EP4792141A1

EP4792141A1 - Biomarkers for therapy management and/or prognosing disease progression, severity and/or outcome for respiratory virus infections

Info

Publication number: EP4792141A1
Application number: EP24783760.2A
Authority: EP
Inventors: Andrej SCHWABE; Sascha JOHANNES; Jan Wiemer; Michael K. Mansour
Original assignee: BRAHMS GmbH
Current assignee: BRAHMS GmbH
Priority date: 2023-10-09
Filing date: 2024-10-09
Publication date: 2026-08-19
Also published as: AU2024360424A1; CN122003606A; WO2025078420A1

Abstract

The invention relates to a method for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection. The method comprises the steps of providing a first sample isolated from said patient and a second sample isolated from said patient at a time point after isolating the first sample from said patient, wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and/or pro-adrenomedullin (proADM) or fragment(s) 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 is not at risk of an adverse clinical outcome within at least 28 days. The invention further relates to a kit for carrying out the methods of the invention.

Description

BIOMARKERS FOR THERAPY MANAGEMENT AND/OR PROGNOSING DISEASE PROGRESSION, SEVERITY AND/OR OUTCOME FOR RESPIRATORY VIRUS INFECTIONS

DESCRIPTION

The invention relates to the field of therapy guidance, medical risk assessment, clinical diagnostics and prognostics, and corresponding methods and products.

The invention relates to a method for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, comprising the steps of: (a.) providing a first sample isolated from said patient, (b.) providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, (c.) wherein the first and the second sample were isolated within 96 h (4 days) after admission and/or after consultation with medical personnel, (d.) determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and/or pro-adrenomedullin (proADM) or fragment(s) 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 is not at risk of an adverse clinical outcome within at least 28 days.

The invention further relates to a method for therapy management of a patient having a respiratory virus infection, comprising assessing whether said patient is in need of oxygen support, comprising the steps of (a.) providing a first sample isolated from said patient, (b.) providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, (c.) wherein the first and the second sample were isolated within 96 h (4 days) after admission and/or after consultation with medical personnel, (d.) determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C- terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and/or pro-adrenomedullin (proADM) or fragment(s) 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 is not in need of oxygen support for at least 28 days.

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

The invention further relates to a method for prognosing disease progression, severity and/or outcome in a patient having a coronavirus infection, wherein a low-risk level of the one or more biomarkers or fragment(s) thereof in said sample at day 1 indicates that the patient is not at risk of an adverse clinical outcome within 28 days, and/or wherein a low-risk ratio of the levels of one or more biomarkers or fragment(s) thereof in said sample determined on day 4 and day 1 (ratio day 4/day 1) indicates that the patient is not at risk of an adverse clinical outcome within 28 days.

The invention further relates to a kit for carrying out the method for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, comprising detection reagents for determining the level of the one or more biomarkers in a sample from said patient, reference data for the risk of a patient of an adverse clinical outcome within 28 days, optionally detection reagents for determining the presence of a viral infection of the respiratory tract, and optionally, detection reagents for determining the level of at least one additional biomarker or fragment(s) thereof.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome (SARS) is a virus mediated respiratory disease which was first observed in 2002, typically caused by a coronavirus (CoV) infection. Based on scientific reports it is assumed that all human CoVs may be of zoonotic origin. Once a human becomes infected, the virus can quickly spread via droplet transmission and close contact between humans, eventually leading to epidemic scenarios or even to a pandemic (Vijaykrishna et al., 2007; Yuefei et al., 2020). Typical symptoms can be fever, chills, dry cough, dyspnea and diarrhea. The appearance and severity of symptoms can be different among infected patients. Some patients also appear to be asymptomatic (Al-Tawfiq et al., 2020). Hence, the clinical course can vary from harmless, self-healing scenarios with no presentation to a medical site, to severe cases where patients do not survive at Intensive Care Units (ICU).

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has placed significant strain on healthcare systems globally and resulted in substantial economic repercussions. The clinical manifestations of SARS-CoV-2 encompass a broad spectrum of symptoms ranging from mild flu-like symptoms to severe complications such as pneumonia, respiratory failure, and, ultimately, death (Ochani et al., 2021 ; Parasher et al., 2021).

Other Coronavirus infections are known from the past or may occur as developments or mutations of SARS-CoV-2, for which similar symptoms or manifestations may occur. SARS as such is also known in combination with various Coronavirus infections and is, as such, not limited to SARS-CoV-2. In 2002/2003, a previously unknown coronavirus was prevalent, and similar to COVID-19, many affected people developed severe acute respiratory syndrome (SARS). At that time, the pathogen known as "SARS-CoV-1" infected more than 8,000 people in 26 countries around the world and led to severe pneumonia in many of those infected; around 800 people died worldwide. Both SARS-CoV-1 and -2 have a significant similarity in genetic basis and variants and both, and additionally future SARS-CoV-variants, are of concern.

From a pathophysiological point of view, SARS is a complex medical condition, where the virus starts replicating in the upper respiratory tract and can further spread to the lower respiratory tract or target more non-respiratory organs and cells. Clinical investigations show that also liver, the endothelium, kidney, heart, intestine, brain and lymphocytes can be affected. It is proposed that the virus can directly promote cell damage, whereby a systemic inflammatory response followed by multiple organ injury is triggered. CoVs also cause a dysfunctional renin-angiotensin system, which increases the pulmonary vascular permeability being a key factor for the development pulmonary edema. Both, systemic inflammatory responses and a dysfunctional renin-angiotensin system contribute to a so-called cytokine storm that triggers the acute respiratory distress syndrome (ARDS). Multi-organ failure and/or the onset of ARDS represent a severe state of SARS patients with a high mortality risk within few weeks or days (Yuefei et al., 2020; Gu et al, 2005).

Infection caused by SARS-CoV-2 infection is known to impact endothelial cells. The virus attaches to the angiotensin-converting enzyme 2 (ACE-2) receptor found on various cell types, including type II alveolar cells, bronchial epithelial cells, and endothelial cells. Accordingly, a cytokine storm is often triggered, which is characterized by the release of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), 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 in the endothelium. Therefore, the utility of gene signature proteins to assess the direct response of the host immune system to various etiologies, including infections, is an evolving field of interest that holds promise for prognostication and guidance of clinical decision-making.

The following risk factors are considered to foster a severe course of the syndrome: age, cardiovascular diseases, cancer, diabetes, immunosuppression, smoking and obesity (Fei et al., 2020).

During epidemic scenarios, there is a clear need to maintain a functional healthcare system to ensure appropriate care for low to high-risk patients. Emergency Departments or Intensive Care Units can quickly be overcrowded with patients showing severe symptoms. For critically ill patients, a biomarker-based assessment can help to identify patients who may require an escalation of therapeutic interventions or who may not require an escalation of therapies and who may be eligible for an early hospital or ICU discharge. Patient outcome prediction is urgently needed to avoid subjecting patients with good prognosis or low risk to unnecessary treatments, e.g., in a hospital setting, and to reserve capacity for patients at higher risk. Patients at low risk for needing oxygen administration, or a low risk of death or a necessary ICU stay, require effective management and fitting treatment options. In view of the increasing shortage of medical structures, cost and personnel, it is important to make the right early decisions for patients who present to the physician, without unnecessary medical treatment for patients at low risk.

Different biomarkers show superior prognostic performance compared to the standard methods, like the experience of the medical personal, that can vary and is not a reliable feature or clinical parameter or scores that show worse prognostic performance as sole feature to make an accurate decision. Biomarkers of interest are Soluble fms-like tyrosine kinase-1 (sFlt-1), Endothelin-1 (ET-1 ), pro-adrenomedullin (proADM), Procalcitonin (PCT) and IL6.

SFlt-1 , is a naturally occurring soluble form of the Vascular Endothelial Growth Factor (VEGF) receptor 1 , and its concentration is increased in the bloodstream when endothelial cells are activated, such as in cases of hypoxia and inflammation. When sFLT-1 is produced and released, it binds to VEGF and the Placenta Growth Factor (PIGF), preventing them from binding to their receptors on endothelial cells. Accordingly, sFlt-1 serves an antiangiogenic role in conditions such as preeclampsia and kidney disease (Maynard et al., 2003; Palmer et al., 2017; Wewers et al., 2021 ; Lecarpentier et al., 2016). The marker sFlt-1 is known as a standard biomarker for prenatal diagnostics over many years but is not established as an infection marker or risk marker in the ICU or hospital setting when dealin with patients presenting with early symptoms of SARS or who may be at risk of a coronavirus infection or SARS. More recently, previous studies demonstrated that SFlt-1 levels were elevated in COVID-19 infection (Mohebbi et al., 2023; Dupont et al., 2021). Several small-scale studies focusing on pregnant patients indicate a significance of sFlt-1 in predicting clinical outcomes among 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).

ET-1 is a potent vasoconstrictor secreted by the endothelium in conditions of inflammation and decreased tissue blood perfusion. C-terminal pro-endothelin-1 (CT-proET-1) is the stable precursor protein of ET-1 which circulates 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 evaluating the association between acute COVID-19 infection and CT- proET-1 demonstrated increased levels of this biomarker (Gregoriano et al., 2021 ; Abraham et al., 2022). Nevertheless, none of the previous studies showed a prognostic effect within 28 days, under determination of the marker in a short period of 1 to 4 days after initial examination. In the prior art, patient "diagnosis" is emphasized and determined at a certain stage. However, it is not possible to derive a prognostic ability from a diagnostic effect.

Another biomarker enabling risk assessment of SARS patients is pro-adrenomedullin (proADM). ProADM is a prohormone generated by multiple tissues in order to stabilize the microcirculation and to protect against endothelial permeability and consequent 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). The biomarker has shown considerable promise, for example in the fields 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). The endothelium and microcirculation is widely acknowledged to play a significant role in the pathophysiological host response to sepsis, with the regulation and distribution of blood flow within each organ of major importance (Tyagi et al., 2009; Hernandez et al., 2013). Measures of the endothelium and microcirculation, such as via proADM, may therefore provide an alternative indication as to the severity of the general host response, compared to scores of individual organ dysfunction.

The use of MR-proADM for risk stratification in Coronavirus patients is also disclosed in W02021/204770 and US2023/160893A1 . Further, Atallah et al ("Mid-regional Proadrenomedullin Biomarker Predicts Coronavirus Disease 2019 Clinical Outcomes: A US-Based Cohort Study", Open Forum Infectious Diseases, 2022) discloses the prediction of the need of mechanical ventilation based on single MR-proADM values. 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) discloses the prediction of 28-day mortality in COVID patients based on measurements of MR-proADM within 48h and on day 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) discloses the repeated measurement of MR- proADM (day 0-3, 7, 14) and the prediction of mortality in COVID patients.

However, considering the potentially fast rate of infection of Corona- or SARS-viruses in the population at large, and the potential for large-scale hospital admissions in epidemic or pandemic scenarios, novel means are needed to assess the risk of disease progression in patients that have or are at risk of developing severe outcomes associated with a coronavirus infection, for example after infection with SARS-CoV-viruses. Further, novel means are required for therapy management of these patients having a respiratory virus infection. Until now, clinicians have insufficiently reliable means to assess whether patients require intensified treatment and/or disease monitoring or can be safely released from clinical care.

SUMMARY OF THE INVENTION

In light of the difficulties in the prior art, one technical problem underlying the present invention is the provision of means for therapy management and predicting disease progression, severity and/or outcome in a patient having a respiratory virus infection.

Another problem of the present invention is the provision of means for a rule-out of the risk of an adverse clinical outcome within at least 28 days in a patient having a respiratory virus infection.

Another problem of the present invention is the provision of means with a high negative predictive value in assessing whether a patient having a respiratory virus infection is at risk of an adverse clinical outcome within at least 28 days.

Another problem underlying the present invention is the provision of means for the management of hospitalization and the need of ICU treatment in a patient having a respiratory virus infection.

Another problem underlying the present invention is the provision of means for assessing whether a patient can be discharged from the hospital or the ICU.

Another problem of the present invention is the provision of means with a high positive predictive value in assessing whether a patient having a respiratory virus infection whether a patient can be discharged from the hospital or the ICU.

In light of the difficulties in the prior art, another problem underlying the present invention is the provision of means for assessing whether a patient having a respiratory virus infection is in need of oxygen support. Another problem underlying the present invention is the provision of means for therapy management in patients with a respiratory virus infection, in particular management of oxygen support. Another objective of the invention is the provision of means with a high negative predictive value in assessing whether a patient having a respiratory virus infection is in need of oxygen support.

Another problem of the present invention is the provision of means for predicting disease progression and/or outcome, in particular adverse outcomes within 28 days in a patient that has a coronavirus infection. Another objective of the invention is the provision of means with a high negative predictive value in determining a low risk of adverse events or death or other adverse disease progression and/or outcome, in particular adverse outcomes within 28 days in a patient that has a coronavirus infection.

These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

In one aspect, the invention relates to a method for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, comprising the steps of: a. providing a first sample isolated from said patient, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, d. determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and/or pro-adrenomedullin (proADM) or fragment(s) 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 is not at risk of an adverse clinical outcome within at least 28 days.

In embodiments, the invention demonstrates surprisingly high negative predictive values (for rule- out of the risk of an adverse clinical outcome), such that medical staff can quickly (within the first 4 days after presentation of the patient) and effectively determine the need for intensified treatment in such patients having a respiratory virus infection.

As outlined above, patient therapy management and risk assessment are urgently needed to avoid subjecting patients with good prognosis or low risk to unnecessary treatments, e.g., in a hospital setting, and to reserve capacity for patients at higher risk and higher need for intensified treatment. Patients at low risk for an adverse clinical outcome and intensified treatment require effective management and suitable treatment options. In view of the increasing shortage of medical structures and personnel, it is important to make the right decisions early on for patients who present to the physician, without unnecessary medical treatment for patients at low risk. The invention enables straightforward, rapid biomarker testing, rather than waiting for the results of complicated score determination, thus introducing objective assessment independent of the level of experience of medical staff.

Previous studies have failed to show a clear effect on risk assessment and therapy management within 28 days, when the level of a biomarker is determined within a short period of time after initial presentation to medical personnel, e.g., determining the biomarker on 1 to 4 days after initial presentation. In the prior art, patient "diagnosis" is emphasized and determined at a certain stage. However, it is typically not possible to derive a prognostic ability from a diagnostic effect and the determination of biomarker levels with a high negative predictive value with a very low false negative rate, as shown in the invention, cannot be assumed from studies indicating the diagnostic potential of biomarkers. The present invention enables primarily effective measurements for negative predictive value, in essence ruling out the risk of clinical adverse events and need for intensified treatment in certain patient populations. In other words, a clinician cannot rule-out an adverse event if the biomarker value is above the threshold value, and cannot automatically expect that the patient will develop an adverse event, if the positive predictive value (PPV) of the biomarker for the threshold level is low, especially if the PPV is below 50%.

Thereby, in particular, determining the level of an endothelial biomarker in two samples obtained within 4 days, preferably on day 1 and day 4, and comparing the level of the biomarker in said samples (ratio of the second to the first sample) provides a surprisingly improved and high negative predictive value compared to single measurements of the biomarker, e.g., on day 1 or day 4. This ratio allows to effectively and safely rule out patients as not being at risk of an adverse clinical outcome and to adapt the treatment of said patients accordingly. To the best knowledge of the inventors there is no disclosure or suggestion in the prior art of such ratio of two samples obtained within 4 days improving the negative predictive value and thus being particularly useful and reliable in the rule out of patients with respiratory virus infections with regards to an adverse event.

In some embodiments, a positive statement is possible, although the primary effect demonstrated by the present invention is an effective rule-out of the risk of an adverse clinical outcome and need of intensified treatment within 28 days for any given patient based on biomarker measurement. In some embodiments a high positive predictive value and thus rule-in is provided with regards to the discharge of the patient from the ICU or hospital. This corresponds to the high negative predictive value and rule-out of the risk of an adverse clinical event and need for intensified treatment, as a discharge from ICU or hospital means that the patient in not or no longer in need of an intensified treatment as provided in the ICU or hospital.

Endothelial biomarkers, such as sFlt-1 , ET-1 , or proADM, are associated with endothelial function and structure and in particular hemostasis of angiogenesis. Adrenomedullin (ADM) is a vasodilator peptide having a variety of physiological functions, including the maintenance of vascular tone and endothelial barrier function. Further, ADM has been shown to promote cardiac angiogenesis after myocardial infarction, indicating its role in tissue repair and regeneration and pro-angiogenic effect by supporting the formation of new blood and lymphatic vessels in hypoxic conditions. ADM may be determined directly or by determining the precursor molecule proADM or other fragments thereof such as the more stable fragment MR-proADM, which is split from proADM in an equimolar ratio to ADM. sFlt-1 is a non-membrane-associated splice variant of the VEGF receptor 1 (Flt-1) and binds to angiogenic factors such as VEGF (vascular endothelial growth factor) and PIGF (placental growth factor). By binding these factors, sFlt-1 reduces the concentration of free VEGF and PIGF, thereby inhibiting the growth of blood vessels and showing an antiangiogenetic effect.

ET-1 is also an endothelial modulator and biomarker, and acts as a vasoconstrictor secreted by endothelial cells and is involved in arteriosclerosis and vascular remodeling. Preproendothelin is precursor of the peptide ET-1. Endothelial cells convert preproendothelin to proendothelin and subsequently to mature endothelin, which the cells release. ET-1 also has an angiogenic effect on endothelial and perivascular cells, and has been shown to have an indirect action through the increased release of the potent pro-angiogenic substance vascular endothelial growth factor (VEGF), via hypoxia inducible factor-1. sFlt-1 , proADM and ET-1 all play a key role in maintaining hemostasis of angiogenesis and thereby the function of the vascular system within a subject. The clinical adverse outcomes in a patient having a respiratory virus infection according to the present invention are usually associated with hypoxia (lack of oxygen) in injured, inflamed and diseased tissues, thereby resulting in the activation of angiogenesis and thus the formation new blood vessels to a certain extent to improve oxygen supply to said tissues. Within angiogenesis, multiple biomarkers with either angiogenic or anti-angiogenic effects are both activated to maintain homeostasis. Thus, such biomarkers involved in angiogenesis, in particular SFlt-1 , ET-1 and proADM or fragments thereof (proportionally representing the levels and activity of ADM) are associated with the adverse clinical outcomes according to the present invention and their determination advantageously allows to assess the risk of a patient having a respiratory virus infection of developing such adverse clinical outcome. Despite covering different endothelial biomarkers, these various embodiments of the invention are thus preferably unified by their role in angiogenesis (endothelial biomarkers) and the ability to effectively and accurately assess the risk of an adverse clinical outcome in a patient having a respiratory virus infection based on these endothelial biomarkers.

In embodiments, a higher level of the one or more endothelial biomarkers in the second sample compared to the first sample is not a determination that the patient is at risk of an adverse clinical outcome. In embodiments, the patients with a higher level in the second sample are those for whom a risk of an adverse clinical outcome cannot be “ruled out” using the biomarker measurement, without necessarily indicating a “rule-in”. In preferred embodiments, the patients with a lower or equal level in the second sample are effectively ruled-out from said risk of an adverse clinical outcome indicated by a strong negative predictive value (NPV), as described in more detail below.

In one embodiment 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 an adverse clinical outcome requiring intensified treatment and/or monitoring within at least 28 days.

In one embodiment 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 an adverse clinical outcome requiring a de-escalation of treatment and/or reduce intense medical monitoring and/or discharge the patient from the ICU or the hospital and/or when a patient will exhibit an improvement in the Covid-19 ordinal scale.

