Determine the levels of one or more biomarkers in the first and second samples, wherein the one or more biomarkers include one or more of CT-proET-1 or a fragment thereof, sFlt-1 or a fragment thereof, proADM or a fragment thereof, and PCT or a fragment thereof, and

[0223] a. Where a low-risk level of one or more biomarkers or fragments thereof in the sample on day 1 indicates a risk of the patient having no adverse clinical outcome within 28 days, and / or

[0224] b. The low risk ratio (day 4 / day 1) of the levels of one or more biomarkers or fragments thereof in the second sample measured on day 4 and the first sample measured on day 1 indicates the risk of the patient having no adverse clinical outcome within 28 days.

[0225] In one embodiment, the low-risk level and / or low-risk ratio of the corresponding biomarker and outcome can be derived from the ratios disclosed in Tables 1 to 49.

[0226] In one embodiment, the biomarker levels and ratios disclosed in Tables 1 to 49 represent the risk level and hazard ratio of the respective biomarker and / or combination of biomarkers, and the corresponding predicted adverse outcomes.

[0227] In one embodiment, the low-risk ratio for the level of one or more biomarkers or fragments thereof is ≤ 1. Such values ​​are typically associated with no increase or decrease in biomarker values ​​from day 1 to day 4. Such values ​​correspond to one or more biomarkers at lower or equal levels in the second sample compared to the first sample, preferably endothelial biomarkers (both isolated within 96 hours (4 days) and the second sample isolated at a time point after the isolation of the first sample, preferably the first sample isolated on day 1 and the second sample isolated on day 4).

[0228] As described in more detail below, the hazard ratio of the level of one or more biomarkers or fragments thereof in the sample represents a comparison of the biomarker values ​​measured on day 4 and day 1 (ratio day 4 / day 1). Preferably, the hazard ratio of the level of one or more biomarkers or fragments thereof in the sample represents a comparison of the biomarker values ​​measured in a second sample obtained on day 4 and a first sample obtained on day 1 (ratio day 4 / day 1). In embodiments, the assessment of these hazard ratios provides an accurate prognosis of the outcome, as illustrated by various statistical analyses presented below.

[0229] In one embodiment, the first sample is obtained after a medical visit. This could be in primary care, with a general practitioner, in a hospital ward, emergency room, or ICU. In one embodiment, the sample is obtained within 24 hours of the first contact with a healthcare professional (day 1), preferably within 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours after the medical visit. The sample can also be obtained upon admission or in any setting within a hospital (ED, ICU, or other).

[0230] In one embodiment, the second sample is obtained on day 4 after the initial medical visit. This sample can be obtained from primary care, general practitioners, hospital wards, emergency departments, or ICUs. In one embodiment, samples are obtained at 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, and 108 hours after the initial contact with healthcare personnel. By comparing the values ​​measured in these samples, the day 4:day 1 ratio can be determined. In this embodiment, this ratio itself indicates a statistically significant result.

[0231] Therefore, as disclosed herein, the Day 4 / Day 1 ratio can be determined for samples obtained within approximate time windows of "Day 1" (e.g., 0-24 hours after the first visit) and "Day 4" (e.g., 3-5 days after the first visit, preferably within 60-120 hours, more preferably within 72-120 hours, 72-108 hours, 72-96 hours, or 84-120 hours, 84-108 hours after the first visit).

[0232] In this embodiment, the second sample may be collected on day 3, day 4, or day 5, preferably on day 4. In this embodiment, the second sample may be collected within 60-120 hours after the first visit, preferably within 72-108 hours after the first visit.

[0233] In one embodiment, a low-risk level of CT-proET-1 or a fragment thereof, sFlt-1 or a fragment thereof, proADM or a fragment thereof, IL-6 and PCT or a fragment thereof, or a low-risk ratio of one or more biomarkers, indicates that the patient has no risk of death and / or ICU admission within 28 days.

[0234] In the embodiments, individual or combined measurements of IL-6 indicate the risk of a patient requiring mechanical ventilation, ICU admission, or death, composite severity endpoint, Day 4 Covid Scale, or death. Therefore, IL-6 values ​​provide additional value in determining the need for mechanical ventilation. For example, in the embodiments, the performance of various biomarkers, such as IL-6, with AUC measurements of 0.73, 0.72, 0.72, and 0.71 (p ≤ 0.003), provides reasonable predictive values ​​in assessing the risk of ICU admission or death, composite severity endpoint, Day 4 Covid Scale, mechanical ventilation, or death, such as IL-6.

[0235] In this embodiment, a “high-risk” level does not necessarily indicate that a patient will suffer the stated medical condition, outcome, or require any given treatment. In this embodiment, a “high-risk” patient is one whose risk cannot be “excluded” using biomarker measurements, without necessarily indicating “inclusion.” Therefore, the term “high-risk” preferably refers to a patient who cannot be effectively “excluded” from any given outcome, medical condition, or treatment. However, in a preferred embodiment, low-risk patients are effectively excluded from any given outcome, medical condition, or treatment due to a reliable low-risk level indicated by a strong NPV, as described in more detail below.

[0236] In one embodiment, the method includes determining the levels of at least two biomarkers in a sample, wherein the at least two biomarkers include at least one of C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, pro-adrenergic medullin (proADM) or a fragment thereof, and procalcitonin (PCT) or a fragment thereof, and optionally one or more of IL6, d-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, and C-reactive protein (CRP).

[0237] In one aspect, the present invention relates to a kit for carrying out the method of any one of the preceding claims, the kit comprising:

[0238] - Assay reagents for determining the levels of one or more of the following in samples from patients: C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, pro-adrenergic medullary kinase (proADM) or a fragment thereof, and procalcitonin (PCT) or a fragment thereof.

[0239] - Reference data for the risk of adverse clinical outcomes in patients within 28 days, particularly for risk thresholds, cutoff values, or hazard ratios, wherein the reference data is preferably stored on a computer-readable medium and / or used in the form of computer-executable code configured to compare the measured levels of one or more of the following in a patient’s sample: C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, pro-adrenergic medullary kinase (proADM) or a fragment thereof, and procalcitonin (PCT) or a fragment thereof, with a risk threshold or cutoff value;

[0240] - Optional diagnostic reagents for determining the presence of respiratory viral infection, preferably for coronavirus infection, and preferably for the presence of SARS-CoV2 virus infection;

[0241] - and optionally, a detection reagent for determining the level of at least one additional biomarker or fragment thereof (preferably IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C-reactive protein (CRP)) in a sample from a patient, and reference data, such as reference levels of the at least one additional biomarker (preferably IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C-reactive protein (CRP)), particularly a risk threshold, cutoff value, or hazard ratio, wherein the reference data is preferably stored on a computer-readable medium and / or used in the form of computer-executable code configured to compare the measured level of the at least one additional biomarker or fragment thereof with a threshold or cutoff value.

[0242] Each of the tables below (Tables 1 to 49), each row of the tables below, and combinations of the tables below and / or the rows below represent embodiments of the present invention with respect to hazard ratios, thresholds, and predictions of specific adverse outcomes and further outcomes, including, for example, discharge before or on day 4, discontinuation of supplemental oxygen, and improvement of COVID-19 using the ordinal scale through these thresholds and / or hazard values. Each of the tables below (Tables 1 to 49), each row of the tables below, and combinations of the tables below and / or the rows below represent embodiments of the present invention with respect to thresholds indicating whether the patient is at risk of adverse clinical outcomes. Each of the tables below (Tables 1 to 49), each row of the tables below, and combinations of the tables below and / or the rows below represent embodiments of the present invention with respect to thresholds indicating whether the patient does not require oxygen support and / or intensive treatment.

[0243] The present invention also relates to a method for detecting and / or determining the levels of biomarkers in a patient suffering from coronavirus infection, wherein the method comprises: (a) providing a sample from the patient; (b) detecting and / or determining the levels of one or more biomarkers in the sample; and (c) wherein the one or more biomarkers comprise one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal endothelin-1 precursor (CT-proET-1) or a fragment thereof, pro-adrenergic medullary kinase (proADM) or a fragment thereof, and / or procalcitonin (PCT) or a fragment thereof. In embodiments, patient groups and / or levels and / or statistics (such determination levels, cutoff values, and / or hazard ratios) as disclosed herein may be used in the method.

[0244] The present invention also relates to a method for detecting and / or determining the levels of biomarkers in a patient suffering from a respiratory viral infection, wherein the method comprises: (a) providing a first sample isolated from the patient; (b) providing a second sample isolated from the patient at a time point following the isolation of the first sample; (c) wherein the first and second samples are isolated within 96 hours (4 days) after consultation with a healthcare professional; and (d) detecting and / or determining the levels of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof and / or pro-adrenergic medullaris (proADM) or a fragment thereof. In embodiments, patient groups and / or levels and / or statistics (such determination levels, cutoff values, and / or hazard ratios) as disclosed herein may be used in the method.

[0245] The present invention also relates to a method for treating a patient, preferably for treating a patient to prevent and / or reduce the risk of adverse events or medical conditions associated with coronavirus infection, the method comprising optionally predicting disease progression, severity and / or outcome in a patient suffering from coronavirus infection, wherein the method comprises: (a) providing a sample from the patient; (b) determining the level of one or more biomarkers in the sample; (c) wherein the one or more biomarkers comprise one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, pro-adrenergic medullary kinase (proADM) or a fragment thereof and / or procalcitonin (PCT) or a fragment thereof; (d) wherein the level of the one or more biomarkers in the sample indicates the risk of adverse clinical outcome within 28 days; the method further comprises (e) treating the patient, preferably treating the patient to prevent and / or reduce the risk of adverse events or medical conditions associated with coronavirus infection. In the embodiments, patient groups and / or levels and / or statistics (such determination levels, thresholds and / or hazard ratios) or any one or more treatments known to those skilled in the art or disclosed herein may be used in the method.

[0246] The present invention also relates to a method for treating a patient, preferably for treating a patient to prevent and / or reduce the risk of adverse events or medical conditions associated with respiratory viral infection, the method comprising optionally predicting disease progression, severity and / or outcome in a patient suffering from a respiratory viral infection, wherein the method comprises (a) providing a first sample isolated from the patient, (b) providing a second sample isolated from the patient at a time point following the isolation of the first sample, (c) wherein the first and second samples are isolated within 96 hours (4 days) after consultation with a healthcare professional, (d) detecting and / or determining the level of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFlt1) or a fragment thereof and / or pro-adrenergic medullary kinase (proADM) or a fragment thereof, and (e) wherein a lower or equal level of the one or more endothelial biomarkers in the second sample compared to the first sample indicates that the patient has at least 28 In the risk of adverse clinical outcomes requiring no supplemental oxygen within 24 days, the method further includes (f) treating the patient, preferably to prevent and / or reduce the risk of adverse events or medical conditions associated with respiratory viral infection. In embodiments, patient groups and / or levels and / or statistics, such determination levels, cutoff values ​​and / or hazard ratios, or any one or more treatments known to a person skilled in the art or disclosed herein may be used in the method.

[0247] Other embodiments related to sFlt-1 or fragments thereof as biomarkers:

[0248] In one embodiment, one or more biomarkers include sFlt-1 or fragments thereof, and

[0249] - A low-risk level of sFlt-1 or a fragment thereof measured on day 1 indicates that the patient has no risk of a composite severity endpoint and / or the patient will be discharged before or on day 4 and / or show improvement on the COVID-19 Ordinal Scale on day 4 compared to day 1, or

[0250] - A high risk level of sFlt-1 or a fragment thereof measured on day 1 indicates that the patient is at risk of a composite severity endpoint and / or that the patient will be admitted to a hospital or ICU before or on day 4 and / or show a worsening of the COVID-19 Sequential Scale on day 4 compared to day 1.

[0251] In one embodiment,

[0252] - The low-risk level of sFlt-1 or its fragments measured on day 1 was ≤ 81.3 pgl / L ± 20%, or

[0253] - A high-risk level of sFlt-1 or its fragments measured on day 1 is > 81.3 pgl / L ± 20%, and optionally...

[0254] - The method includes comparing the assay level of one or more biomarkers in the sample with a critical value of 81.3 pgl / L ± 20%.

[0255] In one embodiment,

[0256] - A level of sFlt-1 or a fragment thereof measured on day 1 ≤ 83.9 pg / L ± 20% indicates that the patient has no risk of ICU admission and / or worsening of the COVID-19 Sequence Scale on day 4 compared to day 1 and / or the patient will be discharged before or on day 4, or

[0257] - A level of sFlt-1 or a fragment thereof measured on day 1 > 83.9 pg / L ± 20% indicates a risk of ICU admission and / or worsening of the COVID-19 Sequence Scale on day 4 compared to day 1, and / or the patient will be admitted to a hospital or ICU before or on day 4, and optionally

[0258] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 83.9 pg / L ± 20%.

[0259] In one embodiment,

[0260] - A level of sFlt-1 or a fragment thereof measured on day 1 ≤ 88.4 pg / L ± 20% indicates that the patient is not at risk of at least one of the composite severity endpoints (death within 28 days, ICU admission, or mechanical ventilation), or

[0261] - A level of sFlt-1 or a fragment thereof measured on day 1 > 88.4 pg / L ± 20% indicates that the patient is at risk of at least one of the composite severity endpoints (death, ICU admission, or mechanical ventilation), and optionally

[0262] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 88.4 pg / L ± 20%.

[0263] Variations in the sFlt-1 cutoff value can be used, such as any value of 60, 65, 70, 75, 80, 81, 81.3, 83.9, 84, 85, 88, 88.4, 90, 95, 100, 105, or 110 pg / L, or any value within these values, or any value within a range formed by any two endpoints of this list. Any cutoff value can also be a variation of ±1, 2, 3, 4, 5, 10, 15, 20, 25, or 30% relative to a specific value. Measurements using these cutoff values ​​are preferably applicable to serum samples in B.R.A.H.M.S. KRYPTOR assays. Such possible variations in cutoff values ​​apply to any given embodiment of the relevant biomarker in this disclosure.

[0264] In one embodiment, a low-risk level of sFlt-1 or a fragment thereof ≤ 81.3 pg / L ± 20% and a low-risk ratio indicate that the patient is not at risk of death, ICU admission, any thrombotic event, and / or composite severity endpoint within 28 days. In another embodiment, a low-risk level of sFlt-1 or a fragment thereof measured on day 1 ≤ 81.3 pg / L ± 20% and a low-risk ratio indicate that the patient is not at risk of death, ICU admission, any thrombotic event, and / or composite severity endpoint within 28 days.

[0265] Other embodiments related to proADM or fragments thereof as biomarkers:

[0266] In one embodiment, one or more biomarkers include proADM or a fragment thereof, and

[0267] - A low-risk level of proADM or its fragments measured on day 1 indicates that the patient has no risk of needing mechanical ventilation, death within 28 days, clinical admission to ICU, worsening of the COVID-19 Sequence Scale on day 4, and / or the patient will be discharged before or on day 4.

[0268] - A high risk level of proADM or a fragment thereof measured on day 1 indicates that the patient is at risk of requiring mechanical ventilation, dying within 28 days, being admitted to the ICU, experiencing clinical deterioration on day 4 of the COVID-19 Sequence Scale, and / or that the patient will be admitted to the hospital or ICU before or on day 4.

[0269] In one embodiment, determining the level of proADM or a fragment thereof includes determining the level of mid-pro-adrenergic medullaris peptide (MR-proADM) in the sample.

[0270] In one embodiment,

[0271] - The low-risk level of proADM or its fragments measured on day 1 was ≤ 0.87 nmol / L ± 20%, or

[0272] - The high-risk level of proADM or its fragments measured on day 1 is > 0.87 nmol / L ± 20%, and optionally...

[0273] - The method includes comparing the assay level of one or more biomarkers in the sample with a critical value of 0.87 nmol / L ± 20%.

[0274] In one embodiment,

[0275] - A proADM or fragment level measured on day 1 ≤ 0.87 nmol / L ± 20% indicates that the patient has no death or venous thrombosis or additional infection >= grade 3 within 28 days, requires continuous supplemental oxygen, is admitted to the ICU, requires mechanical ventilation, has a hospital stay of more than 4 days, or is at risk of worsening on the Covid-19 Sequence Scale, or

[0276] - A proADM or fragment level measured on day 1 > 2.25 nmol / L ± 20% indicates a risk of death or venous thrombosis or additional infection >= grade 3 within 28 days, continuous supplemental oxygen, ICU admission, mechanical ventilation requirement, hospital stay exceeding 4 days, or worsening of the Covid-19 Sequence Scale, and optionally

[0277] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 2.25 nmol / L ± 20%.

