SOLUBLE TREM-1 AS A MARKER OF SEVERITY OR COMPLICATIONS FOR A SUBJECT SUFFERING FROM A CORONAVIRUS INFECTION

Abstract

The use of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as a marker in a method for identifying a subject suffering from a disease caused by a coronavirus such as COVID-19 at risk of having or developing a severe form and/or a complication or at risk of death, in a method for assessing the severity of a disease caused by a coronavirus such as COVID-19, and in a method for monitoring a subject suffering from a disease caused by a coronavirus such as COVID-19.

Claims

1-10. (canceled)

11. An in vitro method for identifying a subject suffering from a disease caused by a coronavirus infection as being at risk of having or developing a severe form and/or a complication of the disease caused by a coronavirus infection, or at risk of death occurring after the coronavirus infection, said method comprising: measuring the level of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in a biological sample from the subject; and comparing the level of sTREM-1 measured in the biological sample from the subject to a reference value.

12. A method for treating a subject suffering from a disease caused by a coronavirus identified as being at risk of having or developing a severe form and/or a complication of the disease caused by a coronavirus, or at risk of death occurring after the coronavirus infection, said method comprising: identifying a subject suffering from a disease caused by a coronavirus as being at risk of having or developing a severe form and/or a complication of the disease caused by a coronavirus, or at risk of death occurring after the coronavirus infection, according to the method of claim 11; and treating the subject suffering from a disease caused by a coronavirus identified as being at risk of having or developing a severe form and/or a complication of the disease caused by a coronavirus, or at risk of death occurring after the coronavirus infection, by administering to said subject at least one of the following: respiratory support, vasopressor therapy, fluid therapy, antimicrobial therapy, antiviral therapy, cardiovascular support, renal replacement therapy, sedation, an antiviral agent, an anti-interleukin 6 (anti-IL-6) agent, or any mixes thereof.

13. The in vitro method according to claim 11, wherein the complication of the disease caused by a coronavirus infection is selected from the group consisting of respiratory failure, including acute respiratory failure or acute respiratory distress syndrome (ARDS); persistence of respiratory failure including the requirement for prolonged mechanical ventilation, and failed extubation; secondary infection or superinfection; thrombotic complications including venous and/or arterial thromboembolism; pulmonary embolism; cardiocirculatory failure (also referred to as cardiovascular failure); renal failure; liver failure; and any combinations thereof.

14. The in vitro method according to claim 11, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the disease caused by a coronavirus infection is coronavirus disease 2019 (COVID-19).

15. The in vitro method according to claim 11, wherein the level of sTREM-1 is a transcription level of sTREM-1 or a translation level of sTREM-1.

16. The in vitro method according to claim 11, wherein the biological sample is a blood sample.

17. The in vitro method according to claim 11, wherein the biological sample is a serum sample.

18. The in vitro method according to claim 11, wherein the subject requires hospitalization.

19. The in vitro method according to claim 18, wherein the subject requires respiratory support.

20. The in vitro method according to claim 18, wherein the level of sTREM-1 is measured at day 3 following hospitalization.

21. An in vitro method for determining the severity of a disease caused by a coronavirus infection in a subject, said method comprising: measuring the level of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in a biological sample from the subject; and comparing the level of sTREM-1 measured in the biological sample from the subject to a reference value.

22. The in vitro method according to claim 21, wherein the method allows the monitoring over time of the disease caused by a coronavirus infection in the subject, and wherein the method comprises measuring the level of sTREM-1 in biological samples from the subject obtained on at least two occasions.

23. The in vitro method according to claim 22, wherein the at least two occasions are separated by at least 24 hours.

24. The in vitro method according to claim 21, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the disease caused by a coronavirus infection is coronavirus disease 2019 (COVID-19).

25. The in vitro method according to claim 21, wherein the level of sTREM-1 is a transcription level of sTREM-1 or a translation level of sTREM-1.