In one embodiment, 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 an adverse clinical outcome associated with the need for supplemental oxygen for at least 28 days. In one embodiment, 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 in need of supplemental oxygen for at least 28 days.

In one embodiment, the first sample and the second sample are obtained by the first medical personnel to consult with the patient after occurrence of symptoms of the respiratory virus infection. This may be a primary care, a general practitioner, at a hospital ward, or in the emergency department or ICU. In embodiment, the first sample may also be obtained upon admission to hospital, or any setting within hospital (ED, ICU, or other).

In one embodiment, 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 an adverse clinical outcome within at least 28 days after first consultation with the medical personnel and/or obtaining the first sample. In one embodiment, 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 an adverse clinical outcome associated with the need for supplemental oxygen for at least 28 days after first consultation with the medical personnel and/or obtaining the first sample. In one embodiment, 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 in need of supplemental oxygen for at least 28 days after first consultation with the medical personnel and/or obtaining the first sample.

In one embodiment, the patient shows at least two symptoms selected from the group consisting of >38°C fever and lung infiltrates at first contact with the medical personnel. In one embodiment the patient shows a COVID-19 ordinal scale < 4 at day 1 after first contact with the medical personnel.

In one embodiment the patient is hospitalized and/or the patient is admitted to an intensive care unit (ICU) after first contact with the medical personnel.

In one embodiment the first sample is obtained within 24 h (1 day) after consultation with the medical personnel, and/or the second sample is obtained within 72 to 96 h (on day 4) after consultation with the medical personnel.

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

In embodiments, the time points provided with respect to “after consultation with medical personnel” may also apply to the same time point, defined in relation to hospital admission, for example a sample is obtained within X h (X days) “after (hospital) admission and/or after consultation” with medical personnel. This terminology may apply to any embodiment regarding time points as disclosed herein.

By way of non-limiting example, in some embodiments, the analysis times may be conducted as follows: Example: Monday, Day 1 , 9:00 am, patient presents to medical personnel, within 24 h the first blood sample is taken, until the beginning of Day 2, Tuesday, 9:00 am (24 h). Day 2 would progress from Tuesday 9.00 am to Wednesday 9.00 am (48h). Day 3 would progress from Wednesday 9.00 am to Thursday 9.00 am (72h). Day 4 would begin on Thursday 9.00 am and progress to Friday 9.00 am (96h). In embodiments, the second sample may be collected on day 2, day 3 or day 4, preferably on day 4. In embodiments, the second sample may be collected within 60-96 hours after first presentation, preferably 72-96h after first presentation.

In embodiments, the invention is not necessarily limited to taking and measuring two samples from a patient. In embodiments, the method as described herein may also enable the diagnostic or prognostic statements described using analysis of one sample, for example a sample from day 1 or from day 4, in order to make the relevant diagnostic or prognostic statements. Any embodiment disclosing a diagnostic or prognostic statement, i.e. on therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, is potentially relevant to both methods using two or one sample. Detailed data is presented below, from which corresponding embodiments may be observed.

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

In one embodiment the adverse event is death within at least 28 days after obtaining the first sample.

In one embodiment the adverse event is an adverse respiratory and/or infectious clinical outcome requiring oxygen support and intensified treatment within at least 28 days.

In one embodiment the adverse respiratory and/or infectious clinical outcome are intensive care unit (ICU) treatment, mechanical ventilation and/or an additional infection.

In one embodiment the patient is infected with a coronavirus, and the adverse event is a worsening of a COVID scale score (for example worsening of a COVID scale score to >4) and/or maintained hospitalization on or beyond day 4 after hospital admission.

In one embodiment the oxygen support comprises administration of low flow oxygen or high flow oxygen. In one embodiment low flow oxygen comprises administration of equal or below 6 L/min oxygen. In one embodiment high flow oxygen comprises the administration of >6 -to <10 L/min oxygen. In one embodiment mechanical ventilation comprises non-invasive mechanical ventilation, invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO).

In one embodiment, the clinical adverse outcome is admission to an intensive care unit (ICU), clinical worsening on the COVID-19 ordinal scale, death within 28 days, day 4 COVID-19 ordinal scale > 4, prolonged hospitalization beyond day 4, day 4 COVID-19 ordinal scale equal or worse than day 1 , mechanical ventilation within 28 days, any thrombotic event such as deep vein thrombosis (DVT), pulmonary embolism (PE), and stroke, composite severity endpoint comprising at least one of death within 28 days, admission to ICU and mechanical ventilation, infection within 28 days (excluding COVID-19 related treatment) and/or infection (grade >= 3) within 28 days.

In one embodiment the patient has an age of 19 to 85 years. In one embodiment the sample is selected from the group consisting of a blood sample, such as a whole blood sample, a serum sample or a plasma sample.

In one embodiment the levels for the respective biomarker in the first and second sample (day 1 , day 4 and day 1 vs. day 4 (also termed risk ratio)) and clinical outcome can be derived from the statistical results disclosed in tables 1 to 49.

In one embodiment the levels and ratios for the respective biomarker in the first and second sample (day 1 , day 4 and day 1 vs. day 4 (also termed risk ratio)) disclosed in tables 1 to 49 represent cut off values and/or risk ratios for the respective maker and/or marker combination and the corresponding clinical outcome.

In one embodiment, the method additionally comprises a. determining a 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 fragment(s) thereof, procalcitonin (PCT) or fragment(s) thereof, IL6, D-Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes and/or C-reactive protein (CRP), and b. wherein the level of the at least one additional biomarker and/or 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 an adverse clinical event within at least 28 days.

In one embodiment the level of the at least one additional biomarker and/or 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 an adverse clinical outcome associated with the need for supplemental oxygen for at least 28 days.

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

In one embodiment the method additionally comprises a. determining a 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 fragment(s) thereof, procalcitonin (PCT) or fragment(s) thereof, IL6, D-Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes and/or C-reactive protein (CRP), and b. wherein the level of the at least one additional biomarker and 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 in need of supplemental oxygen for at least 28 days.

In one aspect the invention relates to a kit for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, comprising: detection reagents for determining the level of one or more 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 fragment(s) thereof and/or pro- adrenomedullin (proADM) or fragment(s) thereof (preferably MR-proADM), and reference data for the risk of a patient of an adverse clinical outcome within 28 days, in particular reference data for a risk threshold, cut-off value or risk ratio, wherein said reference data is preferably stored on a computer readable medium and/or employed in the form of computer executable code configured for comparing the determined levels of one or more of the endothelial biomarkers in a sample from a patient with the risk threshold, cut-off value or risk ratio; optionally detection reagents for determining the presence of a viral infection of the respiratory tract, preferably detection reagents for the presence of an infection with a coronavirus, preferably a SARS-CoV2 virus infection, and optionally, detection reagents for determining the level of at least one additional biomarker or fragment(s) thereof, preferably pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, procalcitonin (PCT) or fragment(s) thereof, IL6, D-Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C-reactive protein (CRP), in a sample from a patient, and reference data, such as reference date for said at least one additional biomarker, preferably pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, procalcitonin (PCT) or fragment(s) thereof, IL6, D-Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C- reactive protein (CRP), in particular a risk threshold, cut-off value or risk ratio, wherein said reference data is preferably stored on a computer readable medium and/or employed in the form of computer executable code configured for comparing the determined levels of the at least one additional biomarker or fragment(s) thereof with the threshold, cut-off value or risk ratio.

Embodiments relating to sFlt-1 or fraqment(s) thereof as biomarker:

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises sFlt-1 or fragment(s) thereof and a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not at risk of an adverse clinical outcome within at least 28 days.

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises sFlt-1 or fragment(s) thereof and a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not at risk of an adverse clinical outcome associated with the need supplemental oxygen for at least 28 days. In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises sFlt-1 or fragment(s) thereof and a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not in need of oxygen support and not at risk of death within at least 28 days.

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises sFlt-1 or fragment(s) thereof and a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not in need of oxygen support and not at risk of death and an additional infection within at least 28 days.

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises sFlt-1 or fragment(s) thereof and a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not in need of oxygen support and not at risk of death and an additional infection within at least 28 days after consultation with the medical personnel.

In one embodiment, the method comprises the steps of a. providing a first sample isolated from said patient, wherein said patient is infected with a coronavirus, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first sample was obtained within 24 h (1 day) after consultation within the medical personnel, and the second sample was obtained within 72 to 96 h (on day 4) after consultation with the medical personnel, d. determining a level of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof in said sample, and e. wherein a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample indicates that the patient is not in need of oxygen support and not at risk of death and an additional infection within at least 28 days.

Variation in the sFlt-1 cutoff may be employed, for example 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 from this list, may be employed. Any cut-off may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these cutoffs are preferably applicable for a serum sample in a B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker.

Embodiments relating to CT- roET-1 or fraqment(s) thereof as biomarker:

In one embodiment, determining a level of proADM or fragment(s) thereof comprises determining a level of CT-proET-1 or fragment(s) thereof in the sample.

In one embodiment the one or more biomarkers comprises CT-proET-1 or fragments thereof, and wherein a low-risk level of CT-proET-1 or fragment(s) thereof determined on day 1 indicates that the patient is not at risk of admission to ICU, requiring mechanical ventilation, death and/or composite severity endpoint, and/or that the patient will be discharged from the hospital before or on day 4, or wherein a high-risk level of CT-proET-1 or fragment(s) thereof determined on day 1 indicates that the patient is at risk of admission to ICU, requiring mechanical ventilation, death and/or composite severity endpoint, and/or that the patient will be admitted to hospital or the ICU before or on day 4.

In one embodiment the low-risk level of CT-proET-1 or fragments thereof determined on day 1 is < 83.6 pmol/L ± 20%, or the high-risk level of CT-proET-1 or fragments thereof determined on day 1 is > 83.6 pmol/L ± 20%, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 83.6 pmol/L ± 20%.

In one embodiment a level of CT-proET-1 or fragments thereof < 87.4 pmol/L ± 20% determined on day 1 indicates that the patient is not at risk of death within 28 days and/or requiring mechanical ventilation, or a level of CT-proET-1 or fragments thereof > 87.4 pmol/L ± 20% determined on day 1 indicates that the patient is at risk of death within 28 days and/or requiring mechanical ventilation, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 87.4 pmol/L ± 20%.

In one embodiment a level of CT-proET-1 or fragments thereof < 91 .8 pmol/L ± 20% determined on day 1 indicates that the patient is not at risk of requiring mechanical ventilation, admission to ICU and/or composite severity endpoint, a level of CT-proET-1 or fragments thereof > 91 .8 pmol/L ± 20% determined on day 1 indicates that the patient is at risk of requiring mechanical ventilation, admission to ICU and/or composite severity endpoint, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 91 .8 pmol/L ± 20%.

In one embodiment a level of CT-proET-1 or fragments thereof < 97.6 pmol/L ± 20%, preferably < 102.7 pmol/L ± 20% determined on day 1 indicates that the patient is not at risk of day 4 COVID-19 ordinal scale > 4, or a level of CT-proET-1 or fragments thereof > 97.6 pmol/L ± 20%, preferably > 102.7 pmol/L ± 20% determined on day 1 indicates that the patient is at risk of day 4 COVID-19 ordinal scale > 4, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 97.6 pmol/L ± 20%, or 102.7 pmol/L ± 20%.

In one embodiment a level of CT-proET-1 or fragments thereof < 78.7 pmol/L ± 20% determined on day 1 indicates discontinuation of supplemental oxygen, or a level of CT-proET-1 or fragments thereof > 78.7 pmol/L ± 20% determined on day 1 indicates continuation of supplemental oxygen, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 78.7 pmol/L ± 20%.

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises C-terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and/or a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome within at least 28 days.

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises CT-proET-1 or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and/or a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome associated with the need supplemental oxygen for at least 28 days.

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises CT-proET-1 or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and/or a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not at risk of death within at least 28 days (and preferably not in need of oxygen support).

In one embodiment the one or more endothelial biomarkers determined in the first and the second sample comprises CT-proET-1 or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and/or a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not in need of mechanical ventilation and not at risk of death within at least 28 days after consultation with the medical personnel.

Variation in the CT-proET-1 cutoff may be employed, for example 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 from this list, may be employed. Any cut-off may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these cutoffs are preferably applicable for a serum sample in a B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker.

In one embodiment a low-risk level of CT-proET-1 or fragment(s) thereof < 83.6 pmol/L ± 20%. and a low-risk ratio indicates that the patient is not at risk of death within 28 days, admission to ICU, requirement of mechanical ventilation and/or composite severity endpoint and/or that supplemental oxygen is discontinued.

Embodiments relating to proADM or fraqment(s) thereof as biomarker:

In one embodiment, determining a level of proADM or fragment(s) thereof comprises determining a level of mid-regional pro-adrenomedullin (MR-proADM) in the sample.

In one embodiment, the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome within at least 28 days.

In one embodiment, the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome associated with the need supplemental oxygen for at least 28 days.

In one embodiment, the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not in need of oxygen support and not at risk of death within at least 28 days.

In one embodiment, the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not in need of oxygen support, not at risk of death, admission to an ICU and mechanical ventilation within at least 28 days.

In one embodiment the method comprises the steps of a. providing a first sample isolated from said patient, wherein said patient is infected with a coronavirus, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first sample was obtained within 24 h (1 day) after consultation within the medical personnel, and the second sample was obtained within 72 to 96 h (on day 4), after consultation with the medical personnel, d. determining a level of pro-adrenomedullin (proADM) or fragment(s) thereof in said sample, and e. wherein a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample indicates that the patient is not in need of oxygen support, not at risk of death, admission to an ICU and mechanical ventilation within at least 28 days.

Variation in the proADM, preferably MR-proADM, cutoff may be employed, for example 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 from this list, may be employed. Any cut-off may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker. In other embodiments, the MR-proADM cutoff employed is 0.87 or 0.9 or 1 nmol/L, which may provide an assessment of clinical risk and values above this amount may indicate the need for hospitalization, or in some cases a cut-off of 2.25 nmol/L may be employed, whereby values below this value may indicate that ICU treatment is no longer necessary. Measurements using these cutoffs are preferably applicable for a serum sample in a B R A H M S KRYPTOR Assay.

Further aspects and embodiments of the invention:

In one aspect the invention relates to a method for therapy management of a patient having a respiratory virus infection, comprising assessing whether said patient is in need of oxygen support, comprising the steps of: a. providing a first sample isolated from said patient, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, d. determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are pro-adrenomedullin (proADM) or fragment(s) thereof (preferably MR-proADM) and/or soluble Fms-like tyrosine kinase-1 (sFItl -) or fragment(s) 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 is not in need of supplemental oxygen for at least 28 days.

In embodiments, the invention demonstrates high negative predictive values (for rule-out of the need of oxygen support), such that medical staff can quickly (within the first 4 days after presentation of the patient) and effectively determine the need for oxygen support and/or intensified treatment in such patients having a respiratory virus infection.

As outlined above, patient therapy management and risk assessment is urgently needed to avoid subjecting patients with good prognosis or low risk to unnecessary treatments, e.g., in a hospital setting, and to reserve capacity for patients at higher risk and higher need for intensified treatment. Patients at low risk for needing oxygen support and intensified treatment require effective management and suitable treatment options. In view of the increasing shortage of medical structures and personnel, it is important to make the right decisions early on for patients who present to the physician, without unnecessary medical treatment for patients at low risk. The invention enables straightforward, rapid biomarker testing, rather than complicated score determination, thus introducing objective assessment independent of the level of experience of medical staff.

Previous studies have failed to show a clear effect on risk assessment and therapy management within 28 days, when the biomarker is determined within a short period of time after initial presentation to medical personnel, e.g., 1 to 4 days after initial presentation. In the prior art, patient "diagnosis" is emphasized and determined at a certain stage. However, it is typically not possible to derive a prognostic ability from a diagnostic effect and the determination of biomarker levels with a high negative predictive value with a very low false negative rate, as shown in the invention, cannot be assumed from studies indicating the diagnostic potential of biomarkers. The present invention enables primarily effective measurements for negative predictive value, in essence ruling out the need of oxygen support in certain patient populations. In some embodiments, a positive statement is possible, although the primary effect demonstrated by the present invention is an effective rule-out of the need of oxygen support within 28 days for any given patient based on biomarker measurement.

In embodiments, a higher level of the one or more endothelial biomarkers in the second sample compared to the first sample is not a determination that the patient will need oxygen support. In embodiments, the patients with a higher level in the second sample are those for whom a need for oxygen support cannot be “ruled out” using the biomarker measurement, without necessarily indicating a “rule-in”. In preferred embodiments, the patients with a lower or equal level in the second sample are effectively ruled-out from said need of oxygen support indicated by a strong NPV, as described in more detail below.

In one embodiment the one or more endothelial biomarkers in the second sample compared to the first sample additionally indicates that the patient is not at risk for death within at least 28 days after obtaining the first sample.

In one embodiment 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 an adverse respiratory and/or infectious clinical outcome requiring oxygen support and intensified treatment within at least 28 days.

In one aspect the invention relates to a method for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, wherein the method comprises: a. providing a sample from said patient, b. determining a level of one or more endothelial biomarkers in said sample, c. wherein the one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal pro-endothelin-1 (CT-proET- 1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and procalcitonin (PCT) or fragment(s) thereof, d. wherein said level of the one or more endothelial biomarkers in the sample indicates the risk of an adverse clinical outcome within 28 days.

In one embodiment, said level of the one or more endothelial biomarkers in the sample indicates that said patient is not at risk of an adverse clinical outcome within 28 days.

In one embodiment, said level of the one or more endothelial biomarkers in the sample below a cut off level indicates that said patient is not at risk of an adverse clinical outcome within 28 days. In one embodiment the one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof and procalcitonin (PCT) or fragment(s) thereof, preferably soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof.

In embodiment the invention relates to a method for prognosing disease progression, severity and/or outcome in a patient having a coronavirus infection, wherein the method comprises: a. providing a sample from said patient, b. determining a level of one or more biomarkers in said sample, c. wherein the one or more biomarkers comprise one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and/or procalcitonin (PCT) or fragment(s) thereof, d. wherein said level of the one or more biomarkers in the sample indicates the risk of an adverse clinical outcome within 28 days.

In one embodiment, the level of the one or more biomarkers indicates whether the patient is or is not at risk of an adverse clinical outcome requiring intensified treatment and/or monitoring.

In one embodiment, the level of the one or more biomarkers indicates whether the patient is at low or no risk of an adverse clinical outcome requiring a de-escalation of treatment and/or reduce intense medical monitoring and/or discharge the patient from the ICU or the hospital and/or when a patient will exhibit an improvement in the Covid-19 ordinal scale.

In one embodiment the level of the one or more biomarkers are determined in a sample obtained by the first medical personnel to consult with the patient after occurrence of symptoms of the infection with a coronavirus.

In one embodiment the sample is obtained within 24 h (day 1) and/or within 96 hours, preferably within 72-96 h (day 4), after first contact with the medical personnel.

In one embodiment the sample is obtained on day 4 and the level of the one or more biomarkers in said sample indicates whether the patient is at low or no risk of an adverse clinical outcome, requiring a de-escalation of treatment and/or reduce intense medical monitoring and/or discharge the patient from the ICU or the hospital. In one embodiment the sample is obtained on day 4 and the level of the one or more biomarkers in said sample indicates a de-escalation of treatment and/or reduction of intense medical monitoring and/or discharge the patient from the ICU or the hospital.

In one embodiment the first contact with the medical personnel is in the hospital.

In one embodiment the patient is hospitalized after first contact with the medical personnel.