[0278] In one embodiment,

[0279] - A proADM level or fragment thereof measured on day 1 ≤ 1.33 nmol / L ± 20% indicates no risk of a composite severity endpoint and / or indicates discontinuation of supplemental oxygen, or

[0280] - A proADM or fragment level measured on day 1 > 1.33 nmol / L ± 20% indicates a risk of a composite severity endpoint and / or indicates continued supplemental oxygen, and optionally

[0281] - The method includes comparing the assay level of one or more biomarkers in the sample with a critical value of 1.33 nmol / L ± 20%.

[0282] In one embodiment,

[0283] - A level of proADM or a fragment thereof measured on day 1 ≤ 1.58 nmol / L ± 20% indicates that the patient has no risk of death within 28 days, or

[0284] - A proADM or fragment level > 1.58 nmol / L ± 20% measured on day 1 indicates a risk of death within 28 days, and optionally...

[0285] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 1.58 nmol / L ± 20%.

[0286] In one embodiment,

[0287] - A proADM or fragment level measured on day 1 ≤ 2.25 nmol / L ± 20% indicates that the patient has no death or venous thrombosis or additional infection >= 3 within 28 days, requires continuous supplemental oxygen, is admitted to the ICU, requires mechanical ventilation, has a hospital stay of more than 4 days, or is at risk of worsening on the Covid-19 Sequence Scale, or

[0288] - A proADM or fragment level measured on day 1 > 2.25 nmol / L ± 20% indicates a risk of death or venous thrombosis or additional infection >= grade 3 within 28 days, continuous supplemental oxygen, ICU admission, mechanical ventilation requirement, hospital stay exceeding 4 days, or worsening of the Covid-19 Sequence Scale, and optionally

[0289] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 2.25 nmol / L ± 20%.

[0290] Variations in the proADM, preferably MR-proADM, threshold values ​​can be employed, such as any value of 0.5, 0.6, 0.7, 0.8, 0.87, 0.9, 1.0, 1.10, 1.20, 1.30, 1.33, 1.40, 1.50, 1.58, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.25, 2.30, 2.40, 2.50 or 3, 4, 5, 6, 7, 8, 9 or 10 nmol / L, or any value within these values, or any value within a range formed by any two endpoints of this list. Any threshold value can also be varied by ±1, 2, 3, 4, 5, 10, 15, 20, 25 or 30% relative to a specific value. Such possible variations in threshold values ​​apply to any given embodiment of the relevant biomarker in this disclosure.

[0291] In other embodiments, the MR-proADM cutoff value used is 0.87, 0.9, or 1 nmol / L, which can provide an assessment of clinical risk, and values ​​above this amount can indicate the need for hospitalization, or in some cases, a cutoff value of 2.25 nmol / L can be used, and values ​​below this value can indicate that ICU treatment is no longer needed.

[0292] Measurements using these critical values ​​are preferably applicable to serum samples in B·R·A·H·M·S KRYPTOR assays.

[0293] In one embodiment, a low-risk level of proADM or a fragment thereof ≤ 0.87 nmol / L ± 20% and a low-risk ratio indicate that the patient has no risk of death within 28 days, ICU admission, additional infection, any thrombotic event and / or any thrombotic event and / or indication of discontinuation of supplemental oxygen.

[0294] Other embodiments related to PCT or fragments thereof as biomarkers:

[0295] In one embodiment, one or more biomarkers include PCT or fragments thereof, and

[0296] - A low-risk level of PCT or its fragment measured on day 1 indicates that the patient is not at risk of ICU admission, worsening of the COVID-19 Sequence Scale on day 4, and / or death within 28 days, or

[0297] - One or more biomarkers including PCT or a fragment thereof, wherein a high-risk level of PCT or a fragment thereof measured on day 1 indicates that the patient is at risk of admission to the ICU, deterioration of the COVID-19 Sequential Scale on day 4, and / or death within 28 days.

[0298] In one embodiment,

[0299] - The low-risk level of PCT or its fragments measured on day 1 is ≤ 0.27 ng / mL ± 20%, or

[0300] - A high-risk level of PCT or its fragments measured on day 1 is >0.27 ng / mL ± 20%, and optionally...

[0301] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 0.27 ng / mL ± 20%.

[0302] In embodiments, when using, for example, the B.R.A.H.M.S. Kryptor assay, the critical level for PCT can be a value in the range of 0.01 to 100.00 ng / mL in a serum sample. In a preferred embodiment, the critical level for PCT can be in the range of 0.01 to 100, 0.05 to 50, 0.1 to 20, or 0.1 to 0.27 ng / mL, and most preferably >0.25 to 0.5 ng / mL. Any value within these ranges can be considered a suitable critical value. For example, concentrations of 0.01, 0.05, 0.1, 0.2, 0.25, 0.27, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 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 may be used. In some embodiments, the PCT level in healthy subjects is approximately 0.05 ng / mL. Such possible variations in cutoff values ​​apply to any given embodiment of the relevant biomarkers in this disclosure.

[0303] In one embodiment,

[0304] - A PCT level of ≤ 0.25 ng / mL ± 20% or a fragment thereof measured on day 1 indicates that the patient has no risk of additional infection (>= grade 3) or hospitalization exceeding 4 days or death within the next 28 days, and / or will be discharged on or around day 4, or

[0305] - A PCT level or fragment level of >0.25 ng / mL ± 20% measured on day 1 indicates that the patient requires hospitalization for more than 4 days or is at risk of additional infection (>= grade 3) or has an increased risk of death within the next 28 days, and / or is admitted to a hospital or ICU, and optionally

[0306] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 0.25 ng / mL ± 20%.

[0307] In one embodiment,

[0308] - A PCT or fragment level of ≤ 0.5 ng / mL ± 20% measured on day 1 or day 4 indicates that the patient has no risk of additional infection (>= grade 3), hospitalization for more than 4 days, or death within the next 28 days, or

[0309] - A PCT level or fragment level of >0.5 ng / mL ± 20% measured on day 1 or day 4 indicates that the patient requires hospitalization for more than 4 days or is at risk of additional infection (>= grade 3) or has an increased risk of death within the next 28 days, and optionally

[0310] - The method involves comparing the assay level of one or more biomarkers in the sample with a critical value of 0.5 ng / mL ± 20%.

[0311] In one embodiment, a low-risk level of PCT or a fragment thereof ≤ 0.25 ng / mL ± 20% and a low-risk ratio indicate that the patient is not at risk of death, ICU admission, additional infection, and / or any thrombotic events within 28 days.

[0312] The embodiments and features of the invention described with respect to the methods and kits are considered to be disclosed with respect to every and all other aspects of this disclosure, such that features of characterizing the methods can be used to characterize the kits, and vice versa. Detailed Implementation

[0313] General terminology:

[0314] As used herein, the terms “comprising” and “including” or their grammatical variations shall be regarded as describing in detail the features, integers, steps, or components described, but do not preclude the addition of one or more additional features, integers, steps, components, or groups thereof. This term encompasses the terms “consisting of” and “substantially consisting of”.

[0315] Therefore, the terms "comprising," "including," or "having" mean that any additional components (or similarly, features, integers, steps, etc.) may be present. The term "consisting of" means that no additional components (or similarly, features, integers, steps, etc.) are present.

[0316] When used herein, the term “consistently of” or its grammatical variations shall be regarded as a detailed description of the stated feature, integer, step or component, but does not preclude the addition of one or more additional features, integers, steps, components or groups thereof, provided that such additional features, integers, steps, components or groups thereof do not substantially alter the essential and novel characteristics of the claimed composition, apparatus or method.

[0317] Therefore, the term "substantially constitutes" means that specific additional components (or similarly, features, integers, steps, etc.) may be present, i.e., those that do not substantially affect the essential characteristics of the composition, apparatus, or method. In other words, the term "substantially constitutes" (which may be used interchangeably with "substantially comprises") allows the presence of other components in the composition, apparatus, or method besides mandatory components (or similarly, features, integers, steps, etc.), provided that the presence of these other components does not substantially affect the essential characteristics of the apparatus or method.

[0318] The term "method" refers to the manner, means, technique, and procedure for accomplishing a given task, including, but not limited to, those manner, means, techniques, and procedures known to practitioners in the fields of chemistry, biology, and biophysics, or readily developed from known manner, means, techniques, and procedures. As used herein, a "patient" or "subject" can be a vertebrate. In the context of this invention, the term "subject" includes humans and animals, particularly mammals and other organisms.

[0319] SARS and coronavirus:

[0320] In some embodiments, symptoms of SARS virus infection include fever, sore throat, cough, muscle pain or fatigue, and in some embodiments, additionally sputum production, headache, hemoptysis and / or diarrhea.

[0321] In some embodiments, symptoms of infection with a SARS coronavirus (such as SARS-CoV-2) include fever, sore throat, cough, loss of taste and / or smell, shortness of breath, and / or fatigue.

[0322] Coronaviruses are a group of related viruses that cause diseases in mammals and birds. The scientific name for coronaviruses is the subfamily Orthocoronavirinae or Coronavirinae. Coronaviruses belong to the family Coronaviridae. This family is divided into the subfamilies Coronavirinae and Coronavirinae, which are further divided into six genera: Acoronavirus, Bcoronavirus, Ccoronavirus, Dcoronavirus, Coronaviridae, and Bafinivirus. While viruses in the Acoronavirus and Bcoronavirus genera primarily infect mammals, Ccoronavirus infects avian species, and members of the Dcoronavirus genus have been found in both mammalian and avian hosts.

[0323] In humans, coronaviruses cause mild respiratory infections, such as some cases of the common cold, as well as other potentially fatal cases, such as SARS, MERS, and COVID-19. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical nucleocapsid. The genome size of coronaviruses ranges from about 27 kb to 34 kb, making them the largest known RNA viruses.

[0324] Many human coronaviruses are known, such as, but not limited to, human coronavirus OC43 (HCoV-OC43) of the β-CoV genus, human coronavirus HKU1 (HCoV-HKU1) of the β-CoV genus, human coronavirus 229E (HCoV-229E) of the α-CoV genus, human coronavirus NL63 (HCoV-NL63) of the α-CoV genus, Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0325] The risk factors for coronaviruses vary widely. Some can kill more than 30% of infected individuals (such as MERS-CoV), while others are relatively harmless, such as the common cold. The common cold caused by coronaviruses has main symptoms such as fever and sore throat (e.g., due to adenoid swelling) and mainly occurs in winter and early spring. Coronaviruses can cause pneumonia (direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (direct viral bronchitis or secondary bacterial bronchitis). Coronaviruses can also cause SARS.

[0326] Advances in nucleic acid sequencing technology (commonly known as next-generation sequencing, NGS) are providing a wealth of sequence data from a wide variety of biological samples and allowing for the characterization of known and novel viral strains. Therefore, established methods can be used to identify coronavirus infections.

[0327] Viruses encode a set of proteins necessary for viral self-replication and persistence. Enzymes for genomic mRNA production and genome replication, proteases for protein maturation, proteins for genome capsid formation, and proteins that disrupt the host's antiviral response can be identified by conserved protein motifs or domains. Possibly due to selective pressure, viral genomes are streamlined, and the number of functionally encoded proteins is far greater than in cellular organisms. Therefore, describing the viral genome by collecting encoded protein domains is a potentially useful classification method. This allows for tracking viral evolution and the identification of new coronavirus strains based on sequence comparisons with known coronavirus strains.

[0328] In some embodiments, the patient is infected, preferably with SARS virus, and more preferably with SARS coronavirus (SARS-CoV).

[0329] As used in this article, SARS virus refers to the virus that causes severe acute respiratory syndrome (SARS). Although coronaviruses have been the leading cause of SARS to date, other viral infections, such as other zoonotic viruses, can also cause SARS.

[0330] As used in this article, SARS coronavirus refers to the coronavirus that causes severe acute respiratory syndrome (SARS). This syndrome is a zoonotic viral respiratory disease that first appeared in the early 2000s and was caused by the first discovered strain of SARS coronavirus (SARS-CoV or SARS-CoV-1).

[0331] SARS is transmitted through droplets, and viral replication can be observed in the mucous membranes of the upper and lower respiratory tracts or the gastrointestinal tract. The virus can also directly invade cells in various organs, such as the liver, kidneys, heart, and brain. Another apparent mechanism appears to be the virus directly invading T cells. Many SARS patients with COVID-19 have clinically low lymphocyte concentrations in their blood, a condition known as lymphopenia. Clinically, patients may present with respiratory symptoms such as dry cough and shortness of breath, as well as fever or diarrhea. Symptoms associated with acute liver, heart, or kidney damage may also occur. In less severe cases of SARS, patients may exhibit mild symptoms or even be asymptomatic.

[0332] Clinical and scientific studies have shown that SARS-CoV binds to epithelial cells via the angiotensin-converting enzyme 2 receptor (ACE2). ACE2 is a cell membrane-bound aminopeptidase expressed in the vascular endothelium, kidneys, bladder, heart, nasal mucosa, bronchi, and lungs. As a result, viral binding leads to damage to epithelial and endothelial cells and vascular leakage, which triggers the secretion of pro-inflammatory cytokines and chemokines. The virus also mediates the downregulation and shedding of ACE2, further contributing to dysfunction of the renin-angiotensin system (RAS). Once the RAS is disrupted, it leads to an inflammatory response and increased vascular permeability. Focusing on the respiratory system, ACE2 shedding can lead to increased pulmonary vascular permeability, resulting in pulmonary edema.

[0333] Another pathophysiological factor known as antibody-dependent enhancement (ADE) is associated with the presence of antibodies that bind to the virus and promote viral entry into cells. Therefore, these antibodies support viral replication and spread in the body rather than exhibiting a neutralizing effect. The presence of ADE in a patient can activate or maintain a systemic inflammatory response.

[0334] Some have proposed that direct cellular damage (multi-organ) and impaired RAS are key factors in local and systemic inflammatory responses, leading to a so-called cytokine storm, which has been associated with adverse outcomes in SARS patients. The adverse consequences of SARS may be based on the development of acute respiratory distress syndrome (ARDS), multi-organ damage, or lymphopenia.

[0335] Elderly patients (over 60 years of age), patients with chronic diseases (e.g., cardiovascular disease, diabetes, cancer, COPD), or patients with weakened immune systems are considered to be at higher risk of severe SARS development. Smoking and obesity are also considered risk factors (1-5, 21).

[0336] Examples of SARS coronaviruses include, but are not limited to, any coronavirus that induces SARS or SARS-like pathology. Specific examples include, but are not limited to, SARS coronavirus (SARS-CoV-1), first discovered in 2003 (as described above), Middle East Respiratory Syndrome (MERS-CoV), first discovered in 2012, and SARS-CoV-2, which cause COVID-19, a disease that led to the 2019-2020 coronavirus pandemic.

[0337] In a preferred embodiment, the SARS coronavirus is SARS-CoV-2 or a SARS-CoV derived therefrom.

[0338] The SARS-CoV-2 strain causes COVID-19, the disease that caused the ongoing coronavirus pandemic of 2019–2020. The disease was discovered in December 2019 and spread globally. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include muscle pain, diarrhea, sore throat, loss of taste and / or smell, and abdominal pain. While most cases result in mild symptoms, some cases develop viral pneumonia and multiple organ failure.

[0339] Coronaviruses can be identified using molecular techniques, such as sequence-based analysis, including PCR-based amplification of viral genetic material. Whole-genome phylogenetic analysis showed that SARS-CoV-2 shared 79.5% and 50% sequence identity with SARS-CoV and MERS-CoV, respectively. However, the seven conserved replicase domains in SARS-CoV-2's ORF1ab showed 94.6% sequence identity with SARS-CoV, while the sequence identity with other coronaviruses was less than 90%, suggesting that SARS-CoV-2 belongs to the β-CoV lineage.