26. The in vitro method according to claim 21, wherein the biological sample is a blood sample.

27. The in vitro method according to claim 21, wherein the biological sample is a serum sample.

28. The in vitro method according to claim 21, wherein the subject requires hospitalization.

29. The in vitro method according to claim 28, wherein the subject requires respiratory support.

30. The in vitro method according to claim 28, wherein the level of sTREM-1 is measured at day 3 following hospitalization.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0317] FIGS. 1A-1B are a combination of graphs comparing the levels of sTREM-1 in healthy volunteers (HV) and in patients suffering from COVID-19. FIG. 1A shows the levels of sTREM-1 at baseline (day 1). FIG. 1B shows the levels of sTREM-1 at day 3 following admission in intensive care unit (ICU). ***: p<0.001; ****: p<0.0001.

[0318] FIGS. 2A-2B are a combination of graphs comparing the levels of sTREM-1 in patients suffering from COVID-19 who survived (survivors—S) and in patients suffering from COVID-19 who did not survive (non-survivors—NS). FIG. 2A shows the levels of sTREM-1 at day 1 (i.e., day of admission in ICU). FIG. 2B shows the levels of sTREM-1 at day 3 following admission in intensive care unit (ICU). *: p<0.05; **: p<0.01.

[0319] FIGS. 3A-3B are a combination of graphs comparing the levels of sTREM-1 in healthy volunteers (HV) and in different groups of patients suffering from COVID-19: all: all patients suffering from COVID-19; MV<10: patients suffering from COVID-19 who were under mechanical ventilation for less than 10 days; MV≥10 and NS: patients suffering from COVID-19 who were under mechanical ventilation for 10 days or more, and patients who did not survive (non-survivors—NS) regardless of the duration of mechanical ventilation; MV<15: patients suffering from COVID-19 who were under mechanical ventilation for less than 15 days; MV≥15 and NS: patients suffering from COVID-19 who were under mechanical ventilation for 15 days or more, and patients who did not survive (non-survivors—NS) regardless of the duration of mechanical ventilation. FIG. 3A shows the levels of sTREM-1 at day 1 (i.e., day of admission in ICU). FIG. 3B shows the levels of sTREM-1 at day 3 following admission in ICU. ns: non-significant; *: p<0.05; ***: p<0.001; ****: p<0.0001.

[0320] FIGS. 4A-4B are a combination of graphs showing the levels of sTREM-1 in patients suffering from COVID-19 over time (from day 1 until day 21 following admission in ICU). FIG. 4A compares the levels of sTREM-1 over time in patients suffering from COVID-19 who were under mechanical ventilation for less than 15 days (MV<15) and in patients suffering from COVID-19 who were under mechanical ventilation for 15 days or more (MV≥15). FIG. 4B compares the levels of sTREM-1 over time in patients suffering from COVID-19 with troponin levels at baseline lower than 12 pg/mL (TROPO<12) and in patients suffering from COVID-19 with troponin levels at baseline higher than 12 pg/mL (TROPO>12). Missing data were treated with the LOCF method (Last Observed Carry Forward value).

[0321] FIG. 5A is a graph showing the ROC curve assessing the ability of sTREM-1 levels at day 3 following admission in ICU to discriminate between a mechanical ventilation (MV) lasting less than 15 days and a mechanical ventilation (MV) lasting 15 days or more. FIG. 5B is a table showing the parameters associated with the ROC curve.

[0322] FIGS. 6A-6D are a combination of incidence curves (Cox proportional-hazard (PH) model for individual classifiers and Kaplan-Meier for the overall cohort) showing the proportion over time of patients suffering from COVID-19 becoming free from invasive mechanical ventilation (i.e., begin extubated) or the proportion over time of patients suffering from COVID-19 exiting the intensive care unit. FIG. 6A is an incidence curve showing the proportion over time of patients suffering from COVID-19 becoming free from invasive mechanical ventilation (i.e., begin extubated) depending on a cut-off sTREM-1 level at day 1 (baseline) of 186 pg/mL. FIG. 6B is an incidence curve showing the proportion over time of patients suffering from COVID-19 becoming free from invasive mechanical ventilation (i.e., begin extubated) depending on a cut-off sTREM-1 level at day 3 of 186 pg/mL. FIG. 6C is an incidence curve showing the proportion over time of patients suffering from COVID-19 exiting the intensive care unit depending on a cut-off sTREM-1 level at day 1 (baseline) of 186 pg/mL. FIG. 6D is an incidence curve showing the proportion over time of patients suffering from COVID-19 exiting the intensive care unit depending on a cut-off sTREM-1 level at day 3 of 186 pg/mL. KM overall: proportion over time for the overall cohort of patients; sTREM-1>186: proportion over time for the patients with a sTREM-1 level at the indicated time (day 1 or day 3)>186 pg/mL; sTREM-1<186: proportion over time for the patients with a sTREM-1 level at the indicated time (day 1 or day 3)<186 pg/mL.