In one embodiment the patient is treated with supplemental oxygen.

In one embodiment the patient is assessed for a requirement of treatment with supplemental oxygen, invasive mechanical ventilation and/or ECMO. In one embodiment the patient is assessed for the risk of developing a thrombotic event.

In one embodiment the patient’s survival prediction is assessed for the next 28 days from the first (blood) sample collection.

In one embodiment the patient is not admitted to an intensive care unit (ICU) at first contact with the medical personnel.

In one embodiment the patient shows a COVID-19 ordinal scale < 4 at day 1.

In one embodiment the level of the one or more biomarkers indicates whether the patient is at risk of admission to an intensive care unit (ICU), clinical worsening on the COVID-19 ordinal scale, death within 28 days, day 4 COVID-19 ordinal scale > 4, prolonged hospitalization beyond day 4, day 4 COVID-19 ordinal scale equal or worse than day 1 , mechanical ventilation within 28 days, any thrombotic event such as deep vein thrombosis (DVT), pulmonary embolism (PE), and stroke, composite severity endpoint comprising at least one of death within 28 days, admission to ICU and mechanical ventilation, infection within 28 days (excluding COVID-19 related treatment) and/or infection (grade >= 3) within 28 days.

In embodiments, the invention demonstrates high negative predictive values (for rule-out of risk or adverse events), such that medical staff can quickly (e.g., within the first 4 days after presentation of the patient), on the first day after presentation, or in connection assessment about 3-4 days after first presentation, may effectively determine risk in such patients.

As outlined above, patient outcome prediction is urgently needed to avoid subjecting patients with good prognosis or low risk to unnecessary treatments, e.g., in a hospital setting, and to reserve capacity for patients at higher risk. Patients at low risk for a thrombotic event, a low risk of needing oxygen administration, or a low risk of death or a necessary ICU stay require effective management and fitting treatment options. In view of the increasing shortage of medical structures and personnel, it is important to make the right early decisions for patients who present to the physician, without unnecessary medical treatment for patients at low risk. The invention enables straightforward, rapid biomarker testing, rather than complicated score determination, thus introducing objective assessment independent of the level of experience of medical staff.

Previous studies have however failed to show a clear prognostic effect within 28 days, under determination of the marker in a short period of e.g., 1 to 4 days after initial examination. In the prior art, patient "diagnosis" is emphasized and determined at a certain stage. However, it is typically not possible to derive a prognostic ability from a diagnostic effect and determining biomarker levels with a high negative predictive value with a very low false negative rate, as shown in the invention, cannot be assumed from studies indicating diagnostic potential of biomarkers or even an association between high risk and high biomarker level. The present invention enables primarily effective measurements for negative predictive value, in essence ruling out risk in certain patient populations.

In some embodiments, a positive statement is possible, although the primary effect demonstrated by the present invention is an effective rule-out of risk within 28 days for any given patient based on biomarker measurement. In some embodiments a high positive predictive value and thus rulein is provided with regards to the discharge of the patient from the ICU or hospital. This corresponds to the high negative predictive value and rule-out of the risk of an adverse clinical event and need for intensified treatment, as a discharge from ICU or hospital means that the patient in not or no longer in need of an intensified treatment as provided in the ICU or hospital.

In one embodiment, the method comprises: providing a sample from said patient, and determining a level of one or more biomarkers in said sample, wherein the one or more biomarkers comprise one or more of CT-proET-1 fragment(s) thereof, sFlt-1 or fragment(s) thereof, proADM or fragment(s) thereof and PCT or fragment(s) thereof, a. wherein a low-risk level of the one or more biomarkers or fragment(s) thereof in said sample at day 1 indicates that the patient is not at risk of an adverse clinical outcome within 28 days, and/or b. wherein a low-risk ratio of the levels of one or more biomarkers or fragment(s) thereof in said sample determined on day 4 and day 1 (ratio day 4/day 1) indicates that the patient is not at risk of an adverse clinical outcome within 28 days.

In one embodiment, the method comprises: providing a first sample from said patient, providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, determining a level of one or more biomarkers in said first and second sample, wherein the one or more biomarkers comprise one or more of CT-proET-1 fragment(s) thereof, sFlt-1 or fragment(s) thereof, proADM or fragment(s) thereof and PCT or fragment(s) thereof, and a. wherein a low-risk level of the one or more biomarkers or fragment(s) thereof in said sample at day 1 indicates that the patient is not at risk of an adverse clinical outcome within 28 days, and/or b. wherein a low-risk ratio of the levels of one or more biomarkers or fragment(s) thereof in the second sample determined on day 4 and the first sample determined on day 1 (ratio day 4/day 1) indicates that the patient is not at risk of an adverse clinical outcome within 28 days.

In one embodiment the low risk level and/or the low-risk ratio for the respective biomarker and outcome can be derived from the ratios disclosed in tables 1 to 49. In one embodiment the biomarker levels and ratios disclosed in tables 1 to 49 represent risk levels and risk ratios for the respective maker and/or marker combination and the corresponding predicted adverse outcome.

In one embodiment the low-risk ratio of the levels of one or more biomarkers or fragment(s) thereof is < 1 . Such values are typically associated with no increase or a decrease of the biomarker value from day 1 to day 4. Such values correspond to a lower or equal level of the one or more biomarkers, preferably endothelial biomarkers, in the second sample compared to the first sample (both isolated within 96 h (4 days) and the second sample being isolated at a time point after isolating the first sample, preferably the first sample being isolated at day 1 and the second sample being isolated at day 4).

As described in more detail below, the risk ratio of the levels of one or more biomarkers or fragment(s) thereof in said sample represents a comparison in marker values determined on day 4 and day 1 (ratio day 4/day 1). The risk ratio of the levels of one or more biomarkers or fragment(s) thereof in said sample preferably represents a comparison in marker values determined in a second sample obtained on day 4 and a first sample obtained on day 1 (ratio day 4/day 1) Assessment of these risk ratios provides, in embodiments, an accurate prognosis of outcome, as shown in the various statistical analyses presented below.

In embodiments, the first sample is obtained upon presentation to medical personnel. This may be a primary care, general practitioner, hospital ward, emergency department or ICU. In one embodiment the sample is obtained within 24 h (day 1) after first contact with the medical personnel, preferably within 1 , 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours after presenting to a medical practitioner. This sample may also be obtained upon admission to hospital, or any setting within hospital (ED, ICU, or other).

In embodiments, the second sample is obtained on day 4 after presentation to medical personnel. This sample may be obtained by primary care, a general practitioner, at a hospital ward, or in the emergency department or ICU. In one embodiment the sample is obtained 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 hours after first contact with the medical personnel. By comparing the values measured in these samples, a ratio of day 4: day 1 can be determined. In embodiments, this ratio is, as such, indicative of outcome, with statistical significance.

The day 4/day 1 ratios therefore may be determined for sample obtained within an approximate time window at “day 1” (e.g., 0-24 hours after first presentation to medical personnel) and “day 4” (e.g., after 3-5 days, preferably within 60-120 hours after first presentation to medical personnel, more preferably 72-120h, 72-108h, 72-96h, or 84-120h, 84-108h after first presentation), as disclosed herein.

In embodiments, the second sample may be collected on day 3, day 4 or day 5, preferably on day 4. In embodiments, the second sample may be collected within 60-120 hours after first presentation, preferably 72-108h after first presentation.

In one embodiment a low-risk level of CT-proET-1 fragment(s) thereof, sFlt-1 or fragment(s) thereof, proADM or fragment(s) thereof, IL-6 and PCT or fragment(s) thereof, or a low-risk ratio of the one or more biomarkers indicates that the patient is not at risk of death within 28 days and/or admission to ICU.

In embodiments, solo or combined measurement of IL-6 indicates whether the patient is at risk of mechanical ventilation, ICU admission or death, composite severity endpoint, Day-4 Covid scale or death. IL-6 values therefore provide added value in determining whether mechanical ventilation is required. For example, in embodiments, the performance of various biomarkers, e.g. on assessing risk of ICU admission or death, composite severity endpoint, Day-4 Covid scale, mechanical ventilation or death, provided a sound predictive value, such as IL-6, with AUC measurements of 0.73, 0.72, 0.72, 0.71 (p<0.003) (see below).

In embodiments, the “high-risk” level is not a determination that the patient will suffer said medical condition, outcome, or require any given treatment. In embodiments, the “high-risk” patients are those for whom a risk cannot be “ruled out” using the biomarker measurement, without necessarily indicating a “rule-in”. Thus, the term “high-risk” preferably relates to patients that cannot be effectively “ruled-out” from any given outcome, medical condition or treatment. In preferred embodiments, the low-risk patients are however effectively ruled-out from said 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.

In one embodiment the method comprises determining a level of at least two biomarkers in the sample, wherein the at least two biomarkers comprise at least one of C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and procalcitonin (PCT) or fragment(s) thereof, and optionally one or more of IL6, d-Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C-reactive protein (CRP).

In one aspect the invention relates to a kit for carrying out the method of any one of the preceding claims, comprising: detection reagents for determining the level of one or more of C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and procalcitonin (PCT) or fragment(s) thereof in a sample from a patient, and reference data for the risk of a patient of an adverse clinical outcome within 28 days, in particular reference data for a risk threshold, cut-off value or risk ratio, wherein said reference data is preferably stored on a computer readable medium and/or employed in the form of computer executable code configured for comparing the determined levels of one or more of C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and procalcitonin (PCT) or fragment(s) thereof in a sample from a patient with the risk threshold or cut-off value; optionally detection reagents for determining the presence of a viral infection of the respiratory tract, preferably detection reagents for the presence of an infection with a coronavirus, preferably a SARS-CoV2 virus infection; and optionally, detection reagents for determining the level of at least one additional biomarker or fragment(s) 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 for said at least one additional biomarker, preferably IL6, d- Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C-reactive protein (CRP), in particular a risk threshold, cut-off value or risk ratio, wherein said reference data is preferably stored on a computer readable medium and/or employed in the form of computer executable code configured for comparing the determined levels of the at least one additional biomarker or fragment(s) thereof with the threshold or cut-off value.

Each of the tables below (tables 1 to 49), each row of the tables below and combinations of the tables and/or rows below, represents an embodiment of the present invention with regards to risk ratios, cut-off values and the prediction of specific adverse outcomes and further outcomes, including by way of example discharge form hospital before or on day 4, discontinuation of supplemental oxygen and improvement of COVID-19 ordinal scale by these cut off and/or risk values. Each of the tables below (tables 1 to 49), each row of the tables below and combinations of the tables and/or rows below, represents an embodiment of the present invention with regards to cut-off values indicating whether said patient is not at risk of an adverse clinical outcome. Each of the tables below (tables 1 to 49), each row of the tables below and combinations of the tables and/or rows below, represents an embodiment of the present invention with regards to cut-off values indicating whether said patient is not in need of oxygen support and/or intensified treatment.

The invention relates further to a method for detecting and/or determining the level of a biomarker in a patient having a coronavirus infection, wherein the method comprises: (a) providing a sample from said patient, (b) detecting and/or determining a level of one or more biomarkers in said sample, (c) wherein the one or more biomarkers comprise one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and/or procalcitonin (PCT) or fragment(s) thereof. In embodiments, the patient groups and/or levels and/or statistical values, such determined levels, cut-offs and/or risk ratios as disclosed herein may be employed in the method.

The invention relates further to a method for detecting and/or determining the level of a biomarker in a patient having a respiratory virus infection, wherein the method comprises: (a) providing a first sample isolated from said patient, (b) providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, (c) wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, (d) detecting and/or determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof and/or pro-adrenomedullin (proADM) or fragment(s) thereof. In embodiments, the patient groups and/or levels and/or statistical values, such determined levels, cut-offs and/or risk ratios as disclosed herein may be employed in the method. The invention relates further 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 a coronavirus infection, the method comprising optionally prognosing disease progression, severity and/or outcome in a patient having a coronavirus infection, wherein the method comprises: (a) providing a sample from said patient, (b) determining a level of one or more biomarkers in said sample, (c) wherein the one or more biomarkers comprise one or more of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, pro-adrenomedullin (proADM) or fragment(s) thereof and/or procalcitonin (PCT) or fragment(s) thereof, (d) wherein said level of the one or more biomarkers in the sample indicates the risk of an adverse clinical outcome within 28 days, the method further comprising € treating said patient, preferably treating said patient to prevent and/or reduce the risk of adverse events or medical conditions associated with a coronavirus infection. In embodiments, the patient groups and/or levels and/or statistical values, such determined levels, cut-offs and/or risk ratios, or any one or more treatments known to a skilled person, or as disclosed herein, may be employed in the method.

The invention relates further 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 a respiratory virus infection, the method comprising optionally prognosing disease progression, severity and/or outcome in a patient having a respiratory virus infection, wherein the method comprises (a) providing a first sample isolated from said patient, (b) providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, (c) wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, (d) detecting and/or determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are one or more of soluble Fms-like tyrosine kinase-1 (sFItl) or fragment(s) thereof and/or pro- adrenomedullin (proADM) or fragment(s) thereof, (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 is not in need at risk of an adverse clinical outcome within supplemental oxygen for at least 28 days, the method further comprising (f) treating said patient, preferably treating said patient to prevent and/or reduce the risk of adverse events or medical conditions associated with a respiratory virus infection. In embodiments, the patient groups and/or levels and/or statistical values, such determined levels, cut-offs and/or risk ratios, or any one or more treatments known to a skilled person, or as disclosed herein, may be employed in the method.

Further embodiments relating to sFlt-1 or fragment(s) thereof as biomarker:

In one embodiment the one or more biomarkers comprises sFlt-1 or fragments thereof, and wherein a low-risk level of sFlt-1 or fragment(s) thereof determined on day 1 indicates that the patient is not at risk of composite severity endpoint and/or that the patient will be discharged from the hospital before or on day 4 and/or shows an improvement of day 4 COVID-19 ordinal scale in comparison to day 1 , or wherein a high-risk level of sFlt-1 or fragment(s) thereof determined on day 1 indicates that the patient is at risk of composite severity endpoint and/or that the patient will be 1 admitted to hospital or the ICU before or on day 4 and/or shows a deterioration of day 4 COVID-19 ordinal scale in comparison to day 1.

In one embodiment the low-risk level of sFlt-1 or fragments thereof determined on day 1 is < 81 .3 pgl/L ± 20%, or the high-risk level of sFlt-1 or fragments thereof determined on day 1 is > 81 .3 pgl/L ± 20%, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 81 .3 pgl/L ± 20%.

In one embodiment a level of sFlt-1 or fragments thereof < 83.9 pg/L ± 20% determined on day 1 indicates that the patient is not at risk of ICU admission and/or day 4 worsening of COVID-19 ordinal scale compared to day 1 and/or that the patient will be discharged from the hospital before or on day 4, or a level of sFlt-1 or fragments thereof > 83.9 pg/L ± 20% determined on day 1 indicates that the patient is at risk of ICU admission and/or day 4 worsening of COVID-19 ordinal scale compared to day 1 and/or that the patient will be admitted to hospital or the ICU before or on day 4, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 83.9 pg/L ± 20%.

In one embodiment a level of sFlt-1 or fragments thereof < 88.4 pg/L ± 20% determined on day 1 indicates that the patient is not at risk of at least one of the composite severity endpoints (death, ICU admission or mechanical ventilation within 28 days), or a level of sFlt-1 or fragments thereof > 88.4 pg/L ± 20% determined on day 1 indicates that the patient is at risk of at least one of the composite severity endpoints (death, ICU admission or mechanical ventilation), and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 88.4 pg/L ± 20%.

In one embodiment, a low-risk level of sFlt-1 or fragment(s) thereof < 81 .3 pg/L ± 20% and a low- risk ratio indicates that the patient is not at risk of death within 28 days, admission to ICU, any thrombotic events and/or composite severity endpoint. In one embodiment, a low-risk level of sFlt-1 or fragment(s) thereof < 81.3 pg/L ± 20% determined on day 1 and a low-risk ratio indicates that the patient is not at risk of death within 28 days, admission to ICU, any thrombotic events and/or composite severity endpoint.

Further embodiments relating to proADM or fragments) thereof as biomarker:

In one embodiment the one or more biomarkers comprises proADM or fragments thereof and wherein a low-risk level of proADM or fragment(s) thereof determined on day 1 indicates that the patient is not at risk of requiring mechanical ventilation, death within 28 days, admission to ICU clinical, worsening on the COVID-19 ordinal scale on day 4 and/or that the patient will be discharged from the hospital before or on day 4, or wherein a high-risk level of proADM or fragment(s) thereof determined on day 1 indicates that the patient is at risk of requiring mechanical ventilation, death within 28 days, admission to ICU, clinical worsening on the COVID-19 ordinal scale on day 4 and/or that the patient will be admitted to hospital or the ICU before or on day 4.

In one embodiment determining a level of proADM or fragment(s) thereof comprises determining a level of mid-regional pro-adrenomedullin (MR-proADM) in the sample.

In one embodiment the low-risk level of proADM or fragments thereof determined on day 1 is < 0.87 nmol/L ± 20%, or the high-risk level of proADM or fragments thereof determined on day 1 is > 0.87 nmol/L ± 20%, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 0.87 nmol/L ± 20%.

In one embodiment a level of proADM or fragments thereof < 0.87 nmol/L ± 20% determined on day 1 indicates that the patient is not at risk of death or venous thrombosis or an additional infection grade >=3, continuing supplemental oxygen, ICU admittance, the need of mechanical ventilation, a longer stay than 4 days in the hospital, worse in the Covid-19 ordinale scale) within 28 days, or a level of proADM or fragments thereof > 2.25 nmol/L ± 20% determined on day 1 indicates that the patient is at risk of death or venous thrombosis or an additional infection grade >=3, continuing supplemental oxygen, ICU admittance, the need of mechanical ventilation, a longer stay than 4 days in the hospital, worse in the Covid-19 ordinale scale) within 28 days, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 2.25 nmol/L ± 20%.

In one embodiment a level of proADM or fragments thereof < 1 .33 nmol/L ± 20% determined on day 1 indicates that the patient is not at risk of composite severity endpoint and/or indicating discontinuation of supplemental oxygen, or a level of proADM or fragments thereof > 1 .33 nmol/L ± 20% determined on day 1 indicates that the patient is at risk of composite severity endpoint and/or indicating continuation of supplemental oxygen, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 1 .33 nmol/L ± 20%.

In one embodiment a level of proADM or fragments thereof < 1 .58 nmol/L ± 20% determined on day 1 indicates that the patient is not at risk of death within 28 days, or a level of proADM or fragments thereof > 1 .58 nmol/L ± 20% determined on day 1 indicates that the patient is at risk of death within 28 days, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 1 .58 nmol/L ± 20%.

In one embodiment a level of proADM or fragments thereof < 2.25 nmol/L ± 20% determined on day 1 indicates that the patient is not at risk of death or venous thrombosis or an additional infection grade >=3, continuing supplemental oxygen, ICU admittance, the need of mechanical ventilation, a longer stay than 4 days in the hospital, worse in the Covid-19 ordinale scale) within 28 days, or a level of proADM or fragments thereof > 2.25 nmol/L ± 20% determined on day 1 indicates that the patient is at risk of death or venous thrombosis or an additional infection grade >=3, continuing supplemental oxygen, ICU admittance, the need of mechanical ventilation, a longer stay than 4 days in the hospital, worse in the Covid-19 ordinale scale) within 28 days, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 2.25 nmol/L ± 20%.

Variation in the proADM, preferably MR-proADM, cutoff may be employed, for example 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 from this list, may be employed. Any cut-off may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker.