[0340] Similar to other CoVs, the SARS-CoV-2 viral particle, with a genome size of 29.9 kb

[13] , has a nucleocapsid consisting of genomic RNA and phosphorylated nucleocapsid (N) protein. The nucleocapsid is embedded in a phospholipid bilayer and is covered by two different types of spike proteins: the spike glycoprotein trimer (S), which is present in all CoVs, and the hemagglutinin esterase (HE), which is common only in some CoVs. The membrane (M) protein and the envelope (E) protein are located between the S protein in the viral envelope. The SARS-CoV-2 genome has 5' and 3' end sequences (265 nt at the 5' end and 229 nt at the 3' end), which is typical of β-CoVs, and the gene sequence is 5'-replicaase open reading frame (ORF) 1ab-S-enveloping (E)-membrane (M)-N-30. The predicted lengths of the S, ORF3a, E, M, and N genes for SARS-CoV-2 are 3822, 828, 228, 669, and 1260 nt, respectively. Similar to SARS-CoV, SARS-CoV-2 carries a predicted ORF8 gene (366 nt in length) located between the M and N ORF genes.

[0341] According to Jin et al. (Viruses 2020, 12, 372), fever (98%), cough (76%), and myalgia or fatigue (44%) were the most common symptoms among the initial 41 patients. Less common symptoms were sputum production (28%), headache (8%), hemoptysis (5%), and diarrhea (3%). More than half of the patients experienced dyspnea. The estimated mean latency period and basic reproduction number (R0) were 5.2 days (95% CI: 4.1–7.0) and 2.2 days (95% CI, 1.4–3.9), respectively. Blood tests showed normal or decreased white blood cell counts (25%) and lymphopenia (65%). Bilateral involvement was observed in 98% of the patients on chest CT. The typical chest CT findings in ICU patients upon admission were bilateral multilobular and subsegmental consolidation. The representative chest CT findings in non-ICU patients showed bilateral ground-glass opacities and subsegmental consolidation. An analysis of 1,324 laboratory-confirmed cases showed that fever (87.9%) and cough (67.7%) remained the most common symptoms, while diarrhea was uncommon. Lymphopenia was present in 82.1% of patients admitted to the ICU.

[0342] In some embodiments, the subject has been tested and determined to have SARS-CoV. SAR-CoV, such as SARS-CoV-2, can be detected using various methods, including nucleic acid testing, serological diagnosis, CRISPR / Cas13-based SHERLOCK technology, or imaging techniques (e.g., chest X-ray or CT scan).

[0343] SARS-CoV-2 is primarily transmitted through respiratory droplets, contact, and potentially the fecal-oral route. It is hypothesized that primary viral replication occurs in the mucosal epithelium of the upper respiratory tract (nasal cavity and pharynx), further proliferating in the mucosa of the lower respiratory tract and gastrointestinal tract, leading to mild viremia. At this stage, infection is rarely controlled and patients remain asymptomatic. Some patients also exhibit non-respiratory symptoms such as acute liver and heart damage, kidney failure, and diarrhea, suggesting multi-organ involvement. ACE2 is widely expressed in the nasal mucosa, bronchi, lungs, heart, esophagus, kidneys, stomach, bladder, and ileum, all organs susceptible to SARS-CoV-2 infection.

[0344] According to Jin et al. (Viruses 2020, 12, 372), the first report of pathological results for severe COVID-19 showed bilateral diffuse alveolar damage with cellular fibrinous exudate in the lungs. The right lung showed significant pulmonary desquamation and hyaline membrane formation, suggestive of acute respiratory distress syndrome (ARDS). The left lung tissue showed pulmonary edema with hyaline membrane formation, suggesting early ARDS. Interstitial mononuclear inflammatory infiltration, predominantly lymphocytic, was observed in both lungs. Multinucleated syncytial cells were found within the alveolar spaces, accompanied by atypically enlarged lung cells characterized by large nuclei, amphipathic granular cytoplasm, and prominent nucleoli, indicating viral cytopathic changes.

[0345] According to Jin et al. (Viruses 2020, 12, 372), these lung pathological findings are remarkably similar to those found in SARS and MERS. Moderate microvascular steatosis and mild lobular and portal venous activity were observed in liver biopsy samples, which may be caused by SARS-CoV-2 infection or drug use. Furthermore, only a small amount of interstitial mononuclear inflammatory infiltration was found in cardiac tissue, suggesting that SARS-CoV-2 may not directly damage the heart. Extensive mucus secretion in both lungs was found in COVID-19 deaths, unlike in SARS and MERS. Acute respiratory distress syndrome (ARDS) is a life-threatening lung condition that prevents sufficient oxygen from reaching the lungs and circulatory system, and is a cause of death in many respiratory illnesses and acute lung injuries. In fatal cases of human SARS-CoV, MERS-CoV, and SARS-CoV-2 infection, individuals exhibited severe respiratory distress requiring mechanical ventilation, and histopathological results also supported ARDS. Immune dysfunction and cytokine storms have also been observed in deaths caused by COVID-19.

[0346] Prognosis and risk assessment:

[0347] Therefore, as used herein, the terms “requiring intensive treatment and / or disease surveillance” or “adverse respiratory and / or infectious clinical outcomes requiring oxygen support and intensive treatment” refer to patients who are at higher risk of disease exacerbation (i.e., at higher risk of progression to more serious health risks to the patient) compared to patients with low risk, preferably in conjunction with the above description of the pathogenesis.

[0348] As used herein, the terms “no need for intensive treatment and / or disease surveillance” or “no risk of adverse respiratory and / or infectious clinical outcomes requiring oxygen support and intensive treatment” refer to a prognostic determination indicating a low risk of adverse events and therefore not requiring such treatment and / or surveillance. This term essentially refers to an effective “exclusion criterion” with sufficient negative predictive value to indicate a low risk of adverse events, preferably within 28 days, as described herein.

[0349] As used herein, the term "risk of disease progression to symptoms requiring hospitalization" refers to patients requiring intensive treatment and / or disease surveillance where, based on the advice of healthcare professionals, the hospital-based treatment options are preferable. In other words, the risk is assessed as follows: treatments typically available outside of a hospital are insufficient to safely care for the patient and prevent unwanted adverse events.

[0350] As used herein, the term “no risk of disease progression to a condition requiring hospitalization” relates to a prognostic statement indicating a low risk of disease progressing to a level requiring hospitalization, and therefore hospitalization is not necessary. This term essentially relates to a valid “exclusion statement” with sufficient negative predictive value to indicate a low risk of adverse events, preferably within 28 days, as described herein.

[0351] As used herein, the term "risk of disease progression to the point requiring treatment and / or monitoring in the ICU" refers to patients requiring intensive care and / or monitoring where, based on the advice of healthcare professionals, the treatment options available in the ICU are preferable. In other words, the risk is assessed as follows: treatments typically available outside the ICU are insufficient to safely care for the patient and prevent unwanted adverse events.

[0352] As used herein, the term “no risk of disease progression to a condition requiring ICU treatment and / or disease monitoring” relates to a prognostic statement indicating a low risk of disease progression to the level requiring ICU admission and therefore not requiring ICU treatment. This term essentially relates to a valid “exclusion statement” with sufficient negative predictive value to indicate a low risk of adverse events, preferably within 28 days, as described herein.

[0353] According to the present invention, therapeutic interventions or treatments may also include type 1 interferon (IFN), IFN-α, IFN-β, immunosuppressive drugs, ribavirin, remdesivir, lopinavir, nelfinavir, chloroquine, convalescent plasma, anti-SARS-CoV antibody, anti-Fc specific antibody, renin-angiotensin system (RAS) inhibitors, ACE2-mesenchymal stem cell (MSC) intravenous transplantation, anti-inflammatory drugs, tumor necrosis factor-α blockers, protease inhibitors, steroids, vaccination, or other therapies to reduce or prevent negative outcomes or adverse events or patient death.

[0354] Patients diagnosed as “critically ill” and requiring oxygen support as described in this article may be diagnosed as intensive care unit (ICU) patients, patients requiring continuous and / or close monitoring of their health status, patients diagnosed with SARS, patients with SARS and sepsis, patients with severe sepsis or septic shock, patients diagnosed with SARS and one or more existing organ failures, pre- or post-operative patients, intra-operative patients, post-traumatic patients, trauma patients (such as accident patients, burn patients), or patients with one or more open lesions. Subjects described in this article may be in the emergency department or intensive care unit, or in other point-of-care settings (such as in an emergency transport vehicle such as an ambulance), or at the home of a general practitioner facing a patient with the described symptoms.

[0355] "Prognosis" involves predicting the outcome or specific risk (high or low) of a subject. This may also include an estimate of the subject's chance of recovery or the chance of an adverse outcome. In an embodiment, prognostic assessments can be made based on the determination of one or more biomarkers over a time frame, enabling a prognostic determination of the outcome to be made within 28 days of the first patient sample collection.

[0356] The method of this invention can also be used for monitoring, treatment monitoring, treatment guidance, and / or treatment control. "Monitoring" involves tracking patients and potential complications to, for example, analyze the progress of the healing process or the impact of a particular treatment or therapy on the patient's health.

[0357] In the context of this invention, the terms "treatment monitoring" or "treatment control" refer to monitoring and / or adjusting the patient's therapeutic treatment, for example, by obtaining feedback on the efficacy of the treatment. As used herein, the terms "treatment guidance" or "treatment management" refer to applying certain treatments, therapeutic procedures, or medical interventions based on the values / levels of one or more biomarkers and / or clinical parameters and / or clinical scores. This includes adjustments to treatment or interruptions of treatment.

[0358] In this invention, the terms "risk assessment" and "risk stratification" refer to grouping subjects into different risk groups based on their further prognosis. Risk assessment also involves stratification for the application of preventive and / or treatment measures. The term "treatment stratification" specifically refers to grouping or classifying patients into different groups, such as risk groups or treatment groups that receive certain different treatment measures based on their classification. The term "treatment stratification" also refers to grouping or classifying patients with infection or symptoms of infectious diseases into groups that do not require certain treatment measures.

[0359] Biomarkers:

[0360] As used herein, terms such as “marker,” “surrogate,” “prognostic marker,” “factor,” or “biomarker” are used interchangeably to refer to measurable and quantifiable biomarkers (e.g., the concentration of a specific protein or enzyme or a fragment thereof, the concentration of a specific hormone or a fragment thereof, or the presence of a biological substance or a fragment thereof) used as indicators of health and physiologically relevant assessments, such as the risk of disease / symptom / clinical condition, preferably adverse events. A marker or biomarker is defined as a property that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenesis, or pharmacological response to therapeutic interventions. Biomarkers can be measured in samples such as blood, plasma, urine, or tissue.

[0361] The terms “endothelial biomarker,” “endothelial marker,” “endothelial functional biomarker,” or “endothelial functional marker” refer to biomarkers that are associated with endothelial function and structure and whose levels indicate physiological function or dysfunction of the endothelium in a subject. Endothelial dysfunction is an endothelial pathological state associated with impaired nitric oxide bioavailability, characterized by vasoconstriction, increased vascular permeability, thrombosis, and inflammation. Biomarkers associated with endothelial dysfunction (endothelial biomarkers) include, but are not limited to, ADM precursors or fragments thereof (such as MR-proADM), sFlt-1, and other biomarkers such as ET1 or fragments thereof (such as CT-proET1), D-dimer, von Willebrand factor, fibrin degradation products, C-reactive protein (CRP), ferritin, interleukin-6 (IL-6), plasma creatinine, angiopoietin-2 (Ang-2), E-selectin, P-selectin, multiligand glycan-1, and vascular endothelial growth factor (VEGF)).

[0362] sFlt-1:

[0363] As used herein, the term "soluble Flt-1 (sFlt-1)" (soluble fms-like tyrosine kinase 1, also known as sVEGF-R1) refers to a soluble form of the Flt-1 receptor that is homologous to the protein defined by GenBank accession number U01134 or UniProtP17948 or entry name VGFR1_HUMAN and possesses sFlt-1 biological activity. The biological activity of the sFlt-1 peptide can be determined using any standard method, such as by measuring the binding of sFlt-1 to VEGF. sFlt-1 lacks the transmembrane domain and cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-1 can bind to VEGF and PIGF with high affinity, but it cannot induce proliferation and angiogenesis, and is therefore functionally distinct from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical cord endothelial cells and was later shown to be produced in vivo by trophoblast cells. As used in this article, sFlt-1 includes any sFlt-1 family member or subtype.

[0364] The methods used to determine sFlt-1 are known to those skilled in the art, for example, by using products obtained from Thermo Fisher Scientific / Brahms GmbH.

[0365] CT-proET-1:

[0366] Endothelin-1 (ET-1) is a 21-amino acid peptide and a potent vasoconstrictor. Since its discovery in 1988, its biosynthesis, mode of action, and association with disease have been extensively studied. Three different genes encode endothelin isoforms (endothelin-1, endothelin-2, and endothelin-3), with endothelin-1 being the most concentrated and potent. Endothelin-1 is found in endothelial cells, the lungs, and the heart, and is synthesized in the kidneys and brain. The primary translation product of the human endothelin-1 gene is a 212-amino acid peptide (see, for example, GenBank accession number: AAA52339.1), called proendothelinogen-1 (proendothelinogen). During secretion, a short N-terminal signal sequence (amino acids 1-17) of proendothelinogen is removed by a signal peptidase. The obtained proendothelin is then processed by furin protease on two amino acid pairs to obtain a bioinactive peptide (large endothelin) containing 38 amino acids. Large endothelin is ultimately converted into mature, bioactive endothelin-1 by endothelin convertase (ECE). Furthermore, the C-terminal proendothelin fragment (CT-proET-1) has the amino acid sequence of proendothelin 93-212 or 168-212.

[0367] Endothelin typically exerts its effects by binding to specific receptors located on myocytes, cardiomyocytes, and fibroblasts. This binding leads to calcium efflux, phospholipase C activation, and Na / K ATPase inhibition. In addition to its vasoconstrictive effects, endothelin also possesses growth-regulating properties. Elevated plasma concentrations of endothelin-1 and macroendothelin have been described in a variety of clinical indications. These risks include cardiovascular diseases (particularly pulmonary hypertension, atherosclerosis, congestive heart failure, and myocardial infarction), sepsis and septic shock, and cancer.

[0368] The methods used to determine CT-proET-1 are known to a person skilled in the art, for example, by using products obtained from Thermo Fisher Scientific / Brahms Ltd.

[0369] ADM:

[0370] It should be understood that, in the context of this invention, "determining the level of proADM or a fragment thereof" refers to any means of determining proADM or a fragment thereof. The fragment may have any length, for example, at least about 5, 10, 20, 30, 40, 50, or 100 amino acids, as long as the fragment allows for the explicit determination of the level of proADM or a fragment thereof.

[0371] In a particularly preferred aspect of the invention, "determining the level of proADM" refers to determining the level of mid-region pro-adrenal medullaris (MR-proADM). MR-proADM is a fragment and / or region of proADM.

[0372] The peptide adrenomedullin (ADM) was discovered to be a 52-amino acid-containing antihypertensive peptide isolated from human pheochromocytoma (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a precursor peptide comprising 185 amino acids (“pro-adrenomedullin precursor” or “pro-ADM”). An exemplary amino acid sequence is given in SEQ ID NO: 1.

[0373] SEQ ID NO: 1: Amino acid sequence of pre-pro-ADM:

[0374] 1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

[0375] 51 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

[0376] 101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

[0377] 151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0378] ADM comprises amino acid positions 95 to 146 of the pre-proADM amino acid sequence and is its splice product. "Pre-adrenal medullaris" ("proADM") refers to pre-proADM without the signal sequence (amino acids 1 to 21), i.e., amino acid residues 22 to 185 of pre-proADM. "Middle pro-adrenal medullaris peptide" ("MR-proADM") refers to amino acids 42 to 95 of pre-proADM. An exemplary amino acid sequence of MR-proADM is given in SEQ ID NO: 2.

[0379] SEQ ID NO: 2: Amino acid sequence of MR-pro-ADM (AS 45-92 of pre-pro-ADM):

[0380] ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

[0381] This document also envisions that peptides or fragments of pre-proADM or MR-proADM could be used in the methods described herein. For example, the peptide or fragment may comprise amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre-proADM (mature adrenal medulla, containing the biologically active form, also known as bio-ADM).

[0382] SEQ ID NO: 3: Amino acid sequence of mature ADM (AS 95-146 of pre-pro-ADM):

[0383] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY

[0384] The C-terminal fragment of proADM (amino acids 153 to 185 of pre-proADM) is referred to as vasopressin. Fragments of the proADM peptide or MR-proADM may comprise, for example, at least about 5, 10, 20, 30, or more amino acids. Therefore, the proADM fragment may be selected, for example, from the group consisting of MR-proADM, PAMP, vasopressin, and mature adrenaline, and preferably herein, the fragment is MR-proADM. In the embodiments, the proADM fragment may be selected from the group consisting of MR-proADM, PAMP, and vasopressin.