[0323] FIG. 7 is a flow chart depicting the validation cohort. Severe illness was defined as the need for ICU admission during hospital stay. Data are presented as median with interquartile range.

[0324] FIGS. 8A-C are a combination of graphs showing the plasma levels of sTREM-1 in different groups of patients with COVID-19. FIG. 8A compares the plasma sTREM-1 levels in moderately ill patients (moderate illness) and in severely ill patients (severe illness). Severe illness was defined as the need for ICU admission during hospital stay. FIG. 8B compares the plasma sTREM-1 levels in survivors and in non-survivors. FIG. 8C compares the plasma sTREM-1 levels in patients with a thromboembolic event (TEE) and in patients without a thromboembolic event (no TEE). Data are presented as median with interquartile ranges. P-values were calculated with Mann-Whitney U tests. *: p<0.05; ***: p<0.001.

[0325] FIGS. 9A-D are a combination of graphs showing the plasma levels of sTREM-1 in different groups of patients with COVID-19. FIG. 9A shows the plasma sTREM-1 levels in moderately ill patients with COVID-19 according to outcome (survivors vs. non-survivors, p-value=0.019). FIG. 9B compares the plasma sTREM-1 levels in severely ill patients with COVID-19 according to outcome (survivors vs. non-survivors, p-value=0.007). FIG. 9C compares the plasma sTREM-1 levels in surviving patients with COVID-19 according to illness severity (moderate illness vs. severe illness, p-value=0.068). FIG. 9D compares the plasma sTREM-1 levels in non-surviving patients with COVID-19 according to illness severity (moderate illness vs. severe illness, p-value=0.737). Severe illness was defined as the need for ICU admission during hospital stay. Data are presented as median with interquartile range. P-values were calculated with Mann-Whitney U tests. ns: non-significant; *: p<0.05; **: p<0.01.

[0326] FIGS. 10A-C are a combination of graphs showing the correlations between sTREM-1 levels and other clinical outcomes in patients with COVID-19. FIG. 10A shows the correlation between sTREM-1 levels and total length of hospital stay. FIG. 10B shows the correlation between sTREM-1 levels and total length of ICU stay. FIG. 10C shows the correlation between sTREM-1 levels and illness duration at time of sampling. Correlation coefficients and p-values, as indicated on the figures, were calculated using Spearman's rank correlation test.

[0327] FIGS. 11A-F are a combination of graphs showing correlations between sTREM-1 levels and inflammatory markers in patients with COVID-19. FIG. 11A shows the correlation between sTREM-1 levels and white blood cell (WBC) count. FIG. 11B shows the correlation between sTREM-1 levels and lymphocyte count. FIG. 11C shows the correlation between sTREM-1 levels and the levels of C-reactive protein (CRP). FIG. 11D shows the correlation between sTREM-1 levels and ferritin levels. FIG. 11E shows the correlation between sTREM-1 levels and D-dimer levels. FIG. 11F shows the correlation between sTREM-1 levels and interleukin-6 (IL-6) circulating concentration. Correlation coefficients and p-values, as indicated on the figures, were calculated using Spearman's rank correlation test.

[0328] FIGS. 12A-D are a combination of receiver-operating characteristic (ROC) curves assessing the ability of the indicated biomarkers (measured in the first sample obtained after COVID-19 diagnosis in the hospital) to discriminate between survivors and non-survivors. FIG. 12A is a ROC curve assessing the ability of sTREM-1 levels to discriminate between survivors and non-survivors. FIG. 12B is a ROC curve assessing the ability of C-reactive protein (CRP) levels to discriminate between survivors and non-survivors. FIG. 12C is a ROC curve assessing the ability of ferritin levels to discriminate between survivors and non-survivors. FIG. 12D is a ROC curve assessing the ability of interleukin-6 (IL-6) circulating concentration to discriminate between survivors and non-survivors. AUC: area under the curve.