In other embodiments, the MR-proADM cutoff employed is 0.87 or 0.9 or 1 nmol/L, which may provide an assessment of clinical risk and values above this amount may indicate the need for hospitalization, or in some cases a cut-off of 2.25 nmol/L may be employed, whereby values below this value may indicate that ICU treatment is no longer necessary.

Measurements using these cutoffs are preferably applicable for a serum sample in a B R A H M S KRYPTOR Assay. In one embodiment a low-risk level of proADM or fragment(s) thereof < 0.87 nmol/L ± 20% and a low-risk ratio indicates that the patient is not at risk of death within 28 days, admission to ICU, an additional infection, any thrombotic event and/or any thrombotic event and/or indicated discontinuation of supplemental oxygen.

Further embodiments relating to PCT or fragment(s) thereof as biomarker:

In one embodiment the one or more biomarkers comprises PCT or fragments thereof and wherein a low-risk level of PCT or fragment(s) thereof determined on day 1 indicates that the patient is not at risk of admission to ICU, worsening on the COVID-19 ordinal scale on day 4 and/or death within 28 days, or the one or more biomarkers comprises PCT or fragments thereof and wherein a high-risk level of PCT or fragment(s) thereof determined on day 1 indicates that the patient is at risk of admission to ICU, worsening on the COVID-19 ordinal scale on day 4 and/or death within 28 days.

In one embodiment the low-risk level of PCT or fragments thereof determined on day 1 is < 0.27 ng/mL ± 20%, the high-risk level of PCT or fragments thereof determined on day 1 is > 0.27 ng/mL ± 20%, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 0.27 ng/mL ± 20%.

In embodiments, the cut-off level for PCT may be a value in the range of 0.01 to 100.00 ng/mL in a serum sample, when using for example a B R A H M S KRYPTOR Assay. In a preferred embodiment the cut-off level of PCT may be in the range of 0.01 to 100, 0.05 to 50, 0.1 to 20, or 0.1 to 0.27 ng/mL, and most preferably >0,25 to 0,5 ng/mL. Any value within these ranges may be considered as an appropriate cut-off value. For example, 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 employed. In some embodiments, PCT levels for healthy subjects are approximately 0.05 ng/mL. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker.

In one embodiment a level of PCT or fragments thereof < 0.25 ng/mL ± 20% determined on day 1 indicates that the patient is not at risk of suffering from an additional infection (>= grade 3) or hospital stay for more than 4 days or of death within the next 28 days, and/or will be discharged from hospital on or around day 4, or a level of PCT or fragments thereof > 0.25 ng/mL ± 20% determined on day 1 indicates that the patient needs to stay in hospital for longer than 4 days or is at risk of suffering from an additional infection (grade >=3) within the next 28 days or has an increased risk of death, and/or is admitted to hospital or the ICU, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 0.25 ng/mL ± 20%.

In one embodiment a level of PCT or fragments thereof < 0.5 ng/mL ± 20% determined on day 1 or 4 indicates that the patient is not at risk of suffering from an additional infection (>= grade 3) or hospital stay for more than 4 days or of death within the next 28 days, or a level of PCT or fragments thereof > 0.5 ng/mL ± 20% determined on day 1 or 4 indicates that the patient need to stay in the hospital for longer than 4 days or is at risk of suffering from an additional infection (grade >=3) within the next 28 days or has an increased risk of death, and optionally the method comprises comparing the determined level of one or more biomarkers in said sample to a cut-off value of 0.5 ng/mL ± 20%.

In one embodiment a low-risk level of PCT or fragment(s) thereof < 0.25 ng/mL ± 20% and a low- risk ratio indicates that the patient is not at risk of death within 28 days, admission to ICU, an additional infection and/or any thrombotic event.

Embodiments and features of the invention described with respect to the method and kits, are considered to be disclosed with respect to each and every other aspect of the disclosure, such that features characterizing the methods, may be employed to characterize the kit, and vice- versa.

DETAILED DESCRIPTION

General terms:

As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components 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 “consisting essentially of’.

Thus, the terms “comprising”/“including”/”having” mean that any further component (or likewise features, integers, steps and the like) can/may be present. The term “consisting of’ means that no further component (or likewise features, integers, steps and the like) is present.

The term “consisting essentially of’ or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

Thus, the term “consisting essentially of’ means those specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of' (which can be interchangeably used herein with the term "comprising substantially"), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts. As used herein, the “patient” or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms.

SARS and Coronavirus:

In some embodiments, symptoms of infection with a SARS-virus are fever, sore throat, cough, myalgia or fatigue, and in some embodiments, additionally, sputum production, headache, hemoptysis and/or diarrhea.

In some embodiments, symptoms of an infection with a SARS-coronavirus, for example SARS- CoV-2, are fever, sore throat, cough, lack of taste and/or smell, shortness of breath and/or fatigue.

Coronaviruses are a group of related viruses that cause diseases in mammals and birds. The scientific name for coronavirus is Orthocoronavirinae or Coronavirinae. Coronavirus belongs to the family of Coronaviridae. The family is divided into Coronavirinae and Torovirinae sub-families, which are further divided into six genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus, Torovirus, and Bafinivirus. While viruses in the genera Alphacoronaviruses and Betacoronaviruses infect mostly mammals, the Gammacoronavirus infect avian species and members of the Deltacoronavirus genus have been found in both mammalian and avian hosts.

In humans, coronaviruses cause respiratory tract infections that can be mild, such as some cases of the common cold, and others that can be lethal, such as SARS, MERS, and COVID-19. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases, the largest among known RNA viruses.

Various species of human coronaviruses are known, such as, without limitation, Human coronavirus OC43 (HCoV-OC43), of the genus P-CoV, Human coronavirus HKU1 (HCoV-HKLH), of the genus P-CoV, Human coronavirus 229E (HCoV-229E), a-CoV, Human coronavirus NL63 (HCoV-NL63), a-CoV, Middle East respiratory syndrome-related coronavirus (MERS-CoV), Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Coronaviruses vary significantly in risk factor. Some can kill more than 30% of those infected (such as MERS-CoV), and some are relatively harmless, such as the common cold.

Coronaviruses cause colds with major symptoms, such as fever, and a sore throat, e.g. from swollen adenoids, occurring primarily in the winter and early spring seasons. Coronaviruses can cause pneumonia (either direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (either direct viral bronchitis or secondary bacterial bronchitis). Coronaviruses can also cause SARS.

Advances in nucleic acid sequencing technology (commonly termed Next-Generation Sequencing, NGS) are providing large sets of sequence data obtained from a variety of biological samples and allowing the characterization of both known and novel virus strains. Established methods are therefore available for determining a Coronavirus infection.

Viruses encode a collection of proteins required to ensure self-replication and persistence of the encoding virus. Enzymes for genome mRNA production and genome replication, proteases for protein maturation, proteins for genome encapsidation, and proteins for undermining the host antiviral responses can be identified conserved protein motifs or domains. Likely because of selective pressures, viral genomes are streamlined and the functional protein content encoded by viruses is much higher than for a cellular organisms. Thus, describing a viral genome by the collection of encoded protein domains is a potentially useful classification method. Viral evolution can therefore be followed and novel strains of coronavirus can be determined based on sequence compairon to known coronavirus strains.

In some embodiments, the patient suffers from an infection, preferably with a SARS-virus, more preferably a SARS-coronavirus (SARS-CoV).

As used herein, SARS-virus refers to a virus that leads to severe acute respiratory syndrome (SARS). Although Coronaviruses have to date been primarily responsible for SARS, other viral infections may lead to SARS, for example other viruses of zoonotic origin.

As used herein, SARS-coronavirus refers to a Coronavirus that leads to severe acute respiratory syndrome (SARS). This syndrome is a viral respiratory disease of zoonotic origin that first surfaced in the early 2000s caused by the first-identified strain of the SARS coronavirus (SARS- CoV or SARS-CoV-1).

SARS is induced via droplet transmission and replication of the virus could be shown in the upper and lower respiratory tract or gastrointestinal mucosa. In parallel the virus is also capable of directly invading cells of different organs such as liver, kidney, heart and brain. Another distinct mechanism appears to be the direct invasion of the virus in T-cells. A significant number of SARS patients, who suffer COVID-19, have a clinically low concentration of lymphocytes in the blood, also known as Lymphocytopenia. Clinically, patients show respiratory symptoms such as dry cough and shortness of breath, fever or diarrhea. But symptoms associated with acute liver injury, heart injury or kidney injury can also occur. In a less severe state of SARS, patients can show mild or even no symptoms.

Clinical and scientific investigations show that SARS-CoVs bind to the epithelial cells via the Angiotensin converting enzyme 2 receptor (ACE2). ACE2 is a cell membrane linked aminopeptidase which is expressed in vascular endothelia, kidney, bladder, heart, nasal mucosa, bronchus and lung. As one consequence binding of the virus leads to an epithelial and endothelial cell damage along with vascular leakage, which triggers a secretion of pro- inflammatory cytokines and chemokines. The virus also mediates ACE2 downregulation and shedding which further promotes a dysfunctional renin-angiotensin system (RAS). Once the RAS is disturbed it can lead an inflammatory response and to vascular permeability. Focusing on the respiratory system, ACE2 shedding can lead to pulmonary vascular permeability and subsequently to pulmonary edema.

Another pathophysiological element which is called antibody dependent enhancement (ADE) is associated with the existence of antibodies that bind to viruses and facilitate the entry of the virus into the cell. Thereby those antibodies support the virus replication and spread of the virus in the body instead of showing a neutralizing effect. If ADE is present in a patient, a systemic inflammatory response can be activated or maintained.

It is proposed that direct cell damage (multiorgan) and an impaired RAS are key factors for a local and systematic inflammatory response resulting in a so called cytokine storm which is related to an adverse outcome of SARS patients. Adverse outcomes of SARS can be based on the development of an Acute Respiratory Distress Syndrome (ARDS), multiorgan damage or Lymphocytopenia.

Older patients (above 60 years), patients with chronic diseases (e.g. cardiovascular diseases, diabetes, cancer, COPD) or immunocompromised patients are considered to be at a higher risk to face a severe deveopment of SARS. Smoking and obesity are also considered as risk factors (1-5, 21).

Embodiments of a SARS-coronavirus include, without limitation, any coronavirus that induces a SARS or SARS-similar pathology. Particular embodiments include, without limitation, the SARS- Coronavirus (SARS-CoV-1) first discovered in 2003 (as described above), the Middle East respiratory syndrome (MERS-CoV) first discovered in 2012, and the SARS-CoV-2, which causes COVID-19, a disease which brought about the 2019-2020 coronavirus pandemic.

In preferred embodiments, the SARS-coronavirus is SARS-CoV-2, or SARS-CoV derived therefrom.

The strain SARS-CoV-2 causes COVID-19, a disease which brought about the ongoing 2019- 2020 coronavirus pandemic. The disease was identified 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 the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure.

Coronaviruses can be determined by molecular techniques, for example sequence-based analysis, for example by PCR-based amplification of viral genetic material. Genome-wide phylogenetic analysis indicates that SARS-CoV-2 shares 79.5% and 50% sequence identity to SARS-CoV and MERS-CoV, respectively. However, there is 94.6% sequence identity between the seven conserved replicase domains in ORFIab of SARS-CoV-2 and SARS-CoV, and less than 90% sequence identity between those of SARS-CoV-2 and other -CoVs, implying that SARS-CoV-2 belongs to the lineage of Beta-CoVs.

Similar to other -CoVs, the SARS-CoV-2 virion with a genome size of 29.9 kb [13] possesses a nucleocapsid composed of genomic RNA and phosphorylated nucleocapsid (N) protein. The nucleocapsid is buried inside phospholipid bilayers and covered by two di erent types of spike proteins: the spike glycoprotein trimmer (S) that exists in all CoVs, and the hemagglutinin- esterase (HE) only shared among some CoVs. The membrane (M) protein and the envelope (E) protein are located among the S proteins in the viral envelope. The SARS-CoV-2 genome has 5’ and 3’ terminal sequences (265 nt at the 5’ terminal and 229 nt at the 3’ terminal region), which is typical of -CoVs, with a gene order 5’-replicase open reading frame (ORF) lab-S-envelope(E)- membrane(M)-N-30. The predicted S, ORF3a, E, M, and N genes of SARS-CoV-2 are 3822, 828, 228, 669, and 1260 nt in length, 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.

According to Jin et al (Viruses 2020, 12, 372), in the initial 41 patients, fever (98%), cough (76%), and myalgia or fatigue (44%) were the most common symptoms. Less common symptoms were sputum production (28%), headache (8%), hemoptysis (5%), and diarrhea (3%). More than half of patients developed dyspnea. The average incubation period and basic reproduction number (RO) were estimated to be 5.2 d (95% Cl: 4.1-7.0) and 2.2 (95% Cl, 1.4-3.9), respectively. Blood test showed normal or reduced (25%) white blood cell count and lymphopenia (65%). A total of 98% of patients had bilateral involvement under chest CT. Typical findings of chest CT images of ICU patients on admission were bilateral multiple lobular and subsegmental areas of consolidation. The representative chest CT findings of non-ICU patients showed bilateral ground-glass opacity and subsegmental areas of consolidation. Analysis of 1324 laboratory confirmed cases showed that fever (87.9%) and cough (67.7%) were still the most common symptoms, while diarrhea is uncommon. Lymphopenia was observed in 82.1 % of patients admitted to ICU.

In some embodiments, subjects have been tested and determined to have a SARS-CoV. Various methods may be employed to detect SAR-CoV, e.g. SARS-CoV-2, such as a nucleic acid test, serologic diagnosis, CRISPR/Cas13-based SHERLOCK technology, or imaging technology, such as chest radiographs or CT-scans.

SARS-CoV-2 is transmitted predominantly via respiratory droplet, contact, and potential in fecal- oral. Primary viral replication is presumed to occur in mucosal epithelium of upper respiratory tract (nasal cavity and pharynx), with further multiplication in lower respiratory tract and gastrointestinal mucosa, giving rise to a mild viremia. Few infections are controlled at this point and remain asymptomatic. Some patients have also exhibited non-respiratory symptoms such as acute liver and heart injury, kidney failure, diarrhea, implying multiple organ involvement. ACE2 is broadly expressed in nasal mucosa, bronchus, lung, heart, esophagus, kidney, stomach, bladder, and ileum, and these human organs are all vulnerable to SARS-CoV-2.

According to Jin et al (Viruses 2020, 12, 372), the first report of pathological findings from severe COVID-19 showed pulmonary bilateral diffuse alveolar damage with cellular fibromyxoid exudates. The right lung showed evident desquamation of pneumocytes and hyaline membrane formation, indicating acute respiratory distress syndrome. The left lung tissue displayed pulmonary edema with hyaline membrane formation, suggestive of early-phase acute respiratory distress syndrome (ARDS). Interstitial mononuclear inflammatory infiltrates, dominated by lymphocytes, could be observed in both lungs. Multinucleated syncytial cells with atypical enlarged pneumocytes characterized by large nuclei, amphophilic granular cytoplasm, and prominent nucleoli were identified in the intra-alveolar spaces, indicating viral cytopathic-like changes. According to Jin et al (Viruses 2020, 12, 372), these pulmonary pathological findings extremely resemble those seen in SARS and MERS. Moderate microvascular steatosis and mild lobular and portal activity were observed in liver biopsy specimens, which might be caused by either SARS- CoV-2 infection or drug use. In addition, only a few interstitial mononuclear inflammatory infiltrates were found in the heart tissue, which means that SARS-CoV-2 might not directly impair the heart. Massive mucus secretion in both lungs was found in death cases with COVID-19, which was different from SARS and MERS. Acute Respiratory Distress Syndrome (ARDS) is a life-threatening lung condition that prevents enough oxygen from getting to the lungs and into the circulation, accounting for mortality of most respiratory disorders and acute lung injury. In fatal cases of human SARS-CoV, MERS-CoV, and SARS-CoV-2 infections, individuals exhibit severe respiratory distress requiring mechanical ventilation, and the histopathology findings also support ARDS. Immune dysfunction and cytokine storms were also seen in COVID-19 induced fatalities.

Prognosis and risk assessment:

As used herein, the term “requires intensifying treatment and/or disease monitoring” or “adverse respiratory and/or infectious clinical outcome requiring oxygen support and intensified treatment” therefore refers to a patient with a higher risk of the disease worsening, i.e. a higher risk of progressing to a more severe health-risk to the patient compared to a patient with a low risk, considered preferably in relation to the description of pathogenesis described above.

As used herein, the term “does not require intensifying treatment and/or disease monitoring” or “not at risk of an adverse respiratory and/or infectious clinical outcome requiring oxygen support and intensified treatment” relates to a prognostic statement indicating low risk of an adverse event and thus the lack of need for such treatment and/or monitoring. The term relates in essence to an effective “rule-out statement” with a sufficient negative predictive value indicating a low risk of adverse events, preferably as described herein, preferably within 28 days.

As used herein, the term “at risk of a disease progression to a condition that requires hospitalization” relates to patients that require intensifying treatment and/or disease monitoring, whereby the treatment options available at a hospital are advisable on recommendation of medical staff. In other words, the risk is assessed as such, that the treatments typically available outside of hospital are insufficient to safely care for the patient and avoid unwanted adverse events.

As used herein, the term “not at risk of disease progression to a condition that requires hospitalization” relates to a prognostic statement indicating low risk of a disease occurring at a level that would require hospitalization, and thus the lack of need hospitalization. The term relates in essence to an effective “rule-out statement” with a sufficient negative predictive value indicating a low risk of adverse events, preferably as described herein, preferably within 28 days.

As used herein, the term “at risk of a disease progression to a condition that requires treatment and/or disease monitoring on an ICU” relates to patients that require intensifying treatment and/or disease monitoring, whereby the treatment options available at the ICU are advisable on recommendation of medical staff. In other words, the risk is assessed as such, that the treatments typically available outside of the ICU are insufficient to safely care for the patient and avoid unwanted adverse events. As used herein, the term “not at risk of a disease progression to a condition that requires treatment and/or disease monitoring on an ICU” relates to a prognostic statement indicating low risk of a disease occurring at a level that would require ICU admission, and thus the lack of need of ICU treatment. The term relates in essence to an effective “rule-out statement” with a sufficient negative predictive value indicating a low risk of adverse events, preferably as described herein, preferably within 28 days.

According to the invention, therapeutic interventions or therapeutics can also comprise Type 1 Interferons (IFN), IFN-a, IFN-[3, immunosuppressive drugs, Ribavirin, Remdesivir, Lopinavir, Nelfinavir, Chloroquine, convalescent plasma, anti-SARS CoV antibodies, anti-Fc specific antibodies, renin-angiotensin system (RAS) inhibitors, intravenous transplantation of ACE2- mesenchymal stem cells (MSCs), anti-inflammatory drugs, Tumor necrosis factor- a blocker, proteinase inhibitors, steroids, vaccination or other therapies that reduces or prevent a negative outcome or an adverse event or death of the patient.

The patient described herein who has been diagnosed as being “critically ill” and requiring oxygen support can be diagnosed as an intensive care unit (ICU) patient, a patient who requires constant and/or intense observation of his health state, a patient diagnosed with SARS, with SARS and sepsis, severe sepsis or septic shock, a patient diagnosed with an SARS and one or more existing organ failure(s), a pre- or post-surgical patient, an intraoperative patient, a posttraumatic patient, a trauma patient, such as an accident patient, a burn patient, a patient with one or more open lesions. The subject described herein can be at the emergency department or intensive care unit, or in other point of care settings, such as in an emergency transporter, such as an ambulance, or at a general practitioner, who is confronted with a patient with said symptoms.