[0385] The identification of these different forms of ADM or proADM and their fragments also encompasses measuring and / or detecting specific subregions of these molecules, for example by employing antibodies or other affinity reagents targeting specific parts of the molecule, or by determining the presence and / or amount of the molecule by means of measuring a portion of the protein using mass spectrometry. Any one or more “ADM peptides or fragments” described herein may be used in this invention.

[0386] Therefore, the methods and kits of the present invention may also include the determination of at least one other biomarker, biomarker and / or parameter other than ADM.

[0387] The methods used to determine proADM (especially MR-proADM) are known to those skilled in the art, for example, by using products obtained from Thermo Fisher Scientific / Brahms Ltd.

[0388] PCT:

[0389] As used herein, “procalcitonin” or “PCT” refers to a peptide spanning amino acid residues 1-116, 2-116, 3-116, or a fragment thereof of the procalcitonin peptide. PCT is a peptide precursor of the hormone calcitonin. Therefore, the procalcitonin fragment is at least 12 amino acids in length, preferably more than 50 amino acids, and more preferably more than 110 amino acids. PCT may include post-translational modifications such as glycosylation, lipidation, or derivatization. Procalcitonin is a precursor of both calcitonin and anticalcitonin. Therefore, under normal conditions, circulating PCT levels are very low (< about 0.05 ng / ml).

[0390] The PCT level in a subject's sample can be determined by an immunoassay as described herein. As used herein, the level of the ribonucleic acid or deoxyribonucleic acid encoding "procalcitonin" or "PCT" can also be determined. Methods for determining PCT are known to those skilled in the art, for example, by using products available from Thermo Fisher Scientific / Brahms Ltd.

[0391] Other biomarkers:

[0392] Therefore, the methods and kits of the present invention may also include the determination of at least one other biomarker, biomarker, clinical score and / or parameter.

[0393] As used herein, parameters are characteristics, features, or measurable factors that help define a particular system. Parameters are important elements in assessments related to health and physiology, such as disease / symptom / clinical symptom risk, preferably organ dysfunction. Furthermore, parameters are defined as characteristics that are objectively measured and assessed as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Exemplary parameters can be selected from the following groups: Pneumonia Severity Index (PSI), Acute Physiology and Chronic Health Assessment II (APACHE II), Simplified Acute Physiology Score (SAPSII), Sequential Organ Failure Assessment Score (SOFA), Rapid Sequential Organ Failure Assessment Score (qSOFA), Body Mass Index (BMI), weight, age, sex, IGS II, fluid intake, white blood cell count, sodium, potassium, temperature, blood pressure, dopamine, bilirubin, respiratory rate, partial pressure of oxygen, World Federation of Neurosurgical Societies (WFNS) classification, Glasgow Coma Scale (GCS), CURB-65 Pneumonia Severity Score, Pneumonia Severity Index (PSI), age, sex, family history, race, weight, body mass index (BMI), cystoscopy report, white blood cell count, lymphocyte count, imaging methods such as CT scan, PET imaging, or X-ray, blood pressure, heart rate, antihypertensive treatment, fluid intake, wheezing, body temperature, presence of diabetes, blood glucose level, and (current) smoking habit.

[0394] Such parameters can be evaluated in conjunction with the methods described herein to improve assay implementation and diagnostic statements.

[0395] The present invention offers the following advantages over conventional methods: the methods and kits of the present invention are rapid, objective, easy to use, and accurate. The methods and kits of the present invention relate to biomarkers and clinical scores that are readily measurable using conventional methods, as levels of proADM, PCT, CT-proET-1, sFlt-1, or IL-6 can be determined in routinely obtained blood samples or other biological fluids or samples obtained from subjects.

[0396] D-dimer is normally absent in human plasma. This biomarker is one of the fibrinogen degradation products (FDPs), released after a thrombus or blood clot is enzymatically degraded by plasmin. Below concentrations (e.g., 0.5 mg / L in a blood sample), D-dimer can help rule out clinical conditions characterized by inappropriate blood clot formation, such as deep vein thrombosis, pulmonary embolism, or disseminated intravascular coagulation. If D-dimer levels are elevated, further testing (such as ultrasound, scintigraphy, or CT scan) is required (see [link to relevant documentation]). Adam SS, Key NS, Greenberg CS (March 2009). "D-dimer" "antigen: current concepts and future prospects". Blood. 113 (13): 2878–87. doi:10.1182 / blood-2008-06-165845).

[0397] C-reactive protein (CRP) is a pentamer protein found in bodily fluids such as plasma. CRP levels can rise in response to inflammation. Measuring and mapping CRP values ​​can be helpful in determining disease progression or the effectiveness of treatment.

[0398] Interleukin-6 (IL-6) is produced rapidly and transiently in response to infection and tissue damage, and contributes to host defense by stimulating acute-phase responses, hematopoiesis, and immune responses. Although IL-6 expression is controlled by transcriptional and post-transcriptional mechanisms, persistent dysregulation of IL-6 synthesis plays a pathological role in chronic inflammation and autoimmunity. IL-6 expression is associated with coronavirus infection and can predict the severity of COVID-19.

[0399] The subject's at least one additional biomarker and / or parameter may be selected from the group consisting of: lactate levels in the sample, the subject's Sequential Organ Failure Assessment (SOFA) score (optionally, Rapid SOFA score), the subject's Simplified Acute Physiology Score (SAPSII), the subject's Acute Physiology and Chronic Health Evaluation II (APACHE II) score, and soluble fms-like tyrosine kinase-1 (sFlt-1) levels, histone H2A, histone H2B, histone H3, histone H4, calcitonin, endothelin-1 (ET-1), arginine vasopressin (AVP), atrial natriuretic peptide (ANP), neutrophil gelatinase-associated lipocalcin (NGAL), delta-like protein 1 (DLL1), troponin, brain natriuretic peptide (BNP), C-reactive protein (CRP), pancreatic stone protein (PSP), myeloid cell-expressed excitation receptor-1 (TREM1), endothelial cell-specific molecule-1 (ESM-1), growth differentiation factor- IL-15 (GDF-15), interleukin-6 (IL-6), interleukin-1, interleukin-24 (IL-24), interleukin-22 (IL-22), interleukin-20 (IL-20), other ILs, presepsin (sCD14-ST), lipopolysaccharide-binding protein (LBP), α-1-antitrypsin, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 7 (MMP7), placental growth factor (PlGF), chromogranin A, S100A protein, S100B protein and tumor necrosis factor α (TNFα), insulin-like growth factor binding protein 7 (=IGFBP7), neopterin, soluble trigger receptor-1 expressed on myeloid cells (STREM-1), α-1-antitrypsin, proarginine vasopressin (AVP, proAVP)Or, along with peptides), 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, C CL3L3, 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, FA M19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, IFNA1, IFNA10, IFNA13, IF NA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, BAFF, 4-1BBL, TNFSF8, CD40L G, CD70, CD95L / CD178, EDA-A1, TNFSF14, LTA / TNFB, LTB, TNFa, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, TRAIL, IP-10, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33 or fragments thereof. Other biomarkers include membrane microparticles, platelet count, mean platelet volume (MPV), sCD14-ST, prothrombinase, antithrombin and antithrombin activity, cationic protein 18 (CAP18), von Willebrand factor (vWF) lysin, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, undenatured fibrin D-dimer, platelet factor 4, histones, and PT assays.

[0400] sample:

[0401] As used herein, the term "sample" refers to a biological sample obtained or isolated from a patient or subject. As used herein, "sample" can mean, for example, a fluid or tissue sample obtained for analysis, diagnosis, prognosis, or evaluation of a subject of interest (e.g., a patient). Preferably, the sample is a fluid sample, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, cells, cell extracts, tissue samples, any tissue sample from the upper or lower respiratory tract, tissue biopsy, stool samples, etc. Specifically, the sample is blood, plasma, or serum.

[0402] In the context of this invention, "plasma" is a nearly cell-free supernatant obtained after centrifugation of blood containing an anticoagulant. Exemplary anticoagulants include calcium ion-binding compounds such as EDTA or citrate, and thrombin inhibitors such as heparin or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (e.g., citrate, EDTA-treated, or heparinized blood) at 2000g to 3000g for, for example, at least 15 minutes.

[0403] In the context of this invention, "serum" is the liquid portion collected from whole blood after it has been allowed to clot. A supernatant, composed of serum, can be obtained by centrifuging the coagulated blood (clotted blood).

[0404] Oxygen support:

[0405] "Oxygen support" or "supplementary oxygen" refers to administering additional oxygen to a patient, where "additional" means applying oxygen beyond the amount the patient is inhaling without any additional treatment. In an embodiment, a patient requires oxygen support when the arterial oxygen saturation in the blood is below 95%, preferably below 92%. Symptoms of arterial oxygen saturation below 95% include, but are not limited to, cyanosis of the nails or lips, cough, confusion, fatigue, shortness of breath, and wheezing. The term "oxygen saturation" (SaO2) refers to the fraction of oxygen-saturated hemoglobin in the blood relative to total hemoglobin. Oxygen saturation can be measured by arterial blood (arterial oxygen saturation, SaO2, e.g., arterial blood gas analysis), venous blood (venous blood saturation, SvO2), and peripheral blood (peripheral blood saturation, SpO2, e.g., by pulse oximetry). Other methods for measuring oxygen saturation are known to those skilled in the art. Various types of delivery systems can be used to administer "oxygen support" or "supplementary oxygen" at different concentrations. Two main types of delivery systems include low-flow systems (administered via nasal cannula, face mask, or non-rebreathing mask) and high-flow systems (administered via high-flow nasal cannula, Venturi mask, or continuous positive airway pressure system). In one embodiment, a low-flow system involves administering oxygen at a rate equal to or less than 6 L / min. In one embodiment, a high-flow system involves administering oxygen at a rate >6 to ≤10 L / min. Supplemental oxygen may also be administered during mechanical ventilation, for example, by bypassing the airway, as in ECMO therapy.

[0406] Infect:

[0407] As used herein, within the scope of this invention, "infection" means a pathological process caused by the invasion of normal sterile tissues or fluids by a pathogen or potential pathogen / pathogen, organism, and / or microorganism, and preferably involves infection by bacteria, viruses, fungi, and / or parasites. In the context of this invention, infection particularly relates to viral infection with the SARS virus. An infection can also be a combination of an infection comprising SARS virus infection (such as SARS-CoV2 infection) and one or more additional infections (such as concurrent or subsequent infections or infections already present at the time of SARS virus infection), wherein said additional infection can be a bacterial infection, a viral infection, and / or a fungal infection. Infection with more than one additional pathogen may also occur, including at least one SARS virus, and one or more other bacteria, viruses, or fungi. A triple infection of SARS virus, a bacterium, and a fungus is conceivable. An infection can be localized or systemic. For the purposes of this invention, viral infection may be considered a microbial infection.

[0408] As used herein, "infectious disease" includes viral infectious diseases or conditions associated with primary viral infections (specifically, SARS virus infection). In the context of this invention, in addition to SARS virus infection, infectious diseases may also include infections with one or more bacteria and / or viruses and / or fungi.

[0409] In addition, infection-related complications can be "hospital-acquired" infections. Hospital-acquired infections (HAIs), also known as hospital-acquired infections, are infections acquired in hospitals or other healthcare settings. To emphasize the hospital and non-hospital settings, they are sometimes referred to as healthcare-associated infections (HAIs or HCAIs). These infections can be acquired in hospitals, nursing homes, rehabilitation facilities, outpatient clinics, or other clinical settings. In clinical settings, hospital-acquired infections can spread to susceptible patients in various ways. Besides contaminated equipment, bed linens, or airborne droplets, healthcare workers can also transmit the infection. The infection may originate from the external environment, another infected patient, a potentially infected staff member, or, in some cases, the source of infection cannot be identified. In some cases, the microorganism originates from the patient's own skin microbiome and becomes an opportunistic pathogen after surgery or other procedures that damage the protective skin barrier. Although the patient may have contracted the infection from their own skin, it is still considered a hospital-acquired infection because it occurred in a healthcare setting.

[0410] Furthermore, subjects with an infection may be exposed to multiple sources of infection simultaneously. For example, a subject with an infection may have a bacterial and a viral infection; a viral and a fungal infection; a bacterial and a fungal infection; a bacterial, a fungal, and a viral infection; or a mixed infection, which includes one or more infections listed herein and may include duplicate infections, such as one or more bacterial infections in addition to one or more viral infections and / or one or more fungal infections.

[0411] Infection levels can be classified as follows:

[0412]

[0413] Regarding procalcitonin, elevated biomarker levels (especially ≥0.25 ng / ml or 0.5 ng / ml) are associated with grade 3 infection, which typically occurs outside of Covid-19 infection and requires antimicrobial treatment and hospitalization.

[0414] The COVID-19 ordinal scale is defined as follows:

[0415]

[0416] Measurement method:

[0417] According to the present invention, sFlt-1, CT-proET, proADM and PCT and / or other biomarkers or clinical scores are used as biomarkers for diagnosis, prognosis, prediction, risk assessment and / or risk stratification of patients who present with symptoms of infectious diseases and develop medical conditions requiring hospitalization.

[0418] Technicians are able to obtain or develop methods for identifying, measuring, determining and / or quantifying any of the above-described molecules or fragments or variants thereof, as well as other markers of the present invention, based on standard molecular biology practices.

[0419] The levels of one or more biomarkers of the present invention can be determined by any assay capable of reliably measuring the concentration of the biomarker. Specifically, mass spectrometry (MS) and / or immunoassays, as illustrated in the appended examples, can be used. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of macromolecules / peptides in solution using antibodies or antibody-binding fragments or immunoglobulins.

[0420] The methods for determining proADM or other biomarkers (such as sFlt-1, CT-proET-1, or PCT) used in the context of this invention are intended for use in this invention. For example, methods selected from the group consisting of: mass spectrometry (MS), luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent immunoassay and fluorescence immunoassay, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), luminescent microbead arrays, magnetic bead arrays, protein microarray assays, rapid detection methods (e.g., immunochromatographic strip assays), rare cavitation compound assays, and automated systems / analyzers can be used.

[0421] Identifying one or more biomarkers based on antibody recognition is a preferred embodiment of the present invention. As used herein, the term "antibody" refers to the immunoglobulin molecule and the immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen-binding site that specifically binds to (responds to) an antigen. According to the present invention, antibodies can be monoclonal antibodies and polyclonal antibodies.

[0422] An antibody is considered specific if its affinity for the molecule of interest is at least 50 times higher, preferably 100 times higher, and most preferably at least 1000 times higher than that of other molecules contained in a sample containing the molecule of interest. How to develop and select antibodies with given specificity is well known in the art. In the context of this invention, monoclonal antibodies are preferred. An antibody or antibody-binding fragment specifically binds to a biomarker or fragment thereof as defined herein. In particular, the antibody or antibody-binding fragment binds to a biomarker as defined herein.

[0423] Exemplary immunoassays may include luminescent immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent immunoassay and fluorescence immunoassay, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), luminescent microbead arrays, magnetic bead arrays, protein microarray assays, rapid test formats, and rare cavitation compound assays. Furthermore, methods suitable for point-of-care and rapid test formats, such as immunochromatographic strip assays, can be employed. Automated immunoassays, such as KRYPTOR assays, are also prepared.

[0424] Alternatively, in place of antibodies, other capture molecules or molecular scaffolds that specifically and / or selectively recognize one or more biomarkers of the present invention may be included within the scope of the invention. Hereinafter, the terms "capture molecule" or "molecular scaffold" include molecules that can be used to bind target molecules or molecules of interest, i.e., analytes from a sample. Therefore, the capture molecule must be sufficiently shaped spatially and in terms of surface characteristics (such as surface charge, hydrophobicity, hydrophilicity, the presence or absence of Lewis donors and / or acceptors) to specifically bind the target molecule or molecule of interest. Thus, binding can be mediated, for example, through ionic, van der Waals, π-π, σ-π, hydrophobic, or hydrogen bonding interactions, or a combination of two or more of the aforementioned interactions or covalent interactions between the capture molecule or molecular scaffold and the target molecule or molecule of interest. In the context of the present invention, the capture molecule or molecular scaffold may be selected, for example, from the group consisting of nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, peptides, and glycoproteins. Capture molecules or molecular scaffolds include, for example, nucleic acid aptamers, DARpins (designed ankylosing protein repeats), and Affimers, etc.