[0329] FIGS. 13A-E are a combination of Kaplan-Meier curves showing the percentage survival over time of patients suffering from COVID-19 depending on the indicated biomarker cut-off values. FIG. 13A shows the percentage survival over time of patients suffering from COVID-19 depending on the indicated sTREM-1 cut-off value. FIG. 13B shows the percentage survival over time of patients suffering from COVID-19 depending on the indicated C-reactive protein (CRP) cut-off value. FIG. 13C shows the percentage survival over time of patients suffering from COVID-19 depending on the indicated ferritin cut-off value. FIG. 13D shows the percentage survival over time of patients suffering from COVID-19 depending on the indicated interleukin-6 (IL-6) cut-off value. FIG. 13E the percentage survival over time of patients suffering from COVID-19 depending on the indicated combinations of sTREM-1 and interleukin-6 (IL-6) cut-off values. Cut-off values were based on the maximal Youden's index derived from the receiver-operating characteristic curves shown on FIGS. 12A-D. Hazard ratios were calculated with the log-rank (Mantel-Cox) test.

[0330] FIG. 14 is a scatter plot of the sTREM-1 plasma concentrations measured with the sTREM-1 Elecsys ECLIA and with the sTREM-1 Quantikine ELISA.

EXAMPLES

[0331] The present invention is further illustrated by the following examples.

Example 1

Materials and Methods

Study Design and Participants

[0332] 27 adult patients diagnosed with COVID-19 according to WHO (World Health Organization) interim guidance and admitted to intensive care unit (ICU) were included between Mar. 15th 2020 and Mar. 31St 2020 in the Centre Hospitalier Régional Universitaire (CHRU) of Nancy, France. All patients were confirmed as positive for SARS-CoV-2 by polymerase chain reaction (nasal/throat swab or pulmonary sample), and were admitted in ICU for acute respiratory distress syndrome. This study was approved by the CHRU Nancy ethic committee (saisine n° 196).

Definitions

[0333] Secondary infection was diagnosed in patients showing clinical signs or symptoms of pneumonia or bacteremia confirmed by positive culture. Acute respiratory distress syndrome (ARDS) was diagnosed according to the Berlin Definition (ARDS Definition Task Force, Ranieri et al., JAMA. 2012; 307(23):2526-2533.). Acute kidney injury was diagnosed according to the kidney disease improving global outcomes (KDIGO) clinical practice guidelines (Khwaja, Nephron Clin Pract. 2012; 120(4):c179-c184). Cardiocirculatory failure was diagnosed if serum levels of troponin were above 12 pg/mL, or if there was a need for vasopressors.

Data Collection and Laboratory Procedures

[0334] Medical records were collected and retrospectively analyzed. Plasma samples were collected from ICU admission to discharge at day 1, 3, 6, 14, and 21. Blood count, hematology, serum biochemical tests, troponins, and interleukin-6 (IL-6) were obtained by routine blood tests. Inflammatory markers were quantified in plasma by enzyme-linked immunosorbent assay (ELISA) and multiplex assay.