“Prognosis” relates to the prediction of an outcome or a specific risk for a subject (high or low risk). This may also include an estimation of the chance of recovery or the chance of an adverse outcome for said subject. In embodiments, the prognosis can be made from one or more biomarker determinations to a timeframe, enabling prognostic statements regarding outcome within 28 days after the first patient sample collection.

The methods of the invention may also be used for monitoring, therapy monitoring, therapy guidance and/or therapy control. “Monitoring” relates to keeping track of a patient and potentially occurring complications, e.g. to analyze the progression of the healing process or the influence of a particular treatment or therapy on the health state of the patient.

The term “therapy monitoring” or “therapy control” in the context of the present invention refers to the monitoring and/or adjustment of a therapeutic treatment of said patient, for example by obtaining feedback on the efficacy of the therapy. As used herein, the term “therapy guidance” or “therapy management” refers to application of certain therapies, therapeutic actions or medical interventions based on the value/level of one or more biomarkers and/or clinical parameter and/or clinical scores. This includes the adjustment of a therapy or the discontinuation of a therapy.

In the present invention, the terms “risk assessment” and “risk stratification” relate to the grouping of subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for applying preventive and/or therapeutic measures. The term “therapy stratification” in particular relates to grouping or classifying patients into different groups, such as risk groups or therapy groups that receive certain differential therapeutic measures depending on their classification. The term “therapy stratification” also relates to grouping or classifying patients with infections or having symptoms of an infectious disease into a group that are not in need to receive certain therapeutic measures.

Biomarkers:

As used herein, terms such as “marker”, “surrogate”, “prognostic marker”, “factor” or “biomarker” or “biological marker” are used interchangeably and relate to measurable and quantifiable biological markers (e.g., specific protein or enzyme concentration or a fragment thereof, specific hormone concentration or a fragment thereof, or presence of biological substances or a fragment thereof) which serve as indices for health- and physiology-related assessments, such as a disease/disorder/clinical condition risk, preferably an adverse event. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers may be measured in a sample (as a blood, plasma, urine, or tissue test).

The term “endothelial biomarker”, “endothelial marker”, “endothelial function biomarker” or “endothelial function marker” refers to a biomarker that is associated with endothelial function and structure and the level of which is indicative of physiological function or dysfunction of the endothelium of a subject. Endothelial dysfunction is a pathological state of the endothelium associated with impaired bioavailability of nitric oxide and characterized by vasoconstriction, increased vascular permeability, thrombosis, and inflammation. Biomarkers associated with endothelial dysfunction (endothelial biomarkers) include without limitation pro ADM or fragment(s) thereof of such as MR-proADM, sFlt-1 and further biomarkers such as ET1 or fragment(s) 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, syndecan-1 , and vascular endothelial growth factor (VEGF).

SFlt-1:

As used herein, the term "soluble Flt-1 (sFlt-1)" (soluble fms-like tyrosine kinase 1 , also known as sVEGF-RI) refers to the soluble form of the Flt-1 receptor, that is homologous to the protein defined by GenBank accession number U01134 or UniProt P17948 or entry name VGFR1_HUMAN and that has sFlt-1 biological activity. The biological activity of an sFlt-1 polypeptide may be assayed using any standard method, for example, by assaying sFlt-1 binding to VEGF. sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-1 can bind to VEGF and P1GF bind with high affinity, but it cannot induce proliferation or angiogenesis and is therefore functionally different from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical endothelial cells and later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1 family member or isoform.

Methods for the determination of sFlt-1 are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific / B R A H M S GmbH. CT-proET-1:

Endothelin-1 (ET-1), a 21 amino acid peptide, is a strong vasoconstrictor. Since its discovery in 1988, its biosynthesis, mode of action and association with disease has been extensively studied. There are three different genes encoding isoforms of endothelin (endothelin-1 , Endothelin-2, endothelin-3), of which endothelin-1 is found in the highest concentrations and is most effective. Endothelin-1 is found in endothelial cells, in the lungs, in the heart, synthesized in the kidney and brain. The primary translation product of the human endothelin-1 gene is a 212 amino acidcomprising peptide (see for example GenBank accession number: AAA52339.1), termed prepro- endothelin-1 (preproendothelin). In the secretion process, a short N-terminal signal sequence (amino acids 1-17) of the preproendothelin is removed by the signal peptidase. The proendothelin obtained is then processed by the protease furin on dibasic amino acid pairs to give a biologically inactive peptide comprising 38 amino acids, big endothelin, from which finally the mature, biologically active endothelin-1 is formed by means of endothelin-converting enzymes (ECEs). In addition, the C-terminal proendothelin fragment (CT-proET-1) has the amino acid sequences 93- 212 or 168-212 of the preproendothelin.

Endothelin typically acts via the bond to specific receptors which are localized on muscle cells, myocytes and fibroblasts. This bond leads to efflux of calcium, activation of phospholipase C and inhibition of Na/K ATPase. In addition to the vasoconstrictive effect, endothelin also has growthregulating properties. Elevated plasma concentrations of endothelin-1 and big endothelin have been described for various clinical indications. These include cardiovascular disease (inter alia pulmonary hypertension, atherosclerosis, congestive heart failure, myocardial infarction), sepsis and septic shock, and cancer.

Methods for the determination of CT-proET-1 are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific / B R A H M S GmbH.

ADM:

It is understood that in the context of the present invention “determining the level of proADM or fragment(s) thereof’ or the like refers to any means of determining proADM or a fragment thereof. The fragment can have any length, e.g. at least about 5, 10, 20, 30, 40, 50 or 100 amino acids, so long as the fragment allows the unambiguous determination of the level of proADM or fragment thereof.

In particular preferred aspects of the invention, “determining the level of proADM” refers to determining the level of midregional proadrenomedullin (MR-proADM). MR-proADM is a fragment and/or region of proADM.

The peptide adrenomedullin (ADM) was discovered as a hypotensive peptide comprising 52 amino acids, which had been isolated from a human phenochromocytome (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a precursor peptide comprising 185 amino acids (“preproadrenomedullin” or “pre proADM”). An exemplary amino acid sequence is given in SEQ ID NO: 1.

SEQ ID NO:1 : amino acid sequence of pre-pro-ADM: 1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

51 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

ADM comprises the positions 95-146 of the pre-proADM amino acid sequence and is a splice product thereof. “Proadrenomedullin” (“proADM”) refers to pre-proADM without the signal sequence (amino acids 1 to 21), i.e. to amino acid residues 22 to 185 of pre-proADM.

“Midregional proadrenomedullin” (“MR-proADM”) refers to the amino acids 42 to 95 of pre- proADM. An exemplary amino acid sequence of MR-proADM is given in SEQ ID NO: 2.

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

ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

It is also envisaged herein that a peptide and fragment thereof of pre-proADM or MR-proADM can be used for the herein described methods. For example, the peptide or the fragment thereof can comprise the amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre- proADM (mature adrenomedullin, including the biologically active form, also known as bio-ADM).

SEQ ID NO:3: amino acid sequence of mature ADM (AS 95-146 of pre-pro-ADM):

YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY

A C-terminal fragment of proADM (amino acids 153 to 185 of pre proADM) is called adrenotensin. Fragments of the proADM peptides or fragments of the MR-proADM can comprise, for example, at least about 5, 10, 20, 30 or more amino acids. Accordingly, the fragment of proADM may, for example, be selected from the group consisting of MR-proADM, PAMP, adrenotensin and mature adrenomedullin, preferably herein the fragment is MR-proADM. In embodiments the fragment of proADM may, be selected from the group consisting of MR-proADM, PAMP and adrenotensin.

The determination of these various forms of ADM or proADM and fragments thereof also encompass measuring and/or detecting specific sub-regions of these molecules, for example by employing antibodies or other affinity reagents directed against a particular portion of the molecules, or by determining the presence and/or quantity of the molecules by measuring a portion of the protein using mass spectrometry. Any one or more of the “ADM peptides or fragments” described herein may be employed in the present invention.

Accordingly, the methods and kits of the present invention can also comprise determining at least one further biomarker, marker and/or parameter in addition to ADM.

Methods for the determination of proADM, in particular MR-proADM, are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific / B R A H M S GmbH.

PCT:

As used herein, “procalcitonin” or “PCT” relates to a peptide spanning amino acid residues 1-116, 2-116, 3-116, or fragments thereof, of the procalcitonin peptide. PCT is a peptide precursor of the hormone calcitonin. Thus the length of procalcitonin fragments is at least 12 amino acids, preferably more than 50 amino acids, more preferably more than 110 amino acids. PCT may comprise post-translational modifications such as glycosylation, liposidation or derivatization. Procalcitonin is a precursor of calcitonin and katacalcin. Thus, under normal conditions the PCT levels in the circulation are very low (< about 0.05 ng/ml).

The level of PCT in the sample of the subject can be determined by immunoassays as described herein. As used herein, the level of ribonucleic acid or deoxyribonucleic acids encoding “procalcitonin” or “PCT” can also be determined. Methods for the determination of PCT are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific I B R A H M S GmbH.

Other biomarkers:

Accordingly, the methods and kits of the present invention can also comprise determining at least one further biomarker, marker, clinical score and/or parameter.

As used herein, a parameter is a characteristic, feature, or measurable factor that can help in defining a particular system. A parameter is an important element for health- and physiology- related assessments, such as a disease/disorder/clinical condition risk, preferably organ dysfunction(s). Furthermore, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. An exemplary parameter can be selected from the group consisting of Pneumonia Severity Index (PSI), Acute Physiology and Chronic Health Evaluation II (APACHE II), the simplified acute physiology score (SAPSII score), sequential organ failure assessment score (SOFA score), quick sequential organ failure assessment score (qSOFA), body mass index, weight, age, sex, IGS II, liquid intake, white blood cell count, sodium, potassium, temperature, blood pressure, dopamine, bilirubin, respiratory rate, partial pressure of oxygen, World Federation of Neurosurgical Societies (WFNS) grading, Glasgow Coma Scale (GCS), CURB-65 pneumonia severity score, Pneumonia Severity Index (PSI), age, gender, family history, ethnicity, body weight, body mass index (BMI), cystoscopy report, white blood cell count, lymphocyte count, imaging methods as such as CT scan, PET imaging or X-ray, blood pressure, heart rate, antihypertensive treatment, liquid intake, wheezing, body temperature, presence of diabetes mellitus, blood gluocose levels, and (current) smoking habits.

Such parameters may additionally be assessed in combination with the methods described herein in order to improve assay implementation and diagnostic statements.

The present invention has the following advantages over the conventional methods: the inventive methods and the kits are fast, objective, easy to use and precise. The methods and kits of the invention relate to markers and clinical scores that are easily measurable in routine methods, because the levels of proADM, PCT, CT-proET-1 , sFlt-1 or IL-6 can be determined in routinely obtained blood samples or further biological fluids or samples obtained from a subject.

D-Dimer is not normally present in human blood plasma. This biomarker is one of the Fibrinogen degradation products (FDP) which is released after a thrombus or blood clot is enzymatically degraded by Plasmin. Below a concentration range (e.g. 0,5 mg/l in a blood sample), D-Dimer can help to rule out clinical conditions that are characterized by inappropriate blood clot formation such as deep vein thrombosis, pulmonary embolism or disseminated intravascular coagulation. In case of increased levels of D-Dimer, further testing (such as ultrasound, scintigraphy, CT scanning) is required (see 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).

C-reactive protein (CRP) is a pentameric protein, which can be found in bodily fluids such as blood plasma. CRP levels can rise in response to inflammation. Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments.

Interleukin 6 (IL-6) is promptly and transiently produced in response to infections and tissue injuries, and contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Although its expression is controlled by transcriptional and posttranscriptional mechanisms, dysregulated continual synthesis of IL-6 plays a pathological effect on chronic inflammation and autoimmunity. IL-6 expression is associated with coronavirus infection and may predict the severity of COVID- 190 .

The at least one further marker and/or parameter of said subject can be selected from the group consisting of a level of lactate in said sample, the sequential organ failure assessment score (SOFA score) of said subject, optionally the quick SOFA score, the simplified acute physiology score (SAPSII) of said subject, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score of said subject and a level of the soluble fms-like tyrosine kinase-1 (sFlt-1), Histone H2A, Histone H2B, Histone H3, Histone H4, calcitonin, Endothelin-1 (ET-1), Arginine Vasopressin (AVP), Atrial Natriuretic Peptide (ANP), Neutrophil Gelatinase-Associated Lipocalin (NGAL), Delta-like protein 1 (DLL1), Troponin, Brain Natriuretic Peptide (BNP), C-Reactive Protein (CRP), Pancreatic Stone Protein (PSP), Triggering Receptor Expressed on Myeloid Cells 1 (TREM1), Endothelial cell-specific molecule 1 (ESM-1), Growth-Differentiation Factor-15 (GDF-15), lnterleukin-6 (IL-6), lnterleukin-1 , Interleukin-24 (IL-24), Interleukin-22 (IL-22), Interleukin (IL-20) other ILs, Presepsin (sCD14-ST), Lipopolysaccharide Binding Protein (LBP), Alpha-1 -Antitrypsin, Matrix Metalloproteinase 2 (MMP2), Metalloproteinase 2 (MMP8), Matrix Metalloproteinase 9 (MMP9), Matrix Metalloproteinase 7 (MMP7, Placental growth factor (PIGF), Chromogranin A, S100A protein, SWOB protein and Tumor Necrosis Factor a (TNFa), Insulin-like growth factorbinding protein 7 (= IGFBP7), Neopterin, soluble Triggering receptor expressed on myeloid cells- 1 (STREM-1), Alpha-1 -Antitrypsin, pro-arginine vasopressin (AVP, proAVP or Copeptin), procalcitonin, atrial natriuretic peptide (ANP, pro-ANP), Endothelin-1 , CCL1/TCA3, CCL11 , CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2/MCP-1 , CCL20, CCL21 , CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 /LAG-1 , CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1 , CXCL1 , CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2/MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7/Ppbp, CXCL9, IL8/CXCL8, XCL1 , XCL2, FAM19A1 , FAM19A2, FAM19A3, FAM19A4, FAM19A5, CLCF1 , CNTF, IL11 , IL31 , IL6, Leptin, LIF, OSM, IFNA1 , IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1 , IFNE, IFNG, IFNZ, IFNA8, IFNA5/IFNaG, IFNw/IFNW1 , BAFF, 4-1 BBL, TNFSF8, CD40LG, CD70, CD95L/CD178, EDA-A1 , TNFSF14, LTA/TNFB, LTB, TNFa, TNFSF10, TNFSF11 , TNFSF12, TNFSF13, TNFSF15, TNFSF4, TRAIL, IP-10, IL18, IL18BP, IL1 A, IL1 B, IL1 F10, IL1 F3/IL1 RA, IL1 F5, IL1 F6, IL1 F7, IL1 F8, IL1 RL2, IL1 F9, IL33 or a fragment thereof. Further markers comprise membrane microparticle, platelet count, mean platelet volume (MPV), sCD14-ST, prothrombinase, antithrombin and/ antithrombin activity, cationic protein 18 (CAP18), von Willebrand factor (vWF)- cleaving proteases, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1 , GPIIb-llla, non-denatured D-dimer of fibrin, platelet factor 4, histones and a PT-Assay.

As used herein, the term “sample” is a biological sample that is obtained or isolated from the patient or subject. “Sample” as used herein may, e.g., refer to a sample of bodily fluid or tissue obtained for the purpose of analysis, diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. Preferably herein, the sample is a sample of a bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusions, cells, a cellular extract, a tissue sample, any tissue sample from the upper or lower respiratory tract, a tissue biopsy, a stool sample and the like. Particularly, the sample is blood, blood plasma, blood serum.

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

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

Oxygen support:

The term “oxygen support” or “supplemental oxygen” refers to administering additional oxygen to a patient, whereby “additional” refers to the administration of oxygen in addition to the amount of oxygen inhaled by the patient without any additional treatment. In embodiments a patient is in need of oxygen support, when having an arterial oxygen saturation in the blood below 95%, preferably below 92%. Symptoms of an arterial oxygen saturation below 95% include without limitation bluish tint to fingernails or lips, coughing, confusion, fatigue, shortness of breath and wheezing. The term “oxygen saturation” (SO2) refers to the fraction of oxygen-saturated haemoglobin relative to total haemoglobin in the blood. The oxygen saturation may be determined in the arterial blood (arterial oxygen saturation, SaO2, for example by an arterial blood gas test), the venous blood (venous blood saturation, SVO2) and the peripheral blood (peripheral blood saturation, SpO2, such as by pulse oximetry) A person skilled in the art is aware of further means for determining the oxygen saturation in blood. “Oxygen support” or supplemental oxygen” can be administered at different concentrations using various types of delivery systems. Two main varieties of delivery system include a low-flow system (administered by a nasal cannula, face masks, or non-rebreathing masks) and a high-flow system (administered by a high-flow nasal cannula, Venturi mask, or a continuous positive airway pressure system). In embodiments a low-flow system comprises administration of equal or below 6 L/min oxygen. In one embodiment a high flow system comprises the administration of >6 -to <10 L/min oxygen. Supplemental oxygen may also be administered during mechanical ventilation such as by bypassing the airway, such as in ECMO therapy.

Infection:

As used herein, “infection” within the scope of the invention means a pathological process caused by the invasion of normally sterile tissue or fluid by pathogenic or potentially pathogenic agents/ pathogens, organisms and/or microorganisms, and relates preferably to infection(s) by bacteria, viruses, fungi, and/or parasites. In the context of the invention, infection relates in particular to viral infections with a SARS-virus. Infection can also be a combination of infections including a SARS-virus infection, such as a SARS-CoV2 infection, and one or more additional infections, such as a simultaneously or subsequently occurring infection or an infection that was already present at the time of SARS-virus infection, wherein the additional infection can be a bacterial infection, viral infection, and/or fungal infection. Infections with more than one additional pathogen can also occur, including at least one SARS-virus, and additionally one or more bacteria, viruses or fungi. For a triple infection with SARS-virus, a bacterium and a fungus can be envisioned. The infection can be a local or systemic infection. For the purposes of the invention, a viral infection may be considered as infection by a microorganism.

As used herein ..infectious disease" comprises a viral infectious diseases or disorders that are associated with a primary viral infection, in particular a SARS-virus infection. In the context of the invention, the infectious disease can comprise in addition to the SARS-virus infection an infection with one or more bacteria and/or viruses and/or fungi.

Furthermore, the infection-related complication can be a “nosocomial” infection. Nosocomial infections are also called hospital-acquired infections (HAI) are infections that are acquired in a hospital or other health care facility. To emphasize both hospital and nonhospital settings, it is sometimes instead called a health care-associated infection (HAI or HCAI). Such an infection can be acquired in hospital, nursing home, rehabilitation facility, outpatient clinic, or other clinical settings. Nosocomial infection may be spread to the susceptible patient in the clinical setting by various means. Health care staff can spread infection, in addition to contaminated equipment, bed linens, or air droplets. The infection can originate from the outside environment, another infected patient, staff that may be infected, or in some cases, the source of the infection cannot be determined. In some cases the microorganism originates from the patient's own skin microbiota, becoming opportunistic after surgery or other procedures that compromise the protective skin barrier. Though the patient may have contracted the infection from their own skin, the infection is still considered nosocomial since it develops in the health care setting.