[0425] Preferably, one antibody can be labeled, while the other antibody can bind to the solid phase or selectively bind to the solid phase. In a particularly preferred aspect of the assay, one antibody is labeled, while the other antibody binds to the solid phase or selectively binds to the solid phase. The first and second antibodies can be dispersed in a liquid reaction mixture, wherein a first labeling component, as part of a fluorescence- or chemiluminescence-based extinction or amplification-based labeling system, binds to the first antibody, and a second labeling component of the labeling system binds to the second antibody, thereby generating a measurable signal after both antibodies have bound to the biomarker or fragment thereof to be detected, which allows the resulting sandwich complex to be detected in the measurement solution. The labeling system may comprise a combination of rare earth cavitation compounds or chelates with fluorescent or chemiluminescent dyes (especially anthocyanin-type).

[0426] In a preferred embodiment, the method is performed as a heterospatial immunoassay, wherein one antibody is immobilized on an arbitrary solid phase, such as the wall of a coated tube (e.g., a polystyrene tube; a coated tube; a CT) or a microtiter plate (e.g., composed of polystyrene), or immobilized on particles (e.g., magnetic particles), such that another antibody has a similar group to a detectable label or is capable of selectively attaching to a label, and is used to detect the formed sandwich structure. It is also possible to temporarily delay immobilization or perform subsequent immobilization by using a suitable solid phase.

[0427] The method according to the invention can also be embodied as a homogeneous method, wherein a sandwich complex formed by the antibody / multiple antibodies to be detected and a label or fragment thereof remains suspended in the liquid phase. In this case, it is preferable that, when using two antibodies, each part of the detection system is labeled with the two antibodies, which causes signal generation or signal triggering when the two antibodies integrate into a single sandwich structure. Such techniques will be specifically embodied as fluorescence enhancement or fluorescence quenching detection methods. A particularly preferred aspect involves the use of detection reagents used in pairs, such as those described in US4882733, EP0180492, or EP0539477 and the prior art referenced therein. In this way, it becomes possible to measure the reaction product containing only two labeled components in a single immune complex, directly in the reaction mixture. For example, such a technique is marketed under the brand name TRACE. ™ (Time Resolved Amplified Cryptate Emission) or KRYPTOR ™ The teachings of the above-described application are provided. Therefore, in a particularly preferred aspect, a diagnostic device is used to implement the methods provided herein. For example, the levels of SFLt-1 or fragments thereof and / or any other markers of the methods provided herein are determined. In a particularly preferred aspect, the diagnostic device is a B.R.A.H.M.S. KRYPTOR analyzer.

[0428] The levels of the biomarkers of the present invention (e.g., sFlt-1 or a fragment thereof, CT-proET1 or a fragment thereof, or other biomarkers) can also be determined by mass spectrometry (MS)-based methods. This method may include detecting the presence, amount, or concentration of one or more modified or unmodified fragment peptides of, for example, sFlt-1 or CT-proET1 in the biological sample or in protein digests (e.g., trypsin digests) from the sample, and optionally separating the sample by chromatography, and performing MS analysis on the prepared and optionally separated samples. For example, selected reaction monitoring (SRM), multiple reaction monitoring (MRM), or parallel reaction monitoring (PRM) mass spectrometry may be used in the MS analysis.

[0429] In this document, the term "mass spectrometry" or "MS" refers to an analytical technique used to identify compounds by their mass. To enhance the mass resolution and mass determination capabilities of mass spectrometry, samples can be pretreated prior to MS analysis. Therefore, this invention relates to MS detection methods that can be combined with immunoenrichment techniques, methods involving sample preparation, and / or chromatographic methods, preferably with liquid chromatography (LC), more preferably with high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC). Sample preparation methods include techniques for lysis, separation, digestion of the sample into peptides, removal, enrichment, dialysis, desalting, alkylation, and / or peptide reduction. However, these steps are optional. Analyte ions can be selectively detected using tandem mass spectrometry (MS / MS). Tandem mass spectrometry is characterized by a mass selection step (as used herein, the term "mass selection" refers to the separation of ions having a specific m / z or a narrow range of m / z), followed by fragmentation of the selected ions and mass analysis of the resulting product (fragment) ions.

[0430] Those skilled in the art know how to quantify the levels of markers in a sample using mass spectrometry. For example, as mentioned above, relative quantification (rSRM) or absolute quantification can be used.

[0431] In addition, levels (including reference levels) can be determined by mass spectrometry-based methods, such as methods for determining the relative quantification of the target protein or its fragments, or methods for determining the absolute quantification.

[0432] Reagent test kit:

[0433] The present invention also relates to reagent kits, uses of the reagent kits, and methods of using such reagent kits.

[0434] The kit may additionally include items for obtaining samples (such as blood samples), for example, the kit may include a container including means for attaching the container to a cannula or syringe, a syringe suitable for blood separation with an internal pressure less than atmospheric pressure, such as suitable for drawing a predetermined volume of sample into the container, and / or additionally include detergents, dissociative salts, ribonuclease inhibitors, chelating agents (such as guanidine isothiocyanate, guanidine hydrochloride, sodium lauryl sulfate, polyoxyethylene sorbitan monolaurate), RNase inhibitor proteins, and mixtures thereof, and / or a filtration system comprising nitrocellulose, a silica matrix, ferromagnetic spheres, a cup for collecting spills, trehalose, fructose, lactose, mannose, polyethylene glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, aniline, pyrrole, citrate, and mixtures thereof.

[0435] As used herein, "detection reagent" and the like are reagents suitable for determining the biomarkers described herein (e.g., sFlt-1, CT-proET1, and / or MR-proADM). Exemplary detection reagents of this kind are, for example, ligands, such as antibodies or fragments thereof, that specifically bind to peptides or epitopes of the biomarkers described herein. Such ligands can be used in the immunoassays described above. Other reagents used in immunoassays to determine biomarker levels may also be included in the kit and are considered detection reagents herein. Detection reagents may also relate to reagents for detecting biomarkers or fragments thereof by MS-based methods. Thus, such detection reagents can also be reagents for preparing samples for MS analysis, such as enzymes, chemicals, buffers, etc. Mass spectrometers can also be considered detection reagents. Detection reagents according to the invention can also be calibration solutions, for example, calibration solutions that can be used to determine and compare biomarker levels.

[0436] In embodiments, the method of the present invention can be implemented in part by a computer. For example, the step of comparing the level of a detected biomarker (e.g., sFlt-1 or a fragment thereof) with a reference level can be performed in a computer system. In the computer system, the measured level of the biomarker can be combined with the subject's other biomarker levels and / or clinical parameters to calculate a score indicating prognosis, risk assessment, and / or risk stratification. For example, the measured values ​​can be entered into the computer system (either manually by a healthcare professional or automatically from a device that has measured the corresponding biomarker level). The computer system can be located directly at the point of care (e.g., a primary care unit, hospital, or home environment) or at a remote location connected via a computer network (e.g., via the Internet or a specialized medical cloud system, optionally in combination with other IT systems or platforms such as a Hospital Information System (HIS)). Typically, computer systems store values ​​(e.g., biomarker levels or clinical parameters such as age, blood pressure, weight, BMI, sex, or scores (e.g., Covid-19 ordinal scales or infection grades) on computer-readable media and calculate scores based on predefined and / or pre-stored reference levels or values. The resulting scores are displayed and / or printed to users (typically healthcare professionals such as physicians) or patients. Alternatively or in addition, associated prognoses, assessments, treatment guidelines, patient management guidelines, or stratifications are displayed and / or printed to users (typically healthcare professionals such as physicians) or patients.

[0437] In one embodiment of the invention, a software system incorporating machine learning algorithms can be employed, preferably using data from electronic health records (EHRs) to identify patients at risk of PE. The machine learning method can be trained on a random forest classifier using EHR data from patients, such as laboratory tests, biomarker expression, vital signs, and demographic data. Machine learning is a form of artificial intelligence that enables computers to learn complex patterns in data without explicit programming, unlike simpler rule-based systems. Early studies have used EHR data to trigger alerts to detect overall clinical deterioration. In one embodiment of the invention, processing of sFlt-1 levels can be incorporated into appropriate software for comparison with existing datasets; for example, sFlt-1, CT-proET-1, proADM, or PCT levels can also be processed in machine learning software to help predict the risk of adverse outcomes.

[0438] Statistical measures:

[0439] Various statistical measures can be used to provide the statistical significance, probability, or level of certainty of any given correlation observed in data, such as demonstrating that the observation is not a random result but based on an underlying biological correlation. For example, positive and negative predictive values ​​(PPV and NPV, respectively) are the proportions of true positive and true negative results, respectively, in statistical and diagnostic tests. PPV and NPV describe the performance of diagnostic tests or other statistical measures. High results can be interpreted as indicating the accuracy of such statistics.

[0440] Positive predictive value: According to Emerg (Tehran). 2015 Summer; 3(3): 87–88, the positive predictive value (PPV) is the proportion of cases that are already patients who have given a positive test result. It is the ratio of patients who are actually diagnosed as positive to all patients with positive test results (including healthy subjects who were misdiagnosed as patients). This characteristic can predict how likely someone is to actually be a patient given a positive test result.

[0441] Positive predictive value = TP / TP + FP

[0442] Negative predictive value: According to Emerg (Tehran). 2015 Summer; 3(3): 87–88, the negative predictive value is the proportion of cases that are already healthy but have given a negative test result. It is the ratio of subjects who are actually diagnosed as negative to all subjects with negative test results (including those who were misdiagnosed as healthy). This characteristic can predict how likely someone is to be actually healthy given a negative test result.

[0443] Negative predictive value = TN / TN + FN

[0444] Therefore, 100% NPV indicates no false negative rate. A biomarker or biomarker value ratio of 100% NPV can correctly classify all patients without adverse outcomes into the low-risk group. A biomarker or biomarker value ratio of 100% NPV can correctly distinguish all patients who do not require oxygen support from those who do.

[0445] "Inclusion" typically identifies a minimum proportion of subjects with a high degree of certainty that they will develop the disease or condition, and ensures that this truly positive group has a sufficiently large proportion of subjects who test positive. The test must achieve minimum sensitivity and minimum positive predictive value (PPV).

[0446] "Exclusion" typically identifies the smallest proportion of subjects with a high degree of certainty that they will not develop the disease or condition, and ensures that a sufficiently small number of subjects who test negative will develop the disease (false negative). Therefore, such a test must achieve minimum specificity and minimum negative predictive value (NPV).

[0447] The sensitivity and specificity of diagnostic and / or prognostic tests depend not only on the "quality" of the test's analysis but also on the definition of abnormal outcomes. Sensitivity equals the true positive rate, and specificity equals the true negative rate. In practice, receiver operating characteristic (ROC) curves are typically calculated by plotting the values ​​of a variable against their relative frequency in "normal" individuals (i.e., individuals who appear healthy and do not experience adverse outcomes) and individuals who experience adverse outcomes (e.g., those who die within 28 days, experience thrombotic events, require supplemental oxygen, develop additional infections requiring antimicrobial therapy and hospitalization, or are hospitalized or stay in the ICU for more than 4 days).

[0448] For any given biomarker (such as sFlt-1, CT-proET-1, proADM, IL6, or PCT), the biomarker level distributions of subjects with and without the disease / condition are likely to overlap. In such cases, the test cannot absolutely distinguish between normal and disease with 100% accuracy, and the overlapping area may indicate a range where the test fails to differentiate between normal and disease. A threshold is chosen below which the test is considered abnormal, above which it is considered normal, or below or above which the test indicates a specific condition, such as infection. The area under the ROC curve is a measure of the probability that the obtained measurement correctly identifies the condition. ROC curves can be used even if the test results do not necessarily provide precise numbers. ROC curves can be created whenever the results can be ranked. For example, test results for “disease” samples can be ranked according to severity (e.g., 1=low, 2=normal, 3=high). This ranking can be correlated with results in the “normal” population, thus creating an ROC curve. These methods are well known in the art; see, for example, Hanley et al., 1982. Radiology 143: 29-36. Preferably, the threshold is selected to provide an ROC curve area greater than about 0.5 (<= 0.5 indicates a test with no discrimination), more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9 (up to 1.0 indicates a test with perfect discrimination). In this context, the term "about" means + / - 5% for a given measurement.

[0449] The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis represents sensitivity, which increases with the true positive rate. Therefore, for a specific cutoff value, the value of (1-specificity) can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is an indicator of the probability that the measured biomarker level can correctly identify a disease or condition. Therefore, the area under the ROC curve can be used to determine the validity of the test.

[0450] Receiver operating characteristic (ROC) curves and concordance (C) statistics (also known as the C-index) are commonly used to assess the ability of risk factors to predict outcomes. For example, biomarkers or risk factors are often incorporated into logistic regression models to predict the likelihood of patients developing a disease of interest. These predictive probabilities or risks can be examined to see their accuracy in identifying which patients will develop the disease or not. For instance, if the predicted probability for individuals with the disease is always higher than that for healthy individuals, then we say the model has perfect discriminant power. Discriminant power is typically measured using ROC curves. To construct an ROC curve, the predicted probability of the outcome of interest is repeatedly divided into above-critical and below-critical values. For each critical value, sensitivity (the probability of a predicted risk above the critical value among patients with the disease) and specificity (the probability of a predicted risk below the critical value among patients without the disease) can be estimated. The critical values ​​can be varied to show a range of sensitivities and specificities. If the model has full discriminative power, the ROC curve should be located in the upper left corner of the graph (100% sensitivity and 100% specificity) (Logan, Medical College of Wisconsin, Biostatistics).

[0451] The area under the ROC curve (AUC) is a useful metric for summarizing ROC curves. If the curve is close to the upper left corner (sensitivity = 100%, specificity = 100%), the AUC should be close to 1. The AUC is equivalent to another statistic commonly used to summarize model discriminative power: the C-statistic or consistency statistic. The C-statistic is interpreted as follows: randomly selected subjects who experienced the outcome have a higher probability of predicting the outcome than randomly selected subjects who did not experience it. Besides calculating the AUC, this probability can also be estimated by obtaining all observation pairs where one patient experienced the event and another did not, and calculating the proportion of pairs where the patient who experienced the event had a higher predictive risk. The C-statistic can also be interpreted as the rank correlation between the predicted probability of the outcome and the observed response (Logan, Medical College of Wisconsin, Biostatistics).

[0452] In other embodiments, ratio data, positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio are used as a measure of the ability to test the predictive risk or diagnose a disease.

[0453] Ratio data are quantitative data with the same properties as interval data, where each data point represents a definite and equal ratio. Negative ratios are impossible. A ratio of 1 indicates no change (100% equal). Ratios less than 1 are associated with a decrease and can be expressed as a percentage or total. For example, a proADM ratio of 0.9 is associated with a 10% decrease, which is typically seen in groups without adverse outcomes. Ratios greater than 1 are associated with an increase and can also be expressed as a percentage or total. For example, a ratio of 1.4 for the sFlt1 sample value from day 1 to day 2 is associated with a rapid slope of 40%.

[0454] In the case of "positive likelihood ratio", a value of 1 indicates that a positive result is equally likely to occur in subjects in the "disease" group and the "control" group; a value greater than 1 indicates that a positive result is more likely to occur in the disease group; and a value less than 1 indicates that a positive result is more likely to occur in the control group. In some preferred embodiments, the markers and / or the set of markers are preferably selected such that the positive or negative likelihood ratio is at least about 1.5 or higher or about 0.67 or lower, more preferably at least about 2 or higher or about 0.5 or lower, even more preferably at least about 5 or higher or about 0.2 or lower, even more preferably at least about 10 or higher or about 0.1 or lower, and most preferably at least about 20 or higher or about 0.05 or lower. In this context, the term "about" refers to + / - 5% of a given measurement.

[0455] In the case of "odds ratio," a value of 1 indicates that a positive result is equally likely in individuals in both the "disease" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the disease group; and a value less than 1 indicates that a positive result is more likely in the control group. In some preferred embodiments, the markers and / or the set of markers are preferably selected such that the odds ratio is at least about 2 or higher or about 0.5 or lower, more preferably at least about 3 or higher or about 0.33 or lower, even more preferably at least about 4 or higher or about 0.25 or lower, even more preferably at least about 5 or higher or about 0.2 or lower, and most preferably at least about 10 or higher or about 0.1 or lower. In this context, the term "about" means + / - 5% of a given measurement.