sTREM-1 Measurement

[0335] sTREM-1 levels were measured using an analytically validated ELISA-based method (EMA Guideline on bioanalytical method validation. EMEA/CHMP/EWP/192217/2009 (2011)) using a commercially available research use only ELISA assay (Human TREM-1 Quantikine® ELISA kit, R&D Systems, reference DTRM10C). The analytical performances and acceptance criteria of this method are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Summary of the analytical performances and acceptance criteria of the sTREM-1 ELISA validated method Analyte sTREM-1 Matrix Human K.sub.2-EDTA plasma Analytical method ELISA Calibration standards 15.6#, 31.3 (LCS), 62.5, 125, 250, 500, 750, 1000, (two wells per CS including 2000 (HCS) and 3000# pg/mL anchoring points#) in assay The imprecision (% CV), must be ≤20% for CS diluent from the kit ranged from 31.3 (LCS) to 2000 pg/mL (HCS). The inaccuracy (% RE) must be within ±20% (±25% at the LCS and the HCS). No more than two CS (30%) may be excluded from the calibration curve, which must contain finally at least 6 calibration concentration levels, including the LCS and HCS. LLOQ in human K25-EDTA 31.3 pg/mL plasma ULOQ in human K2-EDTA 1800 pg/mL plasma QC samples QC.Low (101 pg/mL), QC.Mid (532 pg/mL) and (n = 2 duplicates, unspiked QC.High (1080 pg/mL) or spiked samples prepared % CV must be ≤20% and % RE must be within ±20%. in undiluted or diluted At least 4/6 QC samples must be within acceptance human K.sub.2-EDTA plasma) criteria (2/6 QC samples, not both at the same concentration, may be outside the acceptance criteria). Minimum required dilution None (MRD) Dilution linearity Up to 1/100 in calibrator diluent RD5-18 Specificity against No interference was demonstrated in any of the endogenous matrix blank human K.sub.2-EDTA plasma samples tested. components Stability in human K.sub.2-EDTA Freeze/thaw (F/T) 3 cycles at −24° C. ± 6° C. plasma stability 3 cycles at −75° C. ± 10° C. Short-term stability 24 hours at room temperature 24 hours at +5° C. ± 5° C. Long-term (LT) stability 670 days at −24° C. ± 6° C. 670 days at −75° C. ± 10° C. LCS: lowest calibration standard; HCS: highest calibration standard; CS: calibration standard(s); QC: quality control; RE: relative error; CV: coefficient of variation; LLOQ: lower limit of quantification; ULOQ: upper limit of quantification.

Cytokines Assays

[0336] The Cytometric Bead Array (CBA) Human Soluble Protein Flex Set System (BD Biosciences, San Jose, Calif., USA) was used for the simultaneous detection of 12 cytokines/chemokines and endothelial markers (IL-1β (interleukin-1(3), IL-6 (interleukin-6), IL-8 (interleukin-8), IL-10 (interleukin-10), MCP-1 (monocyte chemoattractant protein-1), INFγ (interferon γ), RANTES (regulated on activation, normal T cell expressed and secreted), VEGF (vascular endothelium growth factor), ICAM-1 (intercellular adhesion molecule 1), E-selectin, P-selectin and VCAM-1 (vascular cell adhesion molecule 1)) in plasma. The CBA technique was performed according to the manufacturer's instructions. The detection sensitivities were 36.06 pg/mL for ICAM-1, E-selectin, P-selectin, VCAM-1 and 9.77 pg/mL for IL-1β, IL-6, IL-8, IL-10, MCP-1, INFγ, RANTES, VEGF. Data were analyzed using the FCAP Array Infinite software (SoftFlow Group, Hungary). Alternatively, plasma concentrations of Ang1 (angiopoietin-1) and Ang2 (angiopoietin-2) were measured by commercially available specific enzyme-linked immunosorbent assay (Human Angiopoietin-1/Angiopoietin-2 Quantikine® ELISA Kit, R&D Systems), according to the manufacturer's instructions. The detection limits were 10.3 pg/mL (Ang 1) and 21.3 pg/mL (Ang2).

Statistical Analysis

[0337] Categorical variables were expressed as number (%) and compared by χ.sup.2 test between groups. Continuous variables were expressed as median (interquartile range) and compared with the Mann-Whitney U test. Receiver-operating characteristic (ROC) curves were constructed to illustrate cut-off values of sTREM-1.

Time-to-event analyses were performed by Kaplan-Meier survival analysis, in which a penalty for death was applied. Missing values were replaced by using the last observation carried forward (LOCF) method when indicated.
Time to ICU exit (exit from intensive care unit) and time to becoming IVM free (extubated from invasive mechanical ventilation, i.e., time to extubation) analyses were conducted separately. Subjects censored due to death were considered as being censored at day 35 for the time-to-event analyses. The Product-Limit Estimates (Kaplan-Meier curves) were estimated first, without any consideration of sTREM-1 class. Subsequently Cox proportional hazard (PH) models were fitted separately for each of these categorical predictors: sTREM-1 classifier at day 1 (baseline), and sTREM-1 classifier at day 3. From each of the Cox PH models, the hazards ratios for the endpoints were produced along with their 95% confidence intervals and p-values from a log-rank test. From each Cox PH model, the cumulative incidence function at each classifier level was plotted and overlaid alongside the Kaplan-Meier curve.
ROC (Receiver Operator Characteristics) curves were used to investigate the sTREM-1 as a classification factor (i) for MV (Mechanical Ventilation) duration of <15 days or (ii) for MV duration of >15 days and non-survivors, as a supportive analysis. The sensitivity (true positive rate) and specificity (true negative rate) were explored using these curves.
The sTREM-1 values at day 1 and day 3 were used in this analysis, separately. The Area Under the Curve (AUC) of the ROC curve was used to assess performance of sTREM-1.