Further, the subject suffering from an infection can suffer from more than one source(s) of infection simultaneously. For example, the subject suffering from an infection can suffer from a bacterial infection and viral infection; from a viral infection and fungal infection; from a bacterial and fungal infection, and from a bacterial infection, fungal infection and viral infection, or suffer from a mixed infection comprising one or more of the infections listed herein, including potentially a superinfection, for example one or more bacterial infections in addition to one or more viral infections and/or one or more fungal infections.

Infection grades can be classified as follows:

Regarding Procalcitonin, increased biomarker level, especially higher or equal 0.25ng/ml or 0.5 ng/ml are connected to >= grade 3 infections, that typically occur in addition to the Covid-19 infection, wherein antimicrobial treatment and hospitalization are required.

The COVID-19 ordinal scale is defined as follows:

Measurement methods:

According to the present invention sFlt-1 , CT-proET, proADM and PCT and/or other markers or clinical scores are employed as markers for the diagnosis, prognosis, prediction, risk assessment and/or risk stratification of a patient in developing a medical condition that requires treatment in hospital who presents with symptoms of an infectious disease.

A skilled person is capable of obtaining or developing means for the identification, measurement, determination and/or quantification of any one of the above molecules, or fragments or variants thereof, as well as the other markers of the present invention according to standard molecular biological practice.

The level of the one or more biomarkers of the present invention can be determined by any assay that reliably determines the concentration of the marker. Particularly, mass spectrometry (MS) and/or immunoassays can be employed as exemplified in the appended examples. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of a macromolecule/polypeptide in a solution through the use of an antibody or antibody binding fragment or immunoglobulin. Methods of determining proADM or other the markers such as sFlt-1 , CT-proET-1 or PCT used in the context of the present invention are intended in the present invention. By way of example, a method may be employed selected from the group consisting of mass spectrometry (MS), luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence- and fluorescence- immunoassays, enzyme immunoassay (EIA), Enzyme-linked immunoassays (ELISA), luminescence-based bead arrays, magnetic beads based arrays, protein microarray assays, rapid test formats such as for instance immunochromatographic strip tests, rare cryptate assay, and automated systems/analyzers.

Determination of the one or more biomarkers based on antibody recognition is a preferred embodiment of the invention. As used herein, the term, "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immuno reacts with) an antigen. According to the invention, the antibodies may be monoclonal as well as polyclonal antibodies.

An antibody is considered to be specific, if its affinity towards the molecule of interest, is at least 50-fold higher, preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to develop and to select antibodies with a given specificity. In the context of the invention, monoclonal antibodies are preferred. The antibody or the antibody binding fragment binds specifically to the herein defined markers or fragments thereof. In particular, the antibody or the antibody binding fragment binds to the herein defined biomarkers.

Exemplary immunoassays can be luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence- and fluorescence- immunoassays, enzyme immunoassay (EIA), Enzyme- linked immunoassays (ELISA), luminescence-based bead arrays, magnetic beads based arrays, protein microarray assays, rapid test formats, rare cryptate assay. Further, assays suitable for point-of-care testing and rapid test formats such as for instance immune-chromatographic strip tests can be employed. Automated immunoassays are also intended, such as the KRYPTOR assay.

Alternatively, instead of antibodies, other capture molecules or molecular scaffolds that specifically and/or selectively recognize the one or more biomarkers of the present invention may be encompassed by the scope of the present invention. Herein, the term “capture molecules” or “molecular scaffolds” comprises molecules which may be used to bind target molecules or molecules of interest, i.e. analytes from a sample. Capture molecules must thus be shaped adequately, both spatially and in terms of surface features, such as surface charge, hydrophobicity, hydrophilicity, presence or absence of lewis donors and/or acceptors, to specifically bind the target molecules or molecules of interest. Hereby, the binding may, for instance, be mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions or a combination of two or more of the aforementioned interactions or covalent interactions between the capture molecules or molecular scaffold and the target molecules or molecules of interest. In the context of the present invention, capture molecules or molecular scaffolds may for instance be selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, a peptide and a glycoprotein. Capture molecules or molecular scaffolds include, for example, aptamers, DARpins (Designed Ankyrin Repeat Proteins). Affimers and the like are included.

Preferably, one of the antibodies can be labeled and the other antibody can be bound to a solid phase or can be bound selectively to a solid phase. In a particularly preferred aspect of the assay, one of the antibodies is labeled while the other is either bound to a solid phase or can be bound selectively to a solid phase. The first antibody and the second antibody can be present dispersed in a liquid reaction mixture, and wherein a first labeling component which is part of a labeling system based on fluorescence or chemiluminescence extinction or amplification is bound to the first antibody, and a second labeling component of said labeling system is bound to the second antibody so that, after binding of both antibodies to said biomarkers or fragments thereof to be detected, a measurable signal which permits detection of the resulting sandwich complexes in the measuring solution is generated. The labeling system can comprise a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

In a preferred embodiment, the method is executed as heterogeneous sandwich immunoassay, wherein one of the antibodies is immobilized on an arbitrarily chosen solid phase, for example, the walls of coated test tubes (e.g. polystyrol test tubes; coated tubes; CT) or microtiter plates, for example composed of polystyrol, or to particles, such as for instance magnetic particles, whereby the other antibody has a group resembling a detectable label or enabling for selective attachment to a label, and which serves the detection of the formed sandwich structures. A temporarily delayed or subsequent immobilization using suitable solid phases is also possible.

The method according to the present invention can furthermore be embodied as a homogeneous method, wherein the sandwich complexes formed by the antibody/antibodies and the marker or a fragment thereof, which is to be detected remains suspended in the liquid phase. In this case it is preferred, that when two antibodies are used, both antibodies are labeled with parts of a detection system, which leads to generation of a signal or triggering of a signal if both antibodies are integrated into a single sandwich. Such techniques are to be embodied in particular as fluorescence enhancing or fluorescence quenching detection methods. A particularly preferred aspect relates to the use of detection reagents which are to be used pair-wise, such as for example the ones which are described in US4882733, EP0180492 or EP0539477 and the prior art cited therein. In this way, measurements in which only reaction products comprising both labeling components in a single immune-complex directly in the reaction mixture are detected, become possible. For example, such technologies are offered under the brand names TRACE™ (Time Resolved Amplified Cryptate Emission) or KRYPTOR™, implementing the teachings of the above-cited applications. Therefore, in particular preferred aspects, a diagnostic device is used to carry out the herein provided method. For example, the level of SFLt-1 or fragments thereof and/or the level of any further marker of the herein provided method, is determined. In particular preferred aspects, the diagnostic device is a B R A H M S KRYPTOR analyzer.

The level of the marker of the present invention, e.g. the sFlt-1 or fragments thereof, CT-proET1 or fragments thereof, or other markers, can also be determined by a mass spectrometric (MS) based methods. Such a method may comprise detecting the presence, amount or concentration of one or more modified or unmodified fragment peptides of e.g. sFlt-1 or the CT-proET 1 in said biological sample or a protein digest (e.g. tryptic digest) from said sample, and optionally separating the sample with chromatographic methods, and subjecting the prepared and optionally separated sample to MS analysis. For example, selected reaction monitoring (SRM), multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) mass spectrometry may be used in the MS analysis.

Herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. In order to enhance the mass resolving and mass determining capabilities of mass spectrometry, the samples can be processed prior to MS analysis. Accordingly, the invention relates to MS detection methods that can be combined with immunoenrichment technologies, methods related to 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 comprise techniques for lysis, fractionation, digestion of the sample into peptides, depletion, enrichment, dialysis, desalting, alkylation and/or peptide reduction.

However, these steps are optional. The selective detection of analyte ions may be conducted with tandem mass spectrometry (MS/MS). Tandem mass spectrometry is characterized by mass selection step (as used herein, the term “mass selection” denotes isolation of ions having a specified m/z or narrow range of m/z's), followed by fragmentation of the selected ions and mass analysis of the resultant product (fragment) ions.

The skilled person is aware how quantify the level of a marker in the sample by mass spectrometric methods. For example, relative quantification “rSRM” or absolute quantification can be employed as described above.

Moreover, the levels (including reference levels) can be determined by mass spectrometric based methods, such as methods determining the relative quantification or determining the absolute quantification of the protein or fragment thereof of interest.

Kits:

The invention further relates to kits, the use of the kits and methods wherein such kits are used.

The kit may additionally comprise items useful for obtaining a sample, such as a blood sample, for example the kit may comprise a container, wherein said container comprises a device for attachment of said container to a cannula or syringe, is a syringe suitable for blood isolation, exhibits an internal pressure less than atmospheric pressure, such as is suitable for drawing a pre-determined volume of sample into said container, and/or comprises additionally detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, and mixtures thereof, and/or A filter system containing nitro-cellulose, silica matrix, ferromagnetic spheres, a cup retrieve spill over, trehalose, fructose, lactose, mannose, poly-ethylen-glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, anilin, pyrol, citrate, and mixtures thereof.

As used herein, the “detection reagent” or the like are reagents that are suitable to determine the herein described marker(s), e.g. of sFlt-1 , CT-proET1 and/or MR-proADM. Such exemplary detection reagents are, for example, ligands, e.g. antibodies or fragments thereof, which specifically bind to the peptide or epitopes of the herein described marker(s). Such ligands might be used in immunoassays as described above. Further reagents that are employed in the immunoassays to determine the level of the marker(s) may also be comprised in the kit and are herein considered as detection reagents. Detection reagents can also relate to reagents that are employed to detect the markers or fragments thereof by MS based methods. Such detection reagent can thus also be reagents, e.g. enzymes, chemicals, buffers, etc, that are used to prepare the sample for the MS analysis. A mass spectrometer can also be considered as a detection reagent. Detection reagents according to the invention can also be calibration solution(s), e.g. which can be employed to determine and compare the level of the marker(s).

The methods of the present invention may in embodiments be partially computer-implemented. For example, the step of comparing the detected level of a biomarker, e.g. the sFlt-1 or fragments thereof, with a reference level can be performed in a computer system. In the computer-system, the determined level of the biomarker(s) can be combined with other biomarker levels and/or clinical parameters of the subject in order to calculate a score, which is indicative for the prognosis, risk assessment and/or risk stratification. For example, the determined values may be entered (either manually by a health professional or automatically from the device(s) in which the respective marker level(s) has/have been determined) into the computer-system. The computersystem can be directly at the point-of-care (e.g. primary care, hospital or home setting) or it can be at a remote location connected via a computer network (e.g. via the internet, or specialized medical cloud-systems, optionally combinable with other IT-systems or platforms such as hospital information systems (HIS)). Typically, the computer-system will store the values (e.g. biomarker level or clinical parameters such as age, blood pressure, weight, BMI, sex or Scores (e.g. The Covid-19 ordinal scale or the infection grade) etc. on a computer-readable medium and calculate the score based-on pre-defined and/or pre-stored reference levels or reference values. The resulting score will be displayed and/or printed for the user (typically a health professional such as a physician or the patient). Alternatively or in addition, the associated prognosis, assessment, treatment guidance, patient management guidance or stratification will be displayed and/or printed for the user (typically a health professional such as a physician or the patient).

In one embodiment of the invention, a software system can be employed, in which a machine learning algorithm is evident, preferably to identify patients at risk for PE using data from electronic health records (EHRs). A machine learning approach can be trained on a random forest classifier using EHR data (such as labs, biomarker expression, vitals, and demographics) from patients. Machine learning is a type of artificial intelligence that provides computers with the ability to learn complex patterns in data without being explicitly programmed, unlike simpler rulebased systems. Earlier studies have used electronic health record data to trigger alerts to detect clinical deterioration in general. In one embodiment of the invention the processing of sFlt-1 levels may be incorporated into appropriate software for comparison to existing data sets, for example sFlt-1 , CT-proET-1 , proADM or PCT levels may also be processed in machine learning software to assist in prognosing the risk of adverse outcomes.

Statistical measures: Various statistical measures may be employed to provide a level of statistical significance or statistical likelihood or certainty for any given correlation observed in the data, for example demonstrating that the observation is not the result of chance but based on an underlying biological correlation. For example, the positive and negative predictive values (PPV and NPV respectively) are the proportions of positive and negative results in statistics and diagnostic tests that are true positive and true negative results, respectively. The PPV and NPV describe the performance of a diagnostic test or other statistical measure. A high result can be interpreted as indicating the accuracy of such a statistic.

Positive predictive value: According to Emerg (Tehran). 2015 Summer; 3(3): 87-88, the positive predictive value (PPV) is the proportion of cases giving positive test results who are already patients. It is the ratio of patients truly diagnosed as positive to all those who had positive test results (including healthy subjects who were incorrectly diagnosed as patient). This characteristic can predict how likely it is for someone to truly be patient, in case of a positive test result.

Positive predictive value =TP / TP+FP

Negative predictive value: According to Emerg (Tehran). 2015 Summer; 3(3): 87-88, the negative predictive value is the proportion of the cases giving negative test results who are already healthy. It is the ratio of subjects truly diagnosed as negative to all those who had negative test results (including patients who were incorrectly diagnosed as healthy). This characteristic can predict how likely it is for someone to truly be healthy, in case of a negative test result.

Negative predictive value = TN / TN+FN

Therefore a 100% NPV shows no false negative rate. A biomarker or a ratio of marker values with a NPV of 100% differentiate all patients without an adverse outcome correctly in the low risk group. A biomarker or a ratio of marker values with a NPV of 100% differentiates all patients without the need of oxygen support correctly from those in need of oxygen support.

A “rule-in” typically identifies a minimal proportion of subjects that with a high certainty will develop the disease or disorder and ensure that this true positive group has a sufficiently large proportion of the subjects testing positive. The test must reach a minimal sensitivity and minimal positive predictive value (PPV).

A “rule-out” typically identifies a minimal proportion of subjects that will with a high certainly not develop the disease or disorder and ensure that of the subjects testing negative, sufficiently few will develop the disease (false negatives). Such a test must therefore reach a minimal specificity and minimal negative predictive value (NPV).

The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. Sensitivity is equal to the true positive rate and specificity is equal to the true negative rate. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency in "normal" (i.e. apparently healthy individuals not having an adverse outcome and suffered from an adverse outcome, e.g. subjects who died within 28 days, had thrombotic events, were in the need of supplemental oxygen, had additional infection with the need of antimicrobials and hospitalization, were hospitalized or admitted to the ICU for more than 4 days etc..

For any particular marker (like sFlt-1 , CT-proET-1 , proADM, IL6 or PCT), a distribution of marker levels for subjects with and without a disease/condition will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap might indicate where the test cannot distinguish normal from disease. A threshold is selected, below which the test is considered to be abnormal and above which the test is considered to be normal or below or above which the test indicates a specific condition, e.g. infection. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results do not necessarily give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (e.g. 1=low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art; see, e.g., Hanley et al. 1982. Radiology 143: 29-36. Preferably, a threshold is selected to provide a ROC curve area of greater than about 0.5 (<= 0.5 means a test without discriminative power, more preferably greater than about 0.7, still 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 means a test with perfect discrimination). The term "about" in this context refers to +/- 5% of a given measurement.

The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

Receiver Operating Characteristic (ROC) curves and the Concordance (C) statistic (also termed C index) are often used to assess the ability of a risk factor to predict outcome. For example, often a biomarker or risk factor is included in a logistic regression model to predict the likelihood a patient will develop a disease of interest. These predictive probabilities or risks can be examined to see how accurate they are at identifying patients who will develop the disease or not. For example, if the predicted probabilities for the diseased individuals are all higher than the predicted probabilities for the healthy individuals, then we say that the model has perfect discrimination. Discrimination is commonly measured using ROC curves. To construct an ROC curve, the predicted probabilities of the outcome of interest are repeatedly dichotomized into above vs. below a cutoff. For each cutoff, one can estimate the sensitivity (probability that the predicted risk is above the cutoff among patients with the disease) and the specificity (probability that the predicted risk is below the cutoff among patients without the disease). One can vary the cutoff to show a range of sensitivities vs. specificities. If the model has perfect discrimination, the ROC curve should hit the upper left corner of the plot (100% sensitivity and 100% specificity) (Logan, Medical College of Wisconsin, Biostatistics). The area under the ROC curve is a useful measure for summarizing the ROC curve. If a curve is close to the upper left corner (Sensitivity=100%, Specificity=100%), then the area under the ROC curve should be close to 1 . The area under the ROC curve is equivalent to another statistic commonly used to summarize model discrimination, the C statistic or Concordance statistic. The C statistic is interpreted as the probability that a randomly selected subject who experienced the outcome will have a higher predicted probability of having the outcome occur than a randomly selected subject who did not experience the outcome. In addition to computing the area under the ROC curve, this probability can be estimated by taking all pairs of observations where one patient experienced the event and the other did not, and computing the proportion of those pairs where the patient experiencing the event had the higher predicted risk. The C statistic can also be interpreted as the rank correlation between predicted probabilities of the outcome occurring and the observed response (Logan, Medical College of Wisconsin, Biostatistics).

In other embodiments, a ratio data, positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease.

The ratio data is a quantitative data with same properties as interval data, with an equal and definitive ratio between each data. A negative ratio is not possible. A ratio of one explains no change (100% equal). A ratio smaller 1 is connected to a decrease and be expressed in percent or as total number. A ratio of proADM of 0,9 for example relates to a decrease of 10%, often seen in the group without suffering from an adverse outcome. A ratio higher than 1 relates to an increase and can be expressed as percent or as total number as well. A ratio of 1 .4 for example for the sFIt 1 sample values day1 to day 2 relates to a fast slope of 40%.

In the case of a "positive likelihood ratio"", a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a "negative likelihood ratio", a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group. In certain preferred embodiments, markers and/or marker panels are preferably selected to exhibit a positive or negative likelihood ratio of at least about 1 .5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less, still more preferably at least about 5 or more or about 0.2 or less, even more preferably at least about 10 or more or about 0.1 or less, and most preferably at least about 20 or more or about 0.05 or less. The term "about" in this context refers to +/- 5% of a given measurement.

In the case of an "odds ratio", a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In certain preferred embodiments, markers and/or marker panels are preferably selected to exhibit an odds ratio of at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less. The term "about" in this context refers to +/- 5% of a given measurement.

An odds ratio per doubling of concentration is a specific way of expressing the association between a biomarker and an outcome. It quantifies the change in odds of the outcome for every doubling of the biomarker concentration. The odds ratio per doubling of concentration provides a standardized measure that allows for easier comparison across different biomarkers or studies. If the odds ratio per doubling of concentration is, for example, 1 .5, it means that for every doubling of the biomarker concentration, the odds of the outcome occurring increase by a factor of 1 .5. This indicates a positive association between the biomarker and the outcome, if the odds ratio per doubling of concentration is less than 1 , such as 0.8, it means that for every doubling of the biomarker concentration, the odds of the outcome occurring decrease by a factor of 0.8. This indicates a negative association between the biomarker and the outcome.

In the case of a "hazard ratio", a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and "control" groups; a value greater than 1 indicates that the risk is greater in the diseased group; and a value less than 1 indicates that the risk is greater in the control group. In certain preferred embodiments, markers and/or marker panels are preferably selected to exhibit a hazard ratio of at least about 1 .1 or more or about 0.91 or less, more preferably at least about 1 .25 or more or about 0.8 or less, still more preferably at least about 1 .5 or more or about 0.67 or less, even more preferably at least about 2 or more or about 0.5 or less, and most preferably at least about 2.5 or more or about 0.4 or less. The term "about" in this context refers to +/5% of a given measurement.

25% percentile and 75% percentile of the median of biomarker concentration, also known as 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 spread or distribution of biomarker values. The median represents the middle value of a dataset when it is arranged in ascending or descending order. It divides the dataset into two equal parts. To determine the 25th and 75th percentiles in relation to the median, the distribution of biomarker values is considered.