[0456] The odds ratio for each doubling of concentration is a specific way of expressing the association between a biomarker and an outcome. It quantifies the change in the outcome odds as the biomarker concentration is doubled. The odds ratio for each doubling of concentration provides a standardized measure that makes it easier to compare different biomarkers or studies. For example, if the odds ratio for each doubling of concentration is 1.5, it means that for each doubling of the biomarker concentration, the odds of the outcome occurring increase by 1.5 times. This indicates a positive correlation between the biomarker and the outcome. If the odds ratio for each doubling of concentration is less than 1, such as 0.8, it means that for each doubling of the biomarker concentration, the odds of the outcome occurring decrease by 0.8 times. This indicates a negative correlation between the biomarker and the outcome.

[0457] In the case of "hazard ratio," a value of 1 indicates that the relative risk of the endpoint (e.g., death) is the same in both the "disease" and "control" groups; a value greater than 1 indicates a greater risk in the disease group; and a value less than 1 indicates a greater risk in the control group. In some preferred embodiments, the markers and / or the set of markers are preferably selected such that the hazard ratio is at least about 1.1 or higher or about 0.91 or lower, more preferably at least about 1.25 or higher or about 0.8 or lower, more preferably at least about 1.5 or higher or about 0.67 or lower, even more preferably at least about 2 or higher or about 0.5 or lower, and most preferably at least about 2.5 or higher or about 0.4 or lower. In this case, the term "about" means + / -5% of the given measurement.

[0458] The 25th and 75th percentiles of the median concentration of a biomarker (also known as the lower quartile (Q1) and upper quartile (Q3)) are measures of dispersion that divide a dataset into four equal parts. These percentiles are commonly used to describe the diffusion or distribution of biomarker values. The median represents the middle value when the dataset is ordered in ascending or descending order. It divides the dataset into two equal parts. To determine the 25th and 75th percentiles relative to the median, the distribution of biomarker values ​​needs to be considered. Attached Figure Description

[0459] The invention is further described with reference to the following accompanying drawings. These drawings are not intended to limit the scope of the invention, but rather represent preferred embodiments of various aspects of the invention provided to better illustrate the invention described herein.

[0460] Brief description of the attached diagram:

[0461] Figure 1: ROC curves of biomarkers used to predict mechanical ventilation or death.

[0462] Figure 2: Comparison of the performance of biomarkers.

[0463] Figure 3: Box plot showing CT-proET1 and MR-proADM data.

[0464] Figure 4: Box plot showing CT-proET1 and MR-proADM data for other outcomes.

[0465] Figure 5: Box plot showing IL6, MR-proADM, PCT, and CT-proET1 data.

[0466] Figure 6: Visualization of the changes of MR-proADM, CT-proET1, and sFlt-1 biomarkers over time.

[0467] Detailed description of the attached diagram:

[0468] Figure 1: ROC curves of biomarkers used to predict mechanical ventilation or death.

[0469] Figure 2: Using a fixed patient set to fairly compare the performance of different biomarkers (performance differences should not be due to patient exclusion from certain analyses due to missing biomarker results). Ten patients in the ICU at enrollment were excluded from the analysis due to differences in disease severity compared to those not in the ICU. Day 1 (=baseline) population (169 patients): Non-ICU patients with available Day 1 results for MR-proADM, PCT, CT-proET1, sFlt-1, CRP, D-dimer, LDH, ferritin, WBC, lymphocytes, and IL-6. Day 4 population (126 patients): Patients in the Day 1 population with available Day 4 results for MR-proADM, PCT, CT-proET1, and sFlt-1.

[0470] Figure 3: Box plot showing (A) CT-proET1 and (B) MR-proADM outcome (endpoint) data: death within 28 days (death2), hospitalization in the ICU or death within 28 days (icu_or_death_bin), mechanical ventilation or death within 28 days (mechvent_death_event).

[0471] Figure 4: Box plot showing outcome (endpoint) data for CT-proET1 (left) and MR-proADM (right): (A) Discharge before or on day 4 (D4), (B) Composite severity endpoint (death_ICU:mechvent), and (C) Discontinuation of supplemental oxygen (off_supp_oxygen_bin) ("NA" group: no supplemental oxygen at randomization).

[0472] Figure 5: Box plots showing outcome (endpoint) data for IL6, MR-proADM, PCT, and CT-proET1 (from left to right): (A) Infection within 28 days (>= grade 3) (ae_infection) and (B) for CT-proET1, Covid scale = 4 versus < 4 for outcome (endpoint) on day 4 (Ord.D4_bin4).

[0473] Figure 6: Visualization of the changes in (A) MR-proADM (nmol / L), (B) CT-proET1 (pmol / L), and (C) sFlt-1 (pg / mL) biomarkers for outcome (endpoint) death within 28 days over time. The mean sFlt-1 concentration in patients who died before day 28 increased from day 1 to day 4, see the gray circle in (B). The mean concentrations and 95% CI are shown for all available patients at time points 1, 4, 7, 14, 21, and 28. Dark gray: died before day 28; light gray: survived on day 28.

[0474] Example

[0475] The invention is further described by way of the following examples. These examples are not intended to limit the scope of the invention, but rather represent preferred examples of various aspects of the invention provided to better illustrate the invention described herein.

[0476] Each of the following tables, each row of the following tables, and / or any combination of the following tables and / or the following rows represents an embodiment of the invention.

[0477] Example 1 – Summary of research characteristics and data related to sFlt-1 and CT-proET-1

[0478] background

[0479] Several novel biomarkers were investigated to predict clinical outcomes in COVID-19 patients. SARS-CoV-2 infection is known to affect endothelial cells. Two biomarkers of interest were endothelin-1 (ET-1) and soluble fms-like tyrosine kinase-1 (sFlt-1).

[0480] Our study aims to investigate C-terminal pro-endothelin-1 (CT-proET-1) and soluble fms-like tyrosine kinase-1 (sFlt-1) as predictors of clinical outcomes in a cohort of COVID-19 patients from a multicenter US trial.

[0481] Assessing the host immune system’s direct response to various pathogens, including infections, using genetically signature proteins is an evolving area of ​​interest with the potential to predict and guide clinical decisions.

[0482] Research objective:

[0483] Primary outcome: In this exploratory analysis, using data from a recently completed multicenter trial, the utility of CT-proET-1 and sFlt-1 in early prediction of COVID-19 disease progression and outcome was evaluated.

[0484] This invention suggests that CT-proET-1 and sFlt-1 have valuable prognostic capabilities for risk stratification of individuals with COVID-19.

[0485] method

[0486] This study used data from a multicenter trial. Patients who underwent biomarker testing and were not admitted to the intensive care unit (ICU) at admission were included. The median concentrations of CT-proET-1 (83.6 pmol / L) and sFlt-1 (81.3 pg / mL) were used as cutoff values ​​for predicting clinical outcomes.

[0487] Hybrid approach design:

[0488] This study was approved by the Institutional Review Board (IRB) of Mass General Brigham. Specimen collection and testing: EDTA plasma samples were collected, separated, and aliquoted into cryovials within 2–12 hours after venipuncture. The cryovials were stored at -80°C until thawed, after which CT-proET-1 and sFlt-1 concentrations were determined on a B.R.A.H.M.S. Kryptonometer. Statistical analysis: Chi-square tests or Fisher's exact tests were used for categorical variables, and Mann-Whitney U tests were used for continuous numerical variables to analyze differences between groups. Prognostic performance measures included sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic (ROC) curve. Estimates and 95% confidence intervals are reported. CIs for sensitivity, specificity, PPV, and NPV were calculated using the Clopper and Pearson methods. Kaplan-Meier curves were plotted and log-rank tests were performed. Patients were stratified into CT-proET-1 (≤83.6 pmol / L vs. >83.6 pmol / L) and sFlt-1 (≤83.9 pg / mL vs. >83.9 pg / mL). Multiple multivariate logistic regression analysis was conducted.

[0489] As shown in more detail below, for CT-proET-1, 83.6 pmol / l is the median concentration; 87.4 pmol / l indicates the endpoint of death within 28 days; and 91.8 pmol / l indicates the endpoint of mechanical ventilation within 28 days; and 102.7 pmol / l indicates the endpoint of Covid scale >=4 versus <4 on day 4.

[0490] As shown in more detail below, for sFlt-1, 81.3 pg / ml is the median concentration; while 83.9 pg / ml represents the endpoints “improvement on the COVID scale (D4 vs. D1)” and “discharge on D4”; and 88.4 pg / ml represents death, ICU admission, and mechanical ventilation within 28 days.

[0491] Research participants:

[0492] This study is a prospective observational cohort study from a multicenter COVID-19 trial. Inclusion criteria were as follows: hospitalized adult patients >18 years of age, diagnosed with SARS-CoV-2 infection via nasopharyngeal swab polymerase chain reaction or serum IgM antibody testing, and exhibiting at least two of the following symptoms: fever >38°C, requiring supplemental oxygen, or pulmonary infiltration. Patients who were not yet admitted to the ICU at enrollment and had complete data for all study biomarkers were included.

[0493] result

[0494] Of the 169 patients, 10.7% received mechanical ventilation or died within 28 days of admission. Among patients who received mechanical ventilation or died within 28 days, CT-proET1 concentrations were significantly higher than those who did not (median 119.6 vs. 79.1 pmol / L, p = 0.001), and even higher in patients admitted to the ICU during hospitalization (median 114.5 vs. 80.6 pmol / L, p = 0.005). Significantly higher sFlt-1 concentrations were observed in ICU patients compared to those who did not (median 96.0 vs. 80.7 pg / mL, p = 0.041).

[0495] Using the Yoden criteria, we derived a cutoff value of 83.6 pmol / L for CT-proET-1 to predict prolonged hospital stays exceeding 4 days. For sFlt-1, we derived a cutoff value of 83.9 pg / mL as the optimal value for predicting prolonged hospital stays exceeding 4 days.

[0496] Using 83.6 pmol / L as the cutoff value, CT-proET-1 showed the following sensitivity, specificity, PPV, and NPV for predicting mechanical ventilation or death: 83%, 54%, 18%, and 96%, respectively, with an AUC of 0.75.

[0497] The critical value for sFlt-1 in predicting mechanical ventilation or death was 81.3 pg / mL, with sensitivity, specificity, PPV, and NPV of 61%, 54%, 14%, and 92%, respectively, and an AUC of 0.59. sFlt-1 showed high statistical value in predicting death and cessation of oxygen supply within 28 days.

[0498] in conclusion

[0499] CT-proET-1 and sFlt-1 are valuable novel biomarkers for predicting clinical outcomes in COVID-19, with lower levels leading to better predictive outcomes.

[0500] Example 2 – Bivariate Results

[0501] patient

[0502] This study used data from a multicenter trial. The inclusion criteria for this trial were patients aged 19 to 85 years who were diagnosed with SARS-CoV-2 infection via nasopharyngeal swab polymerase chain reaction or serum IgM antibody testing and exhibited at least two of the following symptoms: fever >38°C, requiring supplemental oxygen, or pulmonary infiltration (Stone et al., 2020). For this study, patients who were not yet in the ICU at enrollment and had complete data for all study biomarkers were included.

[0503] ending

[0504] The primary outcome of this study was a composite endpoint of mechanical ventilation or death within 28 days of admission, as some patients died without mechanical ventilation. Secondary outcomes within 28 days of admission included: admission to the intensive care unit (ICU), clinical deterioration on the COVID-19 ordinal scale, death, a composite severity endpoint (at least one of the following: death, ICU admission, mechanical ventilation), a COVID-19 ordinal scale score ≥ 4 on day 4, length of hospital stay beyond day 4, a COVID-19 ordinal scale score equal to or worse than day 1 on day 4, mechanical ventilation, deep vein thrombosis (DVT), pulmonary embolism (PE), and stroke. Additional outcomes were also assessed. The “any thrombotic event” outcome was defined as any of the following outcomes occurring within 28 days of admission: DVT, PE, or stroke. The COVID-19 ordinal scale represents the severity of the disease and is based on ICU admission, supplemental oxygen, mechanical ventilation, death, or whether the patient is ready to be discharged home. In the COVID-19 Ordinal Scale, deterioration is defined as an increase of 1 point or more in the score of patients receiving supplemental oxygen, or an increase of 2 points or more in the score of patients not receiving supplemental oxygen (Stone et al., 2020).

[0505] plasma samples

[0506] EDTA plasma samples were collected, separated, and aliquoted into cryovials within 2–12 hours after intravenous puncture. The cryovials were stored at -80°C until thawed for determination of MR-proADM concentration on a B·R·A·H·M·S MR-proADM KRYPTOR (Thermo Fisher Scientific B·R·A·H·M·S MR-proADM).

[0507] The B·R·A·H·M·S KRYPTOR biomarkers analyzed in this study were CT-proET-1, sFlt-1, MR-proADM, and PCT. Additionally, C-reactive protein (CRP), D-dimer, ferritin, interleukin-6 (IL-6), lactate dehydrogenase (LDH), and lymphocytes were also measured.

[0508] Using the Yoden criteria, we derived a cutoff value of 83.6 pmol / L for CT-proET-1 as the optimal value for predicting prolonged hospital stays exceeding 4 days. For sFlt-1, a cutoff value of 83.9 pg / mL was derived as the optimal value for predicting hospital stays exceeding 4 days and for the COVID-19 ordinal scale on day 4 being equal to or worse than that on day 1.

[0509] Biomarker Measurement Schedule

[0510] Day 1 (baseline), days 4, 7, 14, 21, and 28 (days 4 and 7 + / - 1 or 2 days, days 14, 21, and 28 + / - 3 days; 24-hour window for day 1). SFlt-1, CT-proET1, MR-proADM, and PCT biomarker results are available on days 1, 4, 7, 14, 21, and 28.

[0511] Additionally, samples were taken upon discharge. If discharged on days 4, 7, 14, 21, or 28, we used the average of all biomarker measurements for that specific patient on that day for analysis.

[0512] Statistical analysis

[0513] Descriptive statistics were used to summarize patient characteristics. When applicable to categorical variables, chi-square or Fisher's exact test was used to analyze differences between patient groups, and the Mann-Whitney U test was used to analyze continuous numerical variables.

[0514] Prognostic performance was measured by sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristic (AUC). Estimates and 95% confidence intervals (CIs) are reported. CIs for sensitivity, specificity, PPV, and NPV were calculated using the Clopper and Pearson methods.

[0515] Plot Kaplan-Meier curves and perform log-rank tests, stratifying by CT-proET-1 (≤83.6 pmol / L vs. >83.6 pmol / L) and sFlt-1 (≤83.9 pg / mL vs. >83.9 pg / mL).

[0516] Multiple multivariate logistic regression analysis was performed to assess whether CT-proET-1 and sFlt-1 were independent predictors of clinical outcomes. Adjusted variables included age, sex, body mass index, diabetes mellitus, hypertension, heart failure, history of MI, COPD, chronic kidney disease, and the number of days from symptom onset to randomization and CT-proET-1 and sFlt-1 measurements. Odds ratios were reported with 95% confidence intervals and p-values. All statistical tests were two-sided; p-values ​​< 0.05 were considered statistically significant. P-values ​​for multiple tests were not adjusted. R software version 3.5.1 and R package pROC version 1.15.3 were used for statistical analysis (Robin et al., 2011; R Core Team 2018). Kaplan-Meier plots and ROC curves, log-rank tests, and multivariate logistic regression analysis were generated using STATA version 14.1 (StataCorp 2015).

[0517] result:

[0518] Table 1: Outcomes within 28 days stratified by (A) binary CT-proET-1 (critical value 83.6 pmol / L) and (B) binary sFlt-1 (critical value 83.9 pg / mL).

[0519] (A) Binary CT-proET-1 (critical value 83.6 pmol / L)

[0520]

[0521] (B) Binary sFlt-1 (critical value 83.9 pg / mL)

[0522]

[0523] Values ​​expressed as percentages (%) represent the proportion of the total population or the corresponding CT-proET-1 and sFlt-1 strata. Results are expressed as median and interquartile range (first quartile (Q1) - third quartile (Q3)) as appropriate. For categorical factors, statistical significance between CT-proET-1 and sFlt-1 strata was determined by chi-square test or Fisher's exact test, where applicable; for numerical factors, statistical significance was determined by Mann-Whitney U test. No p-value correction was applied for multiple tests.