Results

Healthy Volunteers Characteristics

[0338] Characteristics of the healthy volunteers are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Characteristics of the healthy volunteers Healthy volunteers (n = 21) Age (years) 42 (22-48.5) Gender (n, %) Male 9 (43) Female 12 (57) BMI (kg/m.sup.2) NA Comorbidity (n, %) Diabetes mellitus NA Cardiovascular disease Pulmonary disease Malignancy Length of hospital stay (days) NA MV duration (days) NA Mortality (n, %) NA sTREM-1 (pg/mL) 104 (75-124) IL-6 (pg/mL) NA Data are presented as median (IQR) or n (%). BMI: body mass index. MV: mechanical ventilation. IL-6: interleukin-6.

Patients Characteristics

[0339] Baseline characteristics are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Baseline characteristics of the patients included in the cohort MV ≥ 15 All patients MV < 15 days days and NS p- (n = 27) (n = 11) (n = 16) value Characteristics Age (years) 63 (56-71) 62 (52-71) 64 (59-71) 0.267 BMI (kg/m.sup.2) 28.9 (26.1-31.18) 29.65 (25.95-31.18) 27.95 (26.1-32.55) 0.5845 Male, n (%) 19 (70.37%) 5 (45.45%) 14 (87.50%) 0.0187 Comorbidities Hypertension, n (%) 11 (40.74%) 3 (27.27%) 8 (50.00%) 0.2376 Diabetes, n (%) 4 (14.81%) 0 (0.00%) 4 (25.00%) 0.0724 CV disease, n (%) 1 (3.70%) 0 (0.00%) 1 (6.25%) 0.3981 Pulmonary disease, n (%) 1 (3.70%) 0 (0.00%) 1 (6.25%) 0.3981 Cardiocirculatory failure Vasopressors, n (%) 22 (81.48%) 9 (81.82%) 13 (81.25%) 0.9993 TROPO > 12 pg/mL, n (%) 17 (62.96%) 6 (54.55%) 11 (68.75%) 0.9702 AKI KDIGO 1 (Creat 15-30), n (%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0.223 KDIGO 2 (Creat 30-40), n (%) 0 (0.00%) 0 (0.00%) 0 (0.00%) KDIGO 3 (Creat >40 or RRT), n (%) 2 (7.41%) 0 (0.00%) 2 (12.50%) ARDS PaO.sub.2/FiO.sub.2 ratio <100, n (%) 10 (37.04%) 2 (18.18%) 8 (50.00%) 0.0457 PaO.sub.2/FiO.sub.2 ratio 100-200, n (%) 13 (48.15%) 6 (54.55%) 8 (50.00%) PaO.sub.2/FiO.sub.2 ratio 200-300, n (%) 4 (14.81%) 3 (27.27%) 0 (0.00%) Outcome Secondary Infection, n (%) 9 (33.33%) 1 (9.09%) 8 (50.00%) 0.0267 VAP, n (%) 8 (29.63%) 0 (0.00%) 8 (50.00%) Length of stay (days) 17 (9-19) 10 (8-12) 18 (17-21.75) 0.001 MV duration (days) 15 (8-17) Unless indicated, values are presented as median (interquartile range). P-values are comparisons between mechanical ventilation (MV) < 15 days subgroup and MV ≥ 15 days and non-survivors (NS) subgroup. BMI: body mass index. VAP: ventilator-associated pneumonia. MV: mechanical ventilation. NS: non-survivors. CV disease: cardiovascular disease. Creat: creatine. RRT: renal replacement therapy. ARDS: acute respiratory distress syndrome. TROPO: troponin levels (pg/mL). AKI: acute kidney injury. KDIGO: kidney disease improving global outcomes. PaO.sub.2/FiO.sub.2 ratio: ratio of artery partial pressure of oxygen/inspired oxygen fraction.