FIGURES

The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention and are provided for greater illustration of the invention described herein.

Brief description of the Pictures:

Figure 1 : ROC curves of biomarkers for predicting mechanical ventilation or death.

Figure 2: Compared performance of biomarkers.

Figure 3: Boxplots showing data for CT-proET1 and MR-proADM.

Figure 4: Boxplots showing data for CT-proET1 and MR-proADM for additional outcomes.

Figure 5: Boxplots showing data for IL6, MR-proADM, PCT and CT-proET1.

Figure 6: Visualization of biomarker change over time for MR-proADM, CT-proET 1 , sFlt-1 . Detailed description of the Figures:

Figure 1 : Receiver operating characteristic curves of biomarkers for predicting mechanical ventilation or death.

Figure 2: Fixed sets of patients were used to fairly compare the performance across biomarkers (difference in performance should not be due to patients excluded for some analyses because biomarker results were missing). 10 patients in ICU at enrollment were excluded from analysis due to the different disease severity compared to patients not in ICU. Day-1 (=Baseline) population (169 patients) Non-ICU patients with available day-1 results of MR-proADM, PCT, CT- proETI , sFlt-1 , CRP, D-Dimer, LDH, Ferritin, WBC, Lymphocytes, IL-6. Day-4 population (126 patients) Patients of day-1 population with available day-4 results of MR-proADM, PCT, CT- proET 1 , sFlt-1 .

Figure 3: Boxplots showing data for (A) CT-proET1 and (B) MR-proADM for the outcomes (endpoints) death within 28 days (death2), admission to ICU during hospitalization or death within 28 days (icu_or_death_bin), mechanical ventilation or death within 28 days (mechvent_death_event).

Figure 4: Boxplots showing data for CT-proET1 (left) and MR-proADM (right) for the outcomes (endpoints) (A) discharge from the hospital before or on day 4 (discharge, D4), (B) composite severity endpoint (death_ICU:mechvent) and (C) discontinuation of supplemental oxygen (off_supp_oxygen_bin) (group „NA“: no supplemental oxygen at randomization).

Figure 5: Boxplots showing data for IL6, MR-proADM, PCT and CT-proET1 (left to right) for the outcomes (endpoints) (A) Infection (grade >= 3) within 28 days (aejnfection) and (B) for CT- proETI for the outcome (endpoint) Day 4 Covid scale =4 vs. <4 (Ord.D4_bin4).

Figure 6: Visualization of biomarker change over time for (A) MR-proADM (nmol/L), (B) CT- proETI (pmol/L), (C) sFlt-1 (pg/mL) for the outcome (endpoint) death within 28 days. Mean sFlt-1 concentration of patients died until day 28 increased from day 1 to day 4, see grey circle in (B). Mean concentration of all available patients at time points 1 ,4,7,14,21 ,28 days and 95% Cl shown. Darker gray: died until day 28, lighter gray: alive on day 28.

EXAMPLES

The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention and are provided for greater illustration of the invention described herein.

Each of the tables below, each row of the tables below and/or any combinations of the tables and/or rows below, represents an embodiment of the present invention.

Example 1 - Summary of study characteristics and data relating to sFlt-1 and CT-proET-1

Background

Several novel biomarkers have been studied in predicting clinical outcomes in patients with

COVID-19. Infection caused by SARS-CoV-2 infection is known to impact endothelial cells. Two biomarkers of interest are the Endothelin-1 (ET-1) and the Soluble fms-like tyrosine kinase-1 (sFlt-1).

Our study aimed to study C-terminal proendothelin-1 (CT-proET-1) and Soluble fms-like tyrosine kinase-1 (sFlt-1) as predictors of clinical outcomes in a COVID-19 patient cohort from a US-based multicenter trial.

The utility of gene signature proteins to assess the direct response of the host immune system to various etiologies, including infections, is an evolving field of interest that holds promise for prognostication and guidance of clinical decision-making.

Study aims:

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

The present invention posits that CT-proET-1 and sFlt-1 hold valuable prognostic capabilities for risk stratification among COVID-19 individuals.

Methods

Data from a multicentric trial was used in this study. Patients with biomarker determinations, and not admitted to the intensive care unit (ICU) on admission, were included. Median concentrations of CT-proET-1 (83.6 pmol/L) and sFlt-1 (81.3 pg/mL) were used as cutoffs in predicting clinical outcomes.

Mixed methods design:

The Institutional Review Board (IRB) at Mass General Brigham approved this study. Collection of Specimens and Assay: EDTA plasma samples were collected, isolated, and aliquoted into cryovials within 2-12 hours of venipuncture. Cryovials were stored at -80°C until they were thawed to be assayed on a B R A H M S KRYPTOR for CT-proET-1 and sFlt-1 concentration determination Statistical analysis: Differences between groups were analyzed using the chi- square or Fisher's exact test for categorical variables and the Mann-Whitney U test for numeric continuous variables. Measures of prognostic performance were sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve. Estimates and 95% confidence intervals were reported. Cl of sensitivity, specificity, PPV, NPV were computed according to Clopper and Pearson. Kaplan-Meier curves were plotted, and the log-rank test was performed, stratified 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). Multiple multivariable logistic regression analyses were performed.

As shown in more detail below, for CT-proET-1 , 83.6 pmol/l was the median concentration; whereas 87.4 pmol/l indicated the endpoint death within 28 days; and 91 .8 pmol/l indicated the endpoint mechanical ventilation within 28 days; and 102.7 pmol/l indicated the endpoint on day 4 of a Covid scale >=4 vs <4.

As shown in more detail below, for sFlt-1 , 81.3 pg/ml was the median concentration; whereas 83.9 pg/ml indicated the endpoint ..improvement of COVID scale (D4 vs D1)” and the endpoint ..discharge on d4”; whereas 88.4 pg/ml indicated death within 28 days, ICU admission and mechanical ventilation.

Study participants:

This study is a prospective observational cohort study, from a COVID-19 multicentric trial, with the following inclusion criteria: Hospitalized adult patients>18 years of age with SARS-CoV-2 infection confirmed by nasopharyngeal swab polymerase chain reaction or serum IgM antibody assay, and presenting with at least two of the following symptoms: fever >38°C, needing supplemental oxygen, or lung infiltrates. Patients who were not already admitted to the ICU at enrollment, and had complete data on all studied biomarkers, were included.

Results

Out of 169 patients, 10.7% mechanically ventilated or dead within 28 days of admission. CT- proET 1 concentrations in patients who were mechanically ventilated or dead within 28 days were significantly higher than those who were not (median 119.6 vs 79.1 pmol/L, p=0.001), and higher in patients who were admitted to the ICU during hospitalization compared to those who were not (median 114.5 vs 80.6 pmol/L, p=0.005). Significantly higher sFlt-1 concentrations were seen in patients who were admitted to the ICU compared to those who were not (median 96.0 vs 80.7 pg/mL, p=0.041).

Using Youden’s criterion, we derived a cutoff of 83.6 pmol/L for CT-proET-1 to predict prolonged hospitalization of more than 4 days. For sFlt-1 , a cutoff of 83.9 pg/mL was derived as an optimal value to predict prolonged hospitalization of more than 4 days.

The sensitivity, specificity, PPV and NPV of CT-proET-1 with cutoff 83.6 pmol/L in predicting mechanical ventilation or death were 83%, 54%, 18%, and 96%, respectively, with an AUC of 0.75.

As for the sFlt-1 cutoff of 81.3 pg/mL in predicting mechanical ventilation or death, the sensitivity, specificity, PPV and NPV were 61%, 54%, 14%, and 92%, with an AUC of 0.59. sFlt-1 showed highly statistical values for predicting death within 28 days, and discontinuation of oxygen supply.

Conclusion

CT-proET-1 and sFlt-1 function as valuable novel biomarkers in predicting clinical outcomes in COVID-19, with lower levels predicting better outcomes.

Example 2 - Bivariable results

Patients

The study uses data from a multicenter trial. The inclusion criteria for the trial were patients between the ages of 19 to 85 years, with SARS-CoV-2 infection confirmed by nasopharyngeal swab polymerase chain reaction or serum IgM antibody assay and presenting with at least two of the following symptoms: fever >38°C, needing supplemental oxygen, or lung infiltrates (Stone et al., 2020). For this study, patients who were not already admitted to the ICU at enrollment, and had complete data on all studied biomarkers, were included.

Outcomes The main outcome of this study was the composite endpoint of mechanical ventilation or death within 28 days of admission since some patients had died without being mechanically ventilated. Secondary outcomes within 28 days of admission were: intensive care unit (ICU) admission, clinical worsening on the COVID-19 ordinal scale, death, composite severity endpoint (at least one of the following: death, ICU admission, mechanical ventilation), day 4 COVID-19 ordinal scale > 4, prolonged hospitalization beyond day 4, day 4 COVID-19 ordinal scale equal or worse than day 1 , mechanical ventilation, deep vein thrombosis (DVT), pulmonary embolism (PE), and stroke. Additional outcomes were also assessed. The outcome “any thrombotic events” was defined as patients with any of the following outcomes within 28 days of admission: DVT, PE, or stroke. The COVID-19 ordinal scale represents disease severity and is based on ICU admission, oxygen supplementation, mechanical ventilation, death, or if the patient is ready to be discharged to home. Worsening on the COVID-19 ordinal scale is defined as an increase of one point or more in patients on supplemental oxygen, or an increase of two points or more in patients not receiving supplemental oxygen (Stone et al., 2020).

Plasma samples

EDTA plasma samples were collected, isolated, and aliquoted into cryovials within 2-12 hours of venipuncture. Cryovials were stored at -80°C until they were thawed to be assayed on a B R A H M S MR-proADM KRYPTOR for MR-proADM concentration determination (Thermo Fisher Scientific. B R A H M S MR-proADM).

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

Using Youden’s criterion, we derived a cutoff of 83.6 pmol/L for CT-proET-1 as an optimal value to predict prolonged hospitalization of more than 4 days. As for sFlt-1 , a cutoff of 83.9 pg/mL was derived as an optimal value to predict prolonged hospitalization of more than 4 days and day 4 COVID-19 ordinal scale equal or worse than day 1.

Schedule of biomarker measurements

Day 1 (Baseline), Days 4, 7, 14, 21 and 28 (Days 4 & 7, +/- 1 or 2 days, Days 14, 21 , 28, +/-3 days; 24h window for Day 1). SFlt-1 , CT-proET1 , MR-proADM and PCT biomarker results are available for Days 1 , 4, 7, 14, 21 and 28.

In addition, a sample was drawn at discharge. If discharge was on Day 4, 7, 14, 21 or 28, we used the mean of all biomarker measurements of the specific patient on this day were used for analysis.

Statistical analysis

Patient characteristics were summarized using descriptive statistics. Differences between patient groups were analyzed using the chi-square or Fisher's exact test when applicable for categorical variables and the Mann-Whitney U test for numeric continuous variables.

Measures of prognostic performance were sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the receiver operating characteristic curve (AUC). Estimates and 95% confidence intervals (Cl) were reported. Cl of sensitivity, specificity, PPV, NPV were computed according to Clopper and Pearson.

Kaplan-Meier curves were plotted, and the log-rank test was performed, stratified 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). Multiple multivariable logistic regression analyses were performed to evaluate if CT-proET-1 and sFlt-1 are independent predictors of clinical outcomes. Variables adjusted for included age, gender, body mass index, diabetes, hypertension, heart failure, history of Ml, COPD, chronic kidney disease, and days from symptom onset to randomization and CT-proET-1 and sFlt-1 measurement. Odds ratios were reported with 95% confidence intervals and p-values. All statistical testing was two-sided; p-values < 0.05 were considered statistically significant. P- values were not adjusted for multiple testing. Software R version 3.5.1 and the R package pROC version 1 .15.3 were used for statistical analyses (Robin et al., 2011 ; R Core Team 2018). STATA version 14.1 was used to generate Kaplan-Meier figures and ROC curves, log-rank tests, and multivariable logistic regression analyses (StataCorp 2015). Results:

Table 1 : Demographics and Baseline Characteristics of the Study Population Stratified by (A) Binary CT-proET-1 (cutoff 83.6 pmol/L) and (B) Binary sFlt-1 (cutoff 83.9 pg/mL)

(A) Binary CT-proET-1 (cutoff 83.6 pmol/L)

(B) Binary sFlt-1 (cutoff 81.3 pg/mL)

Values expressed as percentages (%) indicate either the proportion of the total population or the respective CT-proET-1 and sFlt-1 strata. Results are presented as median and interquartile-range (first quartile (Q1) - third quartile (Q3)) where appropriate. Statistical significance between CT- proET-1 and sFlt-1 strata was determined by the chi-square test or the Fisher's exact test when applicable for categorial factors, and the Mann-Whitney U test for numeric factors. P-Values were not corrected for multiple testing.

*One patient was excluded from statistical testing due to missing data on diabetes, heart failure, history of myocardial infarction, chronic obstructive pulmonary disorder, asthma, chronic kidney disease, and history of cancer. t Two patients were excluded from statistical testing due to missing data on smoking status.

Results: Base line characteristics median age 60 years, 58% male,

80% with Covid-19 scale 4 (ICU or non-ICU hospital ward, requiring non-invasive ventilation or high-flow oxygen) median time since symptom onset 9 days, median baseline PCT 0.2 ng/mL, median baseline d-Dimer 884 ng/mL.

Results: Clinical endpoints

Typically, low number of events of the order of 5-20, e.g. 5% mortality (day-1 analysis).

Larger number of events with composite endpoints and lower-severity events

Improvement of Covid-scale 101 events

Discharge before or on day 4 119 events

(both numbers for day-1 analysis)

Results: Time structure of events

Until day 4 90% ICU admissions (% of events in day-1 analysis population), 93% initiations of mechanical ventilation, 49% of supplemental oxygen discontinued. In weeks 1 and 2 Mortality was most pronounced in weeks 1 (38%) and 2 (38%), distributed relatively uniformly up to week 4.

In week 2 50% of mechanical ventilation discontinued.

Table 2: Outcomes Within 28 days Stratified by (A) Binary CT-proET-1 (Cutoff 83.6 pmol/L) and (B) Binary sFlt-1 (cutoff 83.9 pg/mL)

(A) Binary CT-proET-1 (Cutoff 83.6 pmol/L)

(B) Binary sFlt-1 (cutoff 83.9 pg/mL)

Values expressed as percentages (%) indicate either the proportion of the total population or the respective CT-proET-1 and sFlt-1 strata. Statistical significance between CT-proET-1 and sFlt-1 strata was determined by the chi-square test or the Fisher's exact test when applicable. P-Values were not corrected for multiple testing. *Any thrombotic event was defined as patients with any of the following outcomes: Deep vein thrombosis (DVT), pulmonary embolism (PE), or stroke.

Table 3 Distribution of CT-proET1 Levels and of sFlt-1 Stratified by Clinical Outcomes.

Results are presented as median and interquartile-range (first quartile (Q1) - third quartile (Q3)).

Statistical significance between outcome strata was determined by the Mann-Whitney U test. P- Values were not corrected for multiple testing

Table 4: Area Under the Receiver Operating Characteristic Curves (AUC) with 95% Cl of Biomarkers in Predicting Clinical Outcomes

* It was assumed that there was an increased risk associated with lower levels of lymphocytes

Example 3 - Further Results relating to the biomarker levels on day 1, the ratio between the levels of day 4 and day 1 (Day 4/Day1 ratio) and the additional measurement of a second biomarker Results relating to sFItl (Day 1 measurement)

Table 5a: Biomarker concentrations and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 5b: Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 5c: Diagnostic performance - AUC, PPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Results relating sFItl (Ratio day 4 vs day 1)

Table 6a: Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 6b: Diagnostic performance - AUC, NPV, optimized cut off values

Results relating to sFItl (Day 1 cut off (81.3 pg/L) (BM level day 1) + Ratio day 4 vs day 1 (BM2))

Table 7: Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to sFItl (Day 1 (cut off 81.3 pg/L) (BM1) +second Biomarker (BM2))

Table 8: Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to CT-proET1 (Day 1)

Table 9a: Biomarker concentrations and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 9b: Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 9c: Diagnostic performance - AUC, PPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to CT-proET1 (Day 1 (cut off 86.3 pmol/L) (BM1) + Ratio day 4 vs day 1 (BM2))

Table 10: Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to CT-proET1 (Day 1 (cut off 86.3 pmol/L) (BM1) +second Biomarker

(BM2))

Table 11 : Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to MR-proADM (Day 1)

Table 12a: Biomarker concentrations and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 12b: Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 12c: Diagnostic performance - AUC, PPV, optimized cut off values

Results relating to MR-proADM (Ratio day 4 vs day 1)

Table 13a: Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 13b: Diagnostic performance - AUC, NPV, optimized cut off value. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to MR-proADM (Day 1 (cut off 0.87 nmol/L) (BM1) + Ratio day 4 vs day 1 (BM2))

Table 14: Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to MR-proADM (Day 1 (cut off 0.87 nmol/L) (BM1) +second Biomarker (BM2))

Table 15: Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to PCT (Day 1):

Table 16a: Biomarker concentrations and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 16b: Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 16c: Diagnostic performance - AUC, PPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to PCT (Ratio day 4 vs day 1)

Table 17a: Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Tabel 17b: Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 18c: Diagnostic performance - AUC, PPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to PCT (Day 1 (cut off 0.25 ng/mL) (BM1)+ Ratio day 4 vs day 1 (BM2))

Table 18: Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to specific Outcomes (Endpoints) (Day 1)

Table 19a: Admitted to ICU during hospitalization (Outcome) - Biomarker concentration and cut off values (units: CT-proET1 pmol/L; sFlt-1 pg/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 19b: Admitted to ICU during hospitalization (Outcome)- Diagnostic performance - AUC, NVP, optimized cut off values (units: CT-proET1 pmol/L; sFItl pg/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 20a: Composite severity endpoint (at least one the following: Death within 28 days, ICU admission, mechanical ventilation) (Outcome)- Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; sFItl pg/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 20b: Composite severity endpoint (at least one the following: Death within 28 days, ICU admission, mechanical ventilation) (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values (units: CT-proET1 pmol/L; sFItl pg/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 21a: Dav 4 Covid scale =4 vs. <4 (Outcome)- Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 21 b: Dav 4 Covid scale =4 vs. <4 (Outcome)- Diagnostic performance - AUC, NPV, optimized cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 22a: Death within 28 days (Outcome)- Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 22b: Death within 28 days (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 23a: Discharge before or on Dav 4 (yes vs. no) (Outcome)- Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; sFlt-1 pg/L; MR-proADM nmol/L; PCT ng/mL). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 23b: Discharge before or on Dav 4 (yes vs. no) (Outcome) - Diagnostic performance - AUC, PPV, optimized cut off values (units: CT-proET1 pmol/L; SFIt-1 pg/L; MR-proADM nmol/L; PCT ng/mL). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 24a: Discontinuation of supplemental oxygen (Outcome) - Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 24b: Discontinuation of supplemental oxygen (Outcome)- Diagnostic performance - AUC, NPV, optimized cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 25a: Improvement of COVID-scale (Day4 better than Dav 1 vs. Day4 equal or worse than Dav 1) (Outcome) - Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; SFIt- 1 pg/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 25b: Improvement of COVID-scale (Day4 better than Dav 1 vs. Day4 equal or worse than Dav 1) (Outcome)- Diagnostic performance - AUC, PPV, optimized cut off values (units: CT- proETI pmol/L; sFItl pg/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 26a: Infection (grade >= 3) within 28 days (Outcome) - Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 26b: Infection (grade >= 3) within 28 days (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 27a: Mechanical ventilation within 28 days (Outcome)- Biomarker concentrations and cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 27b: Mechanical ventilation within 28 days (Outcome)- Diagnostic performance - AUC, NPV, optimized cut off values (units: CT-proET1 pmol/L; MR-proADM nmol/L). Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to specific Outcomes(Endpoints) (Ratio Day 4 vs. Day 1) Table 28a: Admitted to ICU during hospitalization (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 28b: Admitted to ICU during hospitalization (Outcome) -Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 29a: Composite severity endpoint (at least one the following: Death within 28 days, ICU admission, mechanical ventilation) (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 29b: Composite severity endpoint (at least one the following: Death within 28 days, ICU admission, mechanical ventilation) (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 30a: Dav 4 Covid scale =4 vs. <4 (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 30b: Dav 4 Covid scale =4 vs. <4 (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 31a: Death within 28 days (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 31 b: Death within 28 days (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 32a: Discontinuation of supplemental oxygen (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 32b: Discontinuation of supplemental oxygen (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 33a: Improvement of COVID-scale (Day4 better than Dav 1 vs. Day4 equal or worse than Dav 1) (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 33b: Improvement of COVID-scale (Day4 better than Dav 1 vs. Day4 equal or worse than Dav 1) (outcome) - Diagnostic performance - AUC, PPV, optimized cut off values

Table 34a: Infection (grade >= 3) within 28 days (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 34b: Infection (grade >= 3) within 28 days (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 35a: Mechanical ventilation within 28 days (Outcome) - Biomarker ratios and cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 35b: Mechanical ventilation within 28 days (Outcome) - Diagnostic performance - AUC, NPV, optimized cut off values. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to specific Outcomes (Endpoints) (Day 1 (BM1) + Ratio Day 4 vs. Day 1 (BM2))

Table 36: Admitted to ICU during hospitalization (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 37: Composite severity endpoint (at least one the following: Death within 28 days, ICU admission, mechanical ventilation) (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 38: Death within 28 days (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 39: Discontinuation of supplemental oxygen (Outcome) - Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 40: Infection (grade >= 3) within 28 days (Outcome)-Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 41 : Mechanical ventilation within 28 days (Outcome)-Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Results relating to specific Outcomes (Endpoints) (Day 1 (BM1) + Biomarker 2 (BM2))

Table 42: Admitted to ICU during hospitalization (Outcome) -Diagnostic performance - c index.

Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 43: Composite severity endpoint (at least one the following: Death within 28 days, ICU admission, mechanical ventilation) (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 44: Dav 4 Covid scale =4 vs. <4 (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 45: Discharge before or on Dav 4 (yes vs. no) (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Table 46: Improvement of COVID-scale (Day4 better than Dav 1 vs. Day4 equal or worse than Dav 1) (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention. Table 47: Mechanical ventilation within 28 days (Outcome) -Diagnostic performance - c index. Each endpoint shown represents an embodiment of the present invention, with regards to one or more of prognostic outcome, risk ratio, statistical measure, such as C, NPV or PPV, and/or cut-off value. In embodiments, the endpoint (as prognostic outcome) combined with cut-off represents an embodiment of the invention. In embodiments, the endpoint (as prognostic outcome) combined with risk ratio represents an embodiment of the invention.

Summary of exemplary results shown above:

CT-proET1 had similarly good performance as MR-proADM in predicting clinical outcomes death, ICU admission, mechanical ventilation, composite severity endpoint, discharge on day 4, discontinuation of supplemental oxygen, infection.

CT-proET1 had similarly good performance as MR-proADM, e.q. death AUC 0.79 and 0.86 (p=0.006 and 0.001), mechanical ventilation or death AUC 0.75 and 0.76 (p=0.001 and <0.001), ICU admission or death AUC 0.72 and 0.72 (p=0.001 and <0.001), discharge on day 4 AUC 0.72 and 0.69 (p<0.001 and <0.001), composite severity endpoint AUC 0.71 and 0.71 (p=0.001 and 0.001).

Added value of CT-proET1 and MR-proADM to the conventional biomarker IL6 was observed for clinical outcomes discharge on day 4, improvement of Covid scale from day 1 to day 4 (only CT- proETI), mechanical ventilation or death (only CT-proET1), ICU admission or death, composite severity endpoint, infection or death, ordinal scale on day 4 (only CT-proET1).

Added value of biomarker change from day 1 to day 4 of MR-proADM, CT-proET 1 , sFItl , and PCT to their corresponding day-1 biomarkers was observed for clinical outcomes death, ICU admission (or death), mechanical ventilation (only CT-proET1), composite severity endpoint (only CT-proET1 , sFItl), discontinuation of supplemental oxygen (only CT-proET1 , MR-proADM), infection (only PCT, sFItl).

Further include

Discontinuation of supplemental oxygen:

MR-proADM, CT-proET1 AUC 0.77 and 0.75 (p=0.001 and 0.006).

Infection (grade>3):

IL6, MR-proADM, PCT, CT-proET1 AUC 0.76, 0.75, 0.70, 0.68 (p<0.012).

Day-4 Covid scale

CT-proET 1 AUC 0.71 (p=0.004).

Deep venous thrombosis or death (probably driven by death component) MR-proADM, CT-proET1 AUC 0.79 and 0.69 (p=0.001 and 0.034).

Exemplary cut offs derived for CT-proET 1 and sFLT-1 (derived from A) median concentration and

B) maximal Youden’s J. i.e., maximal sum of sensitivity and specificity):

CT-proET1:

83.6 pmol/L median concentration

87.4 pmol/L for endpoint death within 28 days

91.8 pmol/L for endpoint mechanical ventilation within 28 days

102.7 pmol/L for endpoint Day 4 Covid scale > 4 vs <4 sFItl:

81.3 pg/mL median concentration

83.9 pg/mL for endpoint Improvement of COVID-scale (Day 4 vs Day 1)

83.9 pg/mL for endpoint Discharge on Day 4

88.4 pg/mL for endpoint composite severity endpoint (at least one of the following: death within 28 days, ICU admission, mechanical ventilation) and biomarkers with added value to IL6:

CT-proET1 (C-lndex bivariable vs single biomarker IL6) discharge before or on day 4 0.76 vs 0.67 (p<0.001), improvement of ordinal scale 0.71 vs 0.65 (p=0.003), mechanical ventilation or death 0.79 vs 0.71 (p=0.008), ICU admission or death 0.80 vs 0.73 (p=0.010), composite severity endpoint 0.78 vs 0.72 (p=0.015), infection (grade>3) or death 0.80 vs 0.76 (p=0.027), ordinal COVID-19 scale on day 4 0.77 vs 0.72 (p=0.047)

MR-proADM discharge on day 4 0.73 vs 0.67 (p<0.001) infection (grade>3) or death 0.82 vs 0.76 (p=0.004) ICU admission or death 0.78 vs 0.73 (p=0.016) composite severity endpoint 0.77 vs 0.72 (p=0.029)

PCT infection (grade>3) 0.79 vs 0.77 (p=0.012 and biomarkers with added value to d-Dimer

MR-proADM (C-lndex bivariable vs single biomarker d-Dimer) thrombotic events (MGH definition) or death 0.77 vs 0.66 (p=0.024).

Exemplary endpoints and biomarkers with added value to further biomarkers:

MR-proADM and/or CT-proET1 combined with CRP (ICU admission or death, Day-4 ordinal scale, infection), Ferritin, or LDH (see tables above). sFItl combined with Lymphocytes (see tables above).

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

Exemplary endpoints and biomarkers with added value of biomarker change from day 1 to day 4.

Mortality (C-lndex bivariable vs day-1 biomarker)

MR-proADM 0.95 vs 0.83 (p<0.001), sFItl 0.91 vs 0.70 (p<0.001), PCT 0.86 vs 0.60 (p=0.001), CT-proET1 0.83 vs 0.75 (p=0.039).

ICU admission or death (diagnostic for many patients on day 4)

MR-proADM 0.84 vs 0.71 (p<0.001), sFItl 0.75 vs 0.63 (p=0.003),

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

ICU admission (diagnostic for many patients on day 4) PCT 0.75 vs 0.60 (p<0.001).

Mechanical ventilation or death (diagnostic formost patients on day 4) CT-proET1 0.81 vs 0.75 (p=0.004).

Composite severity endpoint (diagnostic for many patients on day 4) sFItl 0.78 vs 0.65 (p=0.001),

CT-proET1 0.75 vs 0.71 (p=0.013). Discontinuation of supplemental oxygen (diagnostic for many patients on day 4)

MR-proADM 0.89 vs 0.76 (p<0.001),

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

Infection (grade>3) PCT 0.81 vs 0.68 (p=0.001), sFItl 0.73 vs 0.61 (p=0.003).

Any infection (as reported in trial plus infections documented in FU form) or thrombotic events (6 types)

PCT 0.77 vs 0.63 (p<0.001), MR-proADM 0.76 vs 0.74 (p=0.015).

Performance of thrombotic (disbalance) endpoints combined with death is strongly driven by mortality (e.g., deep venous thrombosis or death: 7 out of 8 events came from death).

Further results relating to sFlt-1 and MR-proADM:

Table 48a: Results relating to a single biomarker (level of MR-proADM or SFlt-1 on day 4 vs. day 1 ) including biomarker concentrations and cut off values. Each event shown represents an embodiment of the present invention, with regards to one or more of therapy management, prognostic outcome and statistical measure, such as NPV, and/or cut-off value. In embodiments, the event (such as death within 28 days or mechanical ventilation) combined with a cut-off value represents an embodiment of the invention.

Event: Mechanical ventilation

Table 48b: Diagnostic performance of single biomarkers (level of MR-proADM or SFIt-1 on day 4 vs. day 1) - AUC, NPV, optimized cut off values. Table 49: Results relating to combinations of biomarkers (biomarker 1 (BM1) and biomarker 2 (BM2)) including biomarker concentrations and cut off values. Each event shown represents an embodiment of the present invention, with regards to one or more of therapy management, prognostic outcome and statistical measure, such as NPV, and/or cut-off value. In embodiments, the event (such as death within 28 days or mechanical ventilation) combined with a cut-off value represents an embodiment of the invention.

MR-proADM and sFlt-1 (level of MR-proADM on day 4 vs. day 1) had good performance in guidance on whether a patient is in need of oxygen support, such as supplemental oxygen and in predicting whether a patient is at risk of death within 28 days (see tables 3a and 3b). Patients with a lower or equal level of MR-proADM and/or sFlt-1 in the second sample are effectively ruled-out from the need of supplemental oxygen and the risk of death within 28 days, indicated by a strong NPV for these events.

Further, MR-proADM (level of MR-proADM on day 4 vs. day 1) had good performance in guidance on whether a patient is in need of oxygen support such as supplemental oxygen and mechanical ventilation or whether said patient requires admission to ICU for intensified treatment. Patients with a lower or equal level of MR-proADM in the second sample are effectively ruled-out from the need of oxygen support such as supplemental oxygen, mechanical ventilation and admission to ICU for intensified treatment, indicated by a strong NPV for these events. Further, sFlt-1 (level of sFlt-1 on day 4 vs. day 1) had good performance in guidance on whether a patient is in need of oxygen support such as supplemental oxygen and whether said patient is at risk of an additional infection within at least 28 days. Patients with a lower or equal level of sFlt-1 in the second sample are effectively ruled-out from the need of oxygen support such as supplemental oxygen, and the risk of an additional infection within 28 days.

The combination of two biomarkers (BM1: level of MR-proADM or SFIt-1 on day 4 vs. day 1 ; BM2: level of MR-proADM above a cut-off 0.87 nmol/l on day 1 and/or level of sFItl cutoff 81 .3 pg/L, on day 1 ) further improves the performance in guidance on whether a patient is in need of oxygen support such as supplemental oxygen and other treatment means such as mechanical ventilation or admission to ICU and on the risk of death within 28 days.

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Claims

1 . A method for therapy management and/or prognosing disease progression, severity and/or outcome of a patient having a respiratory virus infection, comprising the steps of: a. providing a first sample isolated from said patient, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first and the second sample were isolated within 96 h (4 days) after consultation with medical personnel, d. determining a level of one or more endothelial biomarkers in the first and the second sample, wherein the one or more endothelial biomarkers are soluble Fms- like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof, C-terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and/or pro-adrenomedullin (proADM) or fragment(s) 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 is not at risk of an adverse clinical outcome within at least 28 days.

2. The method according to 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 an adverse clinical outcome associated with the need for supplemental oxygen for at least 28 days.

3. The method according to any one of the preceding claims, 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 in need of 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 medical personnel to consult with the patient after occurrence 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 and/or wherein the patient is not admitted to an intensive care unit (ICU).

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

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

9. The method according to any one of the preceding claims, wherein the patient is infected with a coronavirus, preferably a SARS-coronavirus, more preferably a 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 intensified treatment within at least 28 days.

12. The method according to the preceding claim, wherein the adverse respiratory and/or infectious clinical outcome are intensive care unit (ICU) treatment, mechanical ventilation and/or an additional infection.

13. The method according to any one of the preceding claims, wherein the patient is infected with a coronavirus, and the adverse event is a worsening of a COVID scale score (for example worsening of a COVID scale score to >4) and/or maintained hospitalization on or beyond day 4 after hospital admission.

14. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises sFlt-1 or fragment(s) thereof and wherein a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFItl or fragment(s) thereof determined in the first sample < 81.3 pg/l ± 20% indicates that the patient is not at risk of an adverse clinical outcome within 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 the second sample comprises sFlt-1 or fragment(s) thereof and wherein a lower or equal level of sFItl or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not at risk of an adverse clinical outcome associated with the need 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 the second sample comprises sFItl or fragment(s) thereof and wherein a lower or equal level of sFItl or fragment(s) thereof in the second sample compared to the first sample, and a level of sFItl or fragment(s) thereof determined in the first sample < 81.3 pg/l ± 20% indicates that the patient is not at risk of death within at least 28 days (and preferably not in need of 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 the second sample comprises sFlt-1 or fragment(s) thereof and wherein a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of sFlt-1 or fragment(s) thereof determined in the first sample < 81 .3 pg/l ± 20% indicates that the patient is not in need of oxygen support and not at risk of death and an additional infection within at least 28 days after consultation with the medical personnel.

18. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises C- terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome within at least 28 days.

19. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises CT- proET-1 or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome associated with the need 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 determined in the first and the second sample comprises CT- proET-1 or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not at risk of death within at least 28 days (and preferably not in need of oxygen support).

21 . The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises CT- proET-1 or fragment(s) thereof and wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample, and a level of CT-proET-1 or fragment(s) thereof determined in the first sample < 87.4 pmol/L ± 20% indicates that the patient is not in need of mechanical ventilation and not at risk of death within at least 28 days after consultation with the medical personnel.

22. The method according to any one of the preceding claims, wherein determining a level of proADM or fragment(s) thereof comprises determining a level of mid-regional pro- adrenomedullin (MR-proADM) in the sample.

23. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and wherein a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome within at least 28 days.

24. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and wherein a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not at risk of an adverse clinical outcome associated with the need 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 determined in the first and the second sample comprises proADM or fragment(s) thereof and wherein a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not at risk of death within at least 28 days (and preferably not in need of oxygen support).

26. The method according to any one of the preceding claims, wherein the one or more endothelial biomarkers determined in the first and the second sample comprises proADM or fragment(s) thereof and wherein a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample, and a level of mid-regional proADM (MR-proADM) or fragment(s) thereof determined in the first and/or second sample < 0.87 nmol/L ± 20% indicates that the patient is not in need of oxygen support, not at risk of death, admission to an ICU and mechanical ventilation within at least 28 days.

27. The method according to any one of the preceding claims, comprising the steps of a. providing a first sample isolated from said patient, wherein said patient is infected with a coronavirus, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first sample was obtained within 24 h (1 day) after consultation within the medical personnel, and the second sample was obtained within 72 to 96 h (on day 4) after consultation with the medical personnel, d. determining a level of soluble Fms-like tyrosine kinase-1 (sFlt-1) or fragment(s) thereof in said sample, and e. wherein a lower or equal level of sFlt-1 or fragment(s) thereof in the second sample compared to the first sample indicates that the patient is not in need of oxygen support and not at risk of death and an additional infection within at least 28 days.

28. The method according to any one of the preceding claims, comprising the steps of a. providing a first sample isolated from said patient, wherein said patient is infected with a coronavirus, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first sample was obtained within 24 h (1 day) after consultation within the medical personnel, and the second sample was obtained within 72 to 96 h (on day 4) after consultation with the medical personnel, d. determining a level of C-terminal proendothelin-1 (CT-proET-1) or fragment(s) thereof in said sample, and e. wherein a lower or equal level of CT-proET-1 or fragment(s) thereof in the second sample compared to the first sample indicates that the patient is not in need of oxygen support and not at risk of death and an additional infection within at least 28 days.

29. The method according to any one of the preceding claims, comprising the steps of a. providing a first sample isolated from said patient, wherein said patient is infected with a coronavirus, b. providing a second sample isolated from said patient at a time point after isolating the first sample from said patient, c. wherein the first sample was obtained within 24 h (1 day) after consultation within the medical personnel, and the second sample was obtained within 72 to 96 h (on day 4), after consultation with the medical personnel, d. determining a level of pro-adrenomedullin (proADM) or fragment(s) thereof in said sample, and e. wherein a lower or equal level of proADM or fragment(s) thereof in the second sample compared to the first sample indicates that the patient is not in need of oxygen support, not at risk of death, admission to an ICU and mechanical ventilation within at least 28 days.

30. The method according to any one of the preceding claims, additionally comprising a. determining a 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 fragment(s) thereof, IL6, D-Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes and/or C-reactive protein (CRP), and b. wherein the level of the at least one additional biomarker and/or 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 an adverse clinical event within at least 28 days.

31 . The method according to the preceding claim, the level of the at least one additional biomarker and/or 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 an adverse clinical outcome associated with the need for supplemental oxygen for at least 28 days.

32. The method according to the preceding claim, the level of the at least one additional biomarker and/or 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 in need of supplemental oxygen for at least 28 days.

33. A kit for carrying out the method of any one of the preceding claims, comprising: detection reagents for determining the level of one or more 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 fragment(s) thereof, C-terminal pro-endothelin-1 (CT-proET-1) or fragment(s) thereof, and/or pro-adrenomedullin (proADM) or fragment(s) thereof (preferably MR-proADM), and reference data for the risk of a patient of an adverse clinical outcome within 28 days, in particular reference data for a risk threshold, cut-off value or risk ratio, wherein said reference data is preferably stored on a computer readable medium and/or employed in the form of computer executable code configured for comparing the determined levels of one or more of the endothelial biomarkers in a sample from a patient with the risk threshold, cut-off value or risk ratio; optionally detection reagents for determining the presence of a viral infection of the respiratory tract, preferably detection reagents for the presence of an infection with a coronavirus, preferably a SARS-CoV2 virus infection, and optionally, detection reagents for determining the level of at least one additional biomarker or fragment(s) thereof, procalcitonin (PCT) or fragment(s) thereof, 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 for said at least one additional biomarker, preferably pro-endothelin-1 (CT-proET- 1) or fragment(s) thereof, procalcitonin (PCT) or fragment(s) thereof, IL6, D- Dimer, lactate dehydrogenase (LDH), ferritin, lymphocytes, C-reactive protein (CRP), in particular a risk threshold, cut-off value or risk ratio, wherein said reference data is preferably stored on a computer readable medium and/or employed in the form of computer executable code configured for comparing the determined levels of the at least one additional biomarker or fragment(s) thereof with the threshold, cut-off value or risk ratio.