[0524] One patient was excluded from the statistical test due to missing data regarding a history of diabetes, heart failure, myocardial infarction, chronic obstructive pulmonary disease, asthma, chronic kidney disease, and cancer.

[0525] Two patients were excluded from the statistical test due to missing data on their smoking status.

[0526] Results: Baseline Features

[0527] Median age 60 years, 58% male

[0528] 80% are COVID-19 Level 4 (ICU or non-ICU hospital wards, requiring non-invasive ventilation or high-flow oxygen).

[0529] The median time to symptom onset was 9 days.

[0530] The median baseline PCT was 0.2 ng / mL, and the median baseline d-dimer was 884 ng / mL.

[0531] Results: Clinical endpoint

[0532] Typically, the number of events is small, around 5-20, for example, a 5% mortality rate (day 1 analysis).

[0533]

[0534] Result: Time structure of events

[0535] By day 4, 90% of ICU admissions (incident percentage in the day 1 population) had been completed, 93% of mechanical ventilation had been initiated, and 49% of supplemental oxygen had been discontinued.

[0536] The mortality rate was most significant in week 1 (38%) and week 2 (38%), and the distribution was relatively even until week 4.

[0537] In the second week, 50% of mechanical ventilation was discontinued.

[0538] Table 2: Outcomes within 28 days stratified by (A) binary CT-proET-1 (critical value 83.6 pmol / L) and (B) binary sFlt-1 (critical value 83.9 pg / mL).

[0539] (A) Binary CT-proET-1 (critical value 83.6 pmol / L)

[0540]

[0541] (B) Binary sFlt-1 (critical value 83.9 pg / mL)

[0542] Values ​​expressed as percentages (%) represent the proportion of the total population or the corresponding CT-proET-1 and sFlt-1 strata. Statistical significance between CT-proET-1 and sFlt-1 strata was determined by chi-square test or Fisher's exact test (if applicable). P-values ​​for multiple tests were not corrected.

[0543] Any thrombotic event is defined as a patient who experiences any of the following outcomes: deep vein thrombosis (DVT), pulmonary embolism (PE), or stroke.

[0544] Table 3 shows the distribution of CT-proET1 and sFlt-1 levels stratified by clinical outcome.

[0545]

[0546] Results are expressed as median and interquartile range (interquartile range of the first quartile (Q1) - interquartile range of the third quartile (Q3)) as appropriate. Statistical significance between outcome strata was determined by the Mann-Whitney U test. P-values ​​for multiple tests were not adjusted.

[0547] Table 4: Area under the receiver operating characteristic (AUC) and its 95% CI of biomarkers for predicting clinical outcomes

[0548] Assuming that lower lymphocyte levels are associated with increased risk

[0549]

[0550] Example 3 – Additional results related to Day 1 biomarker levels, the ratio of Day 4 to Day 1 levels (Day 4 / Day 1 ratio), and additional measurements of the second biomarker.

[0551] Results related to sFlt1 (measured on day 1)

[0552] Table 5a: Biomarker Concentrations and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as NPV or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0553]

[0554] Table 5b: Diagnostic Performance – AUC, NPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as NPV or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0555]

[0556] Table 5c: Diagnostic Performance – AUC, PPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as NPV or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0557]

[0558] Results related to sFlt1 (ratio of day 4 to day 1)

[0559] Table 6a: Biomarker Ratios and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as NPV or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0560]

[0561] Table 6b: Diagnostic Performance - AUC, NPV, Optimization Criterion

[0562]

[0563] Results related to sFlt1 (Day 1 cutoff (81.3 pg / L) (Day 1 BM level) + Day 4 to Day 1 ratio (BM2))

[0564] Table 7: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0565]

[0566] Results related to sFlt1 (Day 1 (cutoff 81.3 pg / L) (BM1) + second biomarker (BM2))

[0567] Table 8: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0568]

[0569] Results related to CT-proET1 (Day 1)

[0570] Table 9a: Biomarker Concentrations and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0571]

[0572]

[0573]

[0574] Table 9b: Diagnostic Performance – AUC, NPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0575]

[0576]

[0577] Table 9c: Diagnostic Performance – AUC, PPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0578]

[0579] Results related to CT-proET1 (Day 1 (cutoff value 86.3 pmol / L) (BM1) + Day 4 to Day 1 ratio (BM2))

[0580] Table 10: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0581]

[0582] Results related to CT-proET1 (Day 1 (cutoff value 86.3 pmol / L) (BM1) + second biomarker (BM2))

[0583] Table 11: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0584]

[0585] Results related to MR-proADM (Day 1)

[0586] Table 12a: Biomarker Concentrations and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0587]

[0588]

[0589] Table 12b: Diagnostic Performance – AUC, NPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0590]

[0591] Table 12c: Diagnostic Performance – AUC, PPV, Optimization Criterion

[0592]

[0593] Results related to MR-proADM (day 4 vs. day 1 ratio)

[0594] Table 13a: Biomarker Ratios and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0595]

[0596] Table 13b: Diagnostic Performance – AUC, NPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0597]

[0598] Results related to MR-proADM (Day 1 (critical value 0.87 nmol / L) (BM1) + Day 4 to Day 1 ratio (BM2))

[0599] Table 14: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0600]

[0601] Results associated with MR-proADM (Day 1 (cutoff value 0.87 nmol / L) (BM1) + second biomarker (BM2))

[0602] Table 15: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0603]

[0604] Results related to PCT (Day 1):

[0605] Table 16a: Biomarker Concentrations and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0606]

[0607] Table 16b: Diagnostic Performance – AUC, NPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0608]

[0609] Table 16c: Diagnostic Performance – AUC, PPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0610]

[0611] Results related to PCT (day 4 vs. day 1 ratio)

[0612] Table 17a: Biomarker Ratios and Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0613]

[0614] Table 17b: Diagnostic Performance – AUC, NPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0615]

[0616] Table 18c: Diagnostic Performance – AUC, PPV, Optimization Criteria. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0617]

[0618] Results related to PCT (Day 1 (cutoff value 0.25 ng / mL) (BM1) + Day 4 compared to Day 1) Rate (BM2)

[0619] Table 18: Diagnostic Performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0620]

[0621] Outcomes associated with a specific outcome (endpoint) (Day 1)

[0622] Table 19a: Admission to ICU during hospitalization (Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; sFlt-1 pg / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0623]

[0624] Table 19b: Admission to ICU during hospitalization (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET1 pmol / L; sFlt1 pg / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention with respect to prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0625]

[0626] Table 20a: Composite severity endpoint (at least one of the following: death within 28 days, admission to ICU, mechanical ventilation) (Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; sFlt1 pg / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0627]

[0628] Table 20b: Composite severity endpoint (at least one of the following: death within 28 days, admission to ICU, mechanical ventilation) (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET1 pmol / L; sFlt1 pg / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention with respect to prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0629]

[0630] Table 21a: Day 4 Covid Scale = 4 vs. < 4(Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0631]

[0632] Table 21b: Day 4 Covid Scale = 4 vs. < 4 (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to prognostic outcome, hazard ratio, statistical measures (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0633]

[0634] Table 22a: Died within 28 days (Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0635]

[0636] Table 22b: Died within 28 days(Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET 1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to prognostic outcome, hazard ratio, statistical measures (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0637]

[0638] Table 23a: Discharged before or on the 4th day (yes or no) (Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; sFlt-1 pg / L; MR-proADM nmol / L; PCT ng / mL). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0639]

[0640] Table 23b: Discharged before or on the 4th day (yes or no) (Outcome) – Diagnostic Performance – AUC, PPV, Optimized Criteria (Units: CT-proET1 pmol / L; SFlt-1 pg / L; MR-proADM nmol / L; PCT ng / mL). Each endpoint shown represents one embodiment of the invention with respect to prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0641]

[0642] Table 24a: Stop supplementing oxygen(Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0643]

[0644] Table 24b: Stop supplementing oxygen (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET 1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to prognostic outcome, hazard ratio, statistical measures (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0645]

[0646] Table 25a: Improvement on the COVID scale (day 4 better than day 1 vs. day 4 equal to or worse than day 1) (Outcome) - Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; SFlt-1 pg / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0647]

[0648] Table 25b: Improvement on the COVID scale (day 4 better than day 1 vs. day 4 equal to or worse than day 1)(Outcome) – Diagnostic Performance – AUC, PPV, Optimized Criteria (units: CT-proET1 pmol / L; sFlt1 pg / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention with respect to prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0649]

[0650] Table 26a Infection within 28 days (>= Level 3) (Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0651]

[0652] Table 26b: Infection within 28 days (>= Level 3) (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to prognostic outcome, hazard ratio, statistical measures (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0653]

[0654] Table 27a: Mechanical ventilation within 28 days(Outcome) – Biomarker concentrations and cutoff values ​​(units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0655]

[0656] Table 27b: Mechanical ventilation within 28 days (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criteria (units: CT-proET1 pmol / L; MR-proADM nmol / L). Each endpoint shown represents one embodiment of the invention, relating to prognostic outcome, hazard ratio, statistical measures (such as C, NPV, or PPV), and / or critical values ​​(one or more). In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0657]

[0658] Outcomes associated with a specific outcome (endpoint) (day 4 vs. day 1 ratio)

[0659] Table 28a: Admission to ICU during hospitalization (Outcome) – Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0660]

[0661] Table 28b: Admission to ICU during hospitalization (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0662]

[0663] Table 29a: Composite severity endpoint (at least one of the following: death within 28 days, admission to ICU, mechanical ventilation) (Outcome) – Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0664]

[0665] Table 29b: Composite severity endpoint (at least one of the following: death within 28 days, admission to ICU, mechanical ventilation) (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0666]

[0667] Table 30a: Day 4 Covid Scale = 4 vs. < 4 (Outcome) - Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0668]

[0669] Table 30b: Day 4 Covid Scale = 4 vs. < 4 (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0670]

[0671] Table 31a: Died within 28 days (Outcome) - Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0672]

[0673] Table 31b: Died within 28 days (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0674]

[0675] Table 32a: Stop supplementing oxygen (Outcome) - Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0676]

[0677] Table 32b: Stop supplementing oxygen (Outcome) - Diagnostic Performance - AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0678]

[0679] Table 33a: Improvement on the COVID scale (day 4 better than day 1 vs. day 4 equal to or worse than day 1)(Outcome) - Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0680]

[0681] Table 33b: Improvement on the COVID scale (day 4 better than day 1 vs. day 4 equal to or worse than day 1) (Outcome) – Diagnostic Performance – AUC, PPV, Optimization Criterion

[0682]

[0683] Table 34a: Infection within 28 days (>= Level 3) (Outcome) - Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0684]

[0685] Table 34b: Infection within 28 days (>= Level 3) (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0686]

[0687] Table 35a: Mechanical ventilation within 28 days (Outcome) - Biomarker ratios and cutoff values. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or cutoff value. In embodiments, the combination of endpoint (as prognostic outcome) and cutoff value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0688]

[0689] Table 35b: Mechanical ventilation within 28 days (Outcome) – Diagnostic Performance – AUC, NPV, Optimized Criterion. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0690]

[0691] With specific The End (The Finale) Related results (Day 1 (BM1) + Day 4 to Day 1 ratio (BM2))

[0692] Table 36: Admission to ICU during hospitalization (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0693]

[0694] Table 37: Composite severity endpoint (at least one of the following: death within 28 days, admission to ICU, mechanical ventilation) (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0695]

[0696] Table 38: Died within 28 days (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0697]

[0698] Table 39: Stop supplementing oxygen (Outcome) – Diagnostic performance – c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as c, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0699]

[0700] Table 40: Infection within 28 days (>= Level 3) (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0701]

[0702] Table 41: Mechanical ventilation within 28 days (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0703]

[0704] With specific The End (The Finale) Related results (Day 1 (BM1) + Biomarker 2 (BM2))

[0705] Table 42: Admission to ICU during hospitalization(Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0706]

[0707] Table 43: Composite severity endpoint (At least one of the following: death within 28 days, admission to ICU, mechanical ventilation) (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of prognostic outcomes, hazard ratios, statistical measures (such as C, NPV, or PPV), and / or cutoff values. In embodiments, the combination of endpoints (as prognostic outcomes) and cutoff values ​​represents one embodiment of the invention. In embodiments, the combination of endpoints (as prognostic outcomes) and hazard ratios represents one embodiment of the invention.

[0708]

[0709] Table 44: Day 4 Covid Scale = 4 vs. < 4 (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0710]

[0711] Table 45: Discharged on or before the 4th day. (True or False) (Outcome) - Diagnostic Performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0712]

[0713] Table 46: Improvement on the COVID scale (day 4 better than day 1 vs. day 4 equal to or worse than day 1)(Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0714]

[0715] Table 47: Mechanical ventilation within 28 days (Outcome) - Diagnostic performance - c-index. Each endpoint shown represents one embodiment of the invention, relating to one or more of the following: prognostic outcome, hazard ratio, statistical measure (such as C, NPV, or PPV), and / or critical value. In embodiments, the combination of endpoint (as prognostic outcome) and critical value represents one embodiment of the invention. In embodiments, the combination of endpoint (as prognostic outcome) and hazard ratio represents one embodiment of the invention.

[0716]

[0717] Summary of the exemplary results shown above:

[0718] In predicting clinical outcomes such as mortality, ICU admission, mechanical ventilation, composite severity endpoint, day 4 discharge, discontinuation of supplemental oxygen, and infection, CT-proET1 and MR-proADM showed similarly good performance.

[0719] CT-proET1 exhibits similar good performance to MR-proADM, for example...

[0720] The AUCs for death were 0.79 and 0.86 (p = 0.006 and 0.001).

[0721] The AUCs for mechanical ventilation or death were 0.75 and 0.76 (p = 0.001 and < 0.001, respectively).

[0722] The AUC for ICU admission or death was 0.72 and 0.72 (p=0.001 and <0.001, respectively).

[0723] The AUCs for patients discharged on day 4 were 0.72 and 0.69 (p<0.001 and <0.001, respectively).

[0724] The composite severity endpoint AUC was 0.71 and 0.71 (p = 0.001 and 0.001).

[0725] Additional values ​​of CT-proET1 and MR-proADM relative to the conventional biomarker IL6 were observed for discharge on day 4, improvement on the Covid scale from day 1 to day 4 (CT-proET1 only), mechanical ventilation or death (CT-proET1 only), ICU admission or death, composite severity endpoint, infection or death, and the day 4 ordinal scale (CT-proET1 only).

[0726] For clinical outcomes including death, ICU admission (or death), mechanical ventilation (CT-proET1 only), composite severity endpoints (CT-proET1 and sFlt1 only), discontinuation of supplemental oxygen (CT-proET1 and MR-proADM only), and infection (PCT and sFlt1 only), additional values ​​were observed for changes in MR-proADM, CT-proET1, sFlt1, and PCT from day 1 to day 4 relative to their corresponding day 1 biomarkers.

[0727] Other potential endpoints include

[0728] Stop supplementing oxygen:

[0729] The AUCs of MR-proADM and CT-proET1 were 0.77 and 0.75 (p = 0.001 and 0.006, respectively).

[0730] Infection (≥ Grade 3):

[0731] IL6, MR-proADM, PCT, CT-proET1 AUC 0.76, 0.75, 0.70, 0.68 (p≤0.012).

[0732] Day 4 Covid Scale

[0733] CT-proET1 AUC 0.71 (p=0.004).

[0734] Deep vein thrombosis or death (possibly driven by factors leading to death).

[0735] The AUCs of MR-proADM and CT-proET1 were 0.79 and 0.69, respectively (p = 0.001 and 0.034).

[0736] Exemplary critical values ​​(derived from A) median concentration and B) maximum concentration for CT-proET1 and sFLT-1 Youden's index (i.e., the maximum sum of sensitivity and specificity):

[0737] CT-proET1:

[0738] Median concentration: 83.6 pmol / L

[0739] 87.4 pmol / L for the endpoint of death within 28 days

[0740] 91.8 pmol / L for the endpoint of mechanical ventilation within 28 days

[0741] 102.7 pmol / L Endpoint for Covid scale ≥ 4 on day 4, compared to < 4.

[0742] sFlt1:

[0743] Median concentration: 81.3 pg / mL

[0744] 83.9 pg / mL Target for improvement endpoint on COVID scale (Day 4 vs. Day 1)

[0745] 83.9 pg / mL was the endpoint for discharge on day 4.

[0746] 88.4 pg / mL for a composite severity endpoint (at least one of the following: death within 28 days, admission to ICU, mechanical ventilation).