[0340] Among the 27 patients of this cohort, 22 patients required vasopressors and 17 had cardiac troponin levels above 12 pg/mL, 2 patients had a KDIGO of 3, 10 patients presented with severe ARDS, 13 with moderate ARDS, and 4 with mild ARDS. 9 patients developed secondary infection, of whom 8 were suffering from ventilator-associated pneumonia (VAP). 3 patients died, and the median duration of mechanical ventilation (MV) was 15 days. This value was unchanged when a penalty of 33 days was attributed for MV duration for non-survivors, which corresponds to the maximum duration of MV observed in that cohort.

11 patients had MV duration below 15 days, and 16 had at least 15 days of MV or died. A higher proportion of male and a higher proportion of patients with severe ARDS had MV duration of at least 15 days or died. These patients had a higher frequency of secondary infections.
No significant differences in baseline and day 3 hematology and biochemistry parameters were observed between these two groups, with an exception for white blood cells count (WBC) and neutrophils at day 3, but values were within normal range.
sTREM-1 Levels were More Elevated in COVID-19 Patients than in Healthy Volunteers (HV), Both at Baseline and at Day 3

[0341] The median sTREM-1 plasma concentration in healthy volunteers (HV) was 103.5 pg/mL (IQR (interquartile range) 75.22-124.4), and median sTREM-1 plasma concentration in COVID-19 patients was 161.1 pg/mL (IQR 129.4-195.7) at baseline, and 142 pg/mL (IQR 108.7-187.3) at day 3 (see FIG. 1). As shown on FIGS. 1A-B, the difference between the sTREM-1 plasma concentration of HV and that of COVID-19 patients was statistically different. Interestingly, even if only 3 patients died, significantly higher sTREM-1 levels were observed in non-survivors (see FIG. 2) as compared to sTREM-1 levels in COVID-19 patients who survived, both at baseline (see FIG. 2A) and at day 3 (see FIG. 2B). The median sTREM-1 plasma concentration in non-survivors (NS) was 422.6 pg/mL at baseline, and 300 pg/mL at day 3.

Higher sTREM-1 Levels were Observed at Baseline (Day 1) and Day 3 in Patients Who Required Longer Duration Under Mechanical Ventilation (MV).

[0342] Indeed, at baseline and day 3, a significant difference in sTREM-1 plasma concentration was observed in the subgroup including patients who did not survive and patients who required 10 days or more under MV, as compared to the subgroup of patients who required less than 10 days under MV. This difference was still present in the subgroup including non-survivors and patients who required 15 days or more under MV, as compared to the subgroup of patients who required less than 15 days under MV (see FIGS. 3A-B and Table 4 below). This was further confirmed when looking at the kinetics of sTREM-1 in these two subgroups (see FIG. 4A). Indeed, non-survivors and patients with 15 days or more under MV showed more elevated levels of sTREM-1 from baseline (day 1) to discharge (day 21), as compared to patients who required less than 15 days under MV.