[0747] Exemplary endpoints and biomarkers with additional values ​​for IL-6:

[0748] CT-proET1 (C-index bivariate comparison with single biomarker IL6)

[0749] The number of patients discharged before or on day 4 was 0.76 compared to 0.67 (p<0.001).

[0750] The improvement in the ordinal scale was 0.71 compared to 0.65 (p=0.003).

[0751] Mechanical ventilation or death: 0.79 vs. 0.71 (p=0.008).

[0752] The ICU admission or death rate was 0.80 compared to 0.73 (p=0.010).

[0753] The composite severity endpoint was 0.78, compared to 0.72 (p=0.015).

[0754] Infection (≥ grade 3) or death: 0.80 vs. 0.76 (p=0.027).

[0755] Day 4 COVID-19 Ordinal Scale score: 0.77 vs. 0.72 (p=0.047).

[0756] MR-proADM

[0757] The discharge rate on day 4 was 0.73, compared to 0.67 (p<0.001).

[0758] Infection (≥ grade 3) or death: 0.82 vs. 0.76 (p=0.004).

[0759] The ICU admission or death rate was 0.78 compared to 0.73 (p=0.016).

[0760] The composite severity endpoint was 0.77, compared to 0.72 (p=0.029).

[0761] PCT

[0762] Infection (≥ grade 3) 0.79 vs. 0.77 (p = 0.012)

[0763] Exemplary endpoints and biomarkers with added value for D-dimer

[0764] MR-proADM (C-index bivariate comparison with single biomarker D-dimer)

[0765] Thrombotic events (MGH definition) or death: 0.77 vs. 0.66 (p=0.024).

[0766] Exemplary endpoints and biomarkers that provide additional value to other biomarkers:

[0767] MR-proADM and / or CT-proET1 combined CRP (ICU admission or death, Day 4 ordinal scale, infection), ferritin or LDH (see table above).

[0768] sFlt1 combined with lymphocytes (see table above).

[0769] MR-proADM combined with CT-proET1 or PCT (infection or death).

[0770] Exemplary endpoints and biomarkers with additional values ​​of biomarker changes from day 1 to day 4.

[0771] Mortality rate (C-index bivariate comparison of day 1 biomarkers)

[0772] MR-proADM 0.95 vs 0.83 (p<0.001).

[0773] sFlt1 0.91 vs 0.70 (p<0.001).

[0774] PCT 0.86 vs. 0.60 (p=0.001).

[0775] CT-proET1 0.83 vs 0.75 (p=0.039).

[0776] Admission to the ICU or death (diagnostic significance for many patients on day 4)

[0777] MR-proADM 0.84 vs. 0.71 (p<0.001).

[0778] sFlt1 0.75 vs 0.63 (p=0.003).

[0779] CT-proET1 0.76 vs 0.72 (p=0.014).

[0780] Admission to the ICU (diagnostic significance for many patients on day 4)

[0781] PCT 0.75 vs. 0.60 (p<0.001).

[0782] Mechanical ventilation or death (most patients are diagnosed on day 4)

[0783] CT-proET1 0.81 vs 0.75 (p=0.004).

[0784] Composite severity endpoint (diagnostic significance for many patients on day 4)

[0785] sFlt1 0.78 vs 0.65 (p=0.001)

[0786] CT-proET1 0.75 vs 0.71 (p=0.013).

[0787] Discontinue supplemental oxygen (this is diagnostically significant for many patients on day 4).

[0788] MR-proADM 0.89 vs 0.76 (p<0.001).

[0789] CT-proET1 0.81 vs 0.76 (p=0.018).

[0790] Infection (≥ Grade 3)

[0791] PCT 0.81 vs. 0.68 (p=0.001).

[0792] sFlt1 0.73 vs 0.61 (p=0.003).

[0793] Any infection (as reported in the trial, plus infections recorded in the FU table) or thrombotic events (6 types)

[0794] PCT 0.77 vs. 0.63 (p<0.001).

[0795] MR-proADM 0.76 vs. 0.74 (p=0.015).

[0796] The combined manifestation of thrombosis (imbalance) endpoint and death was strongly driven by mortality (e.g., deep vein thrombosis or death: 7 out of 8 events were due to death).

[0797] Other results related to sFlt-1 and MR-proADM:

[0798] Table 48a: Results associated with a single biomarker (day 4 vs. day 1 MR-proADM or SFlt-1 level), including biomarker concentration and cutoff values. Each event shown represents one embodiment of the invention, relating to one or more of treatment management, prognostic outcomes, and statistical measures such as NPV and / or cutoff values. In embodiments, a combination of events (such as death or mechanical ventilation within 28 days) with a cutoff value represents one embodiment of the invention.

[0799]

[0800] Table 48b: Diagnostic performance of single biomarkers (day 4 vs. day 1 MR-proADM or SFlt-1 levels) – AUC, NPV, optimized cutoff values.

[0801]

[0802] Table 49: Results associated with the combination of biomarkers (Biomarker 1 (BM1) and Biomarker 2 (BM2)), including biomarker concentrations and cutoff values. Each event shown represents one embodiment of the invention, relating to one or more of treatment management, prognostic outcomes, and statistical measures such as NPV and / or cutoff values. In the embodiments, a combination of events (such as death or mechanical ventilation within 28 days) with a cutoff value represents one embodiment of the invention.

[0803]

[0804] MR-proADM and sFlt-1 (MR-proADM levels on day 4 compared to day 1) performed well in guiding patients on their need for oxygen support (such as supplemental oxygen) and in predicting their risk of death within 28 days (see Tables 3a and 3b). Patients with lower or equal MR-proADM and / or sFlt-1 levels in the second sample effectively excluded the need for supplemental oxygen and the risk of death within 28 days, as indicated by the strong NPV of these events.

[0805] Furthermore, MR-proADM (day 4 vs. day 1 MR-proADM level) performed well in guiding whether a patient required oxygen support (such as supplemental oxygen and mechanical ventilation) or whether the patient needed intensive care unit (ICU) admission. Patients with lower or equal MR-proADM levels in the second sample effectively ruled out the need for oxygen support (such as supplemental oxygen), mechanical ventilation, and ICU admission, as indicated by the strong NPV of these events. Furthermore, sFlt-1 (day 4 vs. day 1 sFlt-1 level) performed well in guiding whether a patient required oxygen support (such as supplemental oxygen) and whether the patient had a risk of additional infection within at least 28 days. Patients with lower or equal sFlt-1 levels in the second sample effectively ruled out the need for oxygen support (such as supplemental oxygen) and the risk of additional infection within 28 days.

[0806] The combination of two biomarkers (BM1: MR-proADM or SFlt-1 levels on day 4 compared to day 1; BM2: MR-proADM levels above the cutoff of 0.87 nmol / L on day 1 and / or sFlt1 levels above the cutoff of 81.3 pg / L on day 1) further improved performance in guiding patients on whether they require oxygen support (such as supplemental oxygen) and other treatments (such as mechanical ventilation or ICU admission) and the risk of death within 28 days.

[0807] References

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Claims

1. A method for managing the treatment of patients with respiratory viral infections and / or predicting disease progression, severity, and / or outcome, the method comprising the following steps: a. Provide a first sample isolated from the patient. b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient. c. The first and second samples were separated within 96 hours (4 days) after consultation with medical personnel. d. Determine the levels of one or more endothelial biomarkers in the first and second samples, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and / or pro-adrenergic medullaris (proADM) or a fragment thereof, and e. Wherein, a lower or equal level of one or more endothelial biomarkers in the second sample, compared to the first sample, indicates a risk of the patient having no adverse clinical outcome for at least 28 days.

2. The method of claim 1, wherein a lower or equal level of the one or more endothelial biomarkers in the second sample, compared to the first sample, indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

3. The method according to the preceding claim, wherein a lower or equal level of the at least one additional biomarker and / or one or more endothelial biomarkers in the second sample, compared to the first sample, indicates that the patient does not require supplemental oxygen for at least 28 days.

4. The method according to any one of the preceding claims, wherein the first sample and the second sample are obtained by the first group of medical personnel who treated the patient after the onset of symptoms of the respiratory virus infection.

5. The method according to any one of the preceding claims, wherein the patient is hospitalized.

6. The method according to any one of the preceding claims, wherein the patient is admitted to and / or not admitted to the intensive care unit (ICU).

7. The method according to any one of the preceding claims, wherein the patient is admitted to the intensive care unit (ICU), preferably after the initial contact with medical staff.

8. The method according to any one of the preceding claims, wherein a. The first sample was obtained within 24 hours (1 day) of admission and / or after consultation with the said medical staff, and / or b. The second sample was obtained within 72 to 96 hours (day 4) after admission and / or after consulting the said medical staff.

9. The method according to any one of the preceding claims, wherein the patient is infected with a coronavirus, preferably SARS coronavirus, more preferably SARS-CoV2 coronavirus.

10. The method according to any one of the preceding claims, wherein the adverse event is death within at least 28 days after obtaining the first sample.

11. The method according to any one of the preceding claims, wherein the adverse event is an adverse respiratory and / or infectious clinical outcome requiring oxygen support and intensive treatment for at least 28 days.

12. The method according to the preceding claims, wherein the adverse respiratory and / or infectious clinical outcome is intensive care unit (ICU) treatment, mechanical ventilation, and / or other infections.

13. The method according to any one of the preceding claims, wherein the patient is infected with coronavirus, and the adverse event is a deterioration in the COVID Scale score (e.g., a deterioration in the COVID Scale score to >4) and / or continued hospitalization on or after day 4 of admission.

14. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and second samples comprise sFlt-1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of sFlt-1 or its fragments, and - A level of sFlt1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient is at risk of no adverse clinical outcome for at least 28 days.

15. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and second samples comprise sFlt-1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of sFlt1 or its fragments, and - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

16. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and second samples comprise sFlt1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of sFlt1 or its fragments, and - A level of sFlt1 or a fragment thereof measured in the first sample of ≤ 81.3 pg / l ± 20% indicates that the patient is not at risk of death for at least 28 days (and preferably does not require oxygen support).

17. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and second samples comprise sFlt-1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of sFlt-1 or its fragments, and - A level of sFlt-1 or a fragment thereof measured in the first sample ≤ 81.3 pg / l ± 20% indicates that the patient does not require oxygen support and is not at risk of death or additional infection for at least 28 days after consultation with the healthcare professional.

18. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and - Wherein, compared to the first sample, the second sample has lower or equal levels of CT-proET-1 or its fragments, and - A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates that the patient is at risk of no adverse clinical outcome for at least 28 days.

19. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise CT-proET-1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has lower or equal levels of CT-proET-1 or its fragments, and - A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

20. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise CT-proET-1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has lower or equal levels of CT-proET-1 or its fragments, and - A level of CT-proET-1 or a fragment thereof measured in the first sample of ≤ 87.4 pmol / L ± 20% indicates that the patient is not at risk of death for at least 28 days (and preferably does not require oxygen support).

21. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise CT-proET-1 or a fragment thereof, and - Wherein, compared to the first sample, the second sample has lower or equal levels of CT-proET-1 or its fragments, and - A level of CT-proET-1 or a fragment thereof measured in the first sample ≤ 87.4 pmol / L ± 20% indicates that the patient does not require mechanical ventilation and is not at risk of death for at least 28 days after consultation with the medical personnel.

22. The method according to any one of the preceding claims, wherein determining the level of proADM or a fragment thereof comprises determining the level of adrenal medullary precursor peptide (MR-proADM) in the sample.

23. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of proADM or its fragments, and - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient is at risk of no adverse clinical outcome for at least 28 days.

24. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of proADM or its fragments, and - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, as measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

25. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of proADM or its fragments, and - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient has no risk of death for at least 28 days (and preferably does not require oxygen support).

26. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers measured in the first and second samples comprise proADM or a fragment thereof, and - Wherein, compared to the first sample, the second sample has a lower or equal level of proADM or its fragments, and - A level of proADM mid-peptide (MR-proADM) or a fragment thereof, measured in the first and / or second sample, ≤ 0.87 nmol / L ± 20% indicates that the patient is not at risk of oxygen support, death, ICU admission, or mechanical ventilation for at least 28 days.

27. The method according to any one of the preceding claims, comprising the following steps: a. Provide a first sample isolated from the patient, wherein the patient is infected with coronavirus. b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient. c. Wherein the first sample was obtained within 1 day after consultation with the medical personnel, and the second sample was obtained within 72 to 96 hours (day 4) after consultation with the medical personnel. d. Determine the level of soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in the sample, and e. Wherein, a lower or equal level of sFlt-1 or a fragment thereof in the second sample, compared to the first sample, indicates that the patient does not require oxygen support for at least 28 days and is not at risk of death or additional infection.

28. The method according to any one of the preceding claims, comprising the following steps: a. Provide a first sample isolated from the patient, wherein the patient is infected with coronavirus. b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient. c. Wherein the first sample was obtained within 1 day after consultation with the medical personnel, and the second sample was obtained within 72 to 96 hours (day 4) after consultation with the medical personnel. d. Determine the level of C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof in the sample, and e. Wherein, a lower or equal level of CT-proET-1 or a fragment thereof in the second sample, compared to the first sample, indicates that the patient does not require oxygen support for at least 28 days and is not at risk of death or additional infection.

29. The method according to any one of the preceding claims, comprising the following steps: a. Provide a first sample isolated from the patient, wherein the patient is infected with coronavirus. b. Provide a second sample separated from the patient at a time point after the first sample has been separated from the patient. c. Wherein the first sample was obtained within 24 hours (1 day) after consulting with the medical personnel, and the second sample was obtained within 72 to 96 hours (4th day) after consulting with the medical personnel. d. Determine the level of pro-adrenomedullin (proADM) or a fragment thereof in the sample, and e. Wherein, a lower or equal level of proADM or a fragment thereof in the second sample compared to the first sample indicates that the patient does not require oxygen support for at least 28 days and is not at risk of death, ICU admission, or mechanical ventilation.

30. The method according to any one of the preceding claims, further comprising: a. Determine the level of at least one additional biomarker in the first and / or second sample, wherein the at least one additional biomarker is procalcitonin (PCT) or a fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, and / or C-reactive protein (CRP), and b. Wherein, compared with the first sample, the lower or equal level of the at least one additional biomarker and / or the one or more endothelial biomarkers in the second sample indicates the risk that the patient has no adverse clinical events for at least 28 days.

31. The method according to the preceding claim, wherein a lower or equal level of the at least one additional biomarker and / or the one or more endothelial biomarkers in the second sample, compared to the first sample, indicates that the patient is not at risk of adverse clinical outcomes related to the need for supplemental oxygen for at least 28 days.

32. The method according to the preceding claim, wherein a lower or equal level of the at least one additional biomarker and / or one or more endothelial biomarkers in the second sample, compared to the first sample, indicates that the patient does not require supplemental oxygen for at least 28 days.

33. A kit for carrying out the method according to any one of the preceding claims, the kit comprising: A detection reagent for determining the levels of one or more endothelial biomarkers in samples from patients, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof, C-terminal pro-endothelin-1 (CT-proET-1) or a fragment thereof, and / or pro-adrenergic medullary kinase (proADM) or a fragment thereof (preferably MR-proADM), and Reference data on the risk of adverse clinical outcomes in patients within 28 days, particularly reference data on risk thresholds, critical values, or hazard ratios, wherein said reference data is preferably stored on a computer-readable medium and / or in the form of computer-executable code configured to compare the measured levels of one or more endothelial biomarkers in a sample from the patient with said risk thresholds, critical values, or hazard ratios. Optional diagnostic reagents for determining the presence of respiratory viral infection, preferably for coronavirus infection, and preferably for the presence of SARS-CoV2 virus infection, and Optionally, a assay reagent is used to determine the levels of at least one additional biomarker or fragment thereof in a sample from a patient, namely procalcitonin (PCT) or fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, and C-reactive protein (CRP), and reference data, such as reference levels of said at least one additional biomarker, preferably pro-endothelin-1 (CT-proET-1) or fragment thereof, procalcitonin (PCT) or fragment thereof, IL6, D-dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, and C-reactive protein (CRP), particularly risk thresholds, cutoff values, or hazard ratios, wherein said reference data is preferably stored on a computer-readable medium and / or in the form of computer-executable code configured to compare the measured levels of said at least one additional biomarker or fragment thereof with said thresholds, cutoff values, or hazard ratios.

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