TABLE-US-00004 TABLE 4 Hematology data, biochemistry data, and inflammatory markers in all COVID-19 patients, and in the two subgroups defined by either less than 15 days under MV, or by death or at least 15 days under MV All patients MV < 15 days MV ≥ 15 days and NS p- (n = 27) (n = 11) (n = 16) value Hematology WBC (G/L) D 1 7380 (6400-9640) 6830 (6310-7630) 8405 (6518-10375) 0.1244 D 3 7690 (6493-9393) 7310 (5020-8500) 8390 (6940-11370) 0.0428 Monocytes (G/L) D 1 300 (170-410) 340 (240-440) 215 (170-402.5) 0.3108 D 3 435 (277.5-827.5) 420 (270-880) 450 (280-810) 0.8284 Ly (G/L) D 1 6403 (440-1010) 640 (400-1010) 655 (492.5-1050) 0.8179 D 3 870 (485-1368) 1010 (530-1620) 860 (350-1200) 0.4127 PMN (G/L) D 1 6420 (4950-8080) 5570 (4840-6820) 7310 (4985-8215) 0.1988 D 3 5830 (5005-7225) 4990 (4320-5830) 6450 (5700-8620) 0.0009 PLT (G/L) D 1 230500 (165500-257250) 240500 (206750-257250) 197000 (146750-261500) 0.1654 D 3 252500 (185750-344500) 290000 (205000-335000) 251000 (163000-352000) 0.413 Biochemistry Hg (g/dL) D 1 12.4 (10.9-13.8) 12.4 (10.7-13.4) 12.75 (10.95-13.88) 0.4731 D 3 11.3 (10.38-12.05) 10.7 (10-11.6) 11.8 (10.5-12.8) 0.0666 Bilirubin (mg/L) D 1 7 (5-8) 5 (5-7) 8 (6-8.75) 0.0931 D 3 6 (4-12) 4.5 (4-8.5) 7 (5-13) 0.2867 AST (U/L) D 1 71 (47-106) 74 (24-116) 70 (52.75-100.3) 0.8557 D 3 56.5 (43.25-117.5) 52 (39-159) 64 (47-103) 0.5232 Creatinine kinase (U/L) D 1 198.5 (74.25-535.3) 105 (46.5-341) 275 (106-836) 0.2093 D 3 138 (33-399) 61 (34-289) 149.5 (31.5-598.8) 0.9048 TP (g/L) D 1 56 (51-59) 56 (51-59) 56 (51.25-59) 0.7793 D 3 54.5 (51.25-57) 54 (51.5-55) 56 (51-58) 0.3393 Lactate (mmol/L) D 1 1.2 (0.6-1.8) 1 (0.7-1.7) 1.2 (0.9-1.575) 0.7611 D 3 1.1 (0.9-1.6) 1.35 (0.8-1.6) 1.1 (1-1.7) 0.88 Inflammatory markers (pg/mL) sTREM-1 D 1 161.1 (129.4-195.7) 150 (94.48-194.9) 176.8 (147-195.9) 0.1383 D 3 142 (108.7-187.3) 117.4 (106.8-129.9) 163.4 (142-263.7) 0.0375 ICAM-1 D 1 79141 (64361-118402) 79337 (67209-152074) 78945 (64004-120051) 0.7702 D 3 109646 (78067-149311) 110651 (66034-185491) 109646 (84864-148100) 0.935 E-Selectin D 1 8047 (5467-12088) 6945 (4133-10925) 8330 (6698-12224) 0.2384 D 3 7676 (4590-10856) 6284 (4308-9382) 7676 (6257-11019) 0.4865 P-Selectin D 1 7387 (5297-20841) 8965 (5366-23250) 7342 (4840-20436) 0.482 D 3 9227 (7206-16571) 10649 (7117-21825) 8019 (7079-15062) 0.683 VCAM-1 D 1 404614 (356629-470779) 393329 (356685-409567) 446181 (313891-478054) 0.446 D 3 375892 (293199-455877) 377341 (308508-453248) 366593 (241842-475257) 0.683 IL-6 D 1 172.2 (94.57-231.7) 145.7 (97.2-342.4) 184.4 (61.2-221.2) 0.9534 D 3 194.6 (112.4-372) 126.2 (95.47-335.5) 216.7 (119.4-395.8) 0.3669 IL-8 D 1 49.37 (39.04-64.63) 49.28 (33.77-72.63) 53.24 (44.55-64.96) 0.6824 D 3 43.91 (25.48-63.12) 34.62 (22.97-54.91) 46.86 (25.61-83.87) 0.311 IL-10 D 1 5.225 (1.583-18.38) 5.1 (2.28-21.03) 5.35 (0-19.97) 0.6077 D 3 0 (0-8.52) 4.265 (0-9.708) 0 (0-7.53) 0.4605 MCP-1 D 1 294.5 (189.4-515.3) 197.9 (108-363.8) 400.7 (266.8-571.6) 0.0732 D 3 194 (81.23-506.1) 168.2 (48.07-258.2) 282.3 (165.2-566) 0.091 RANTES D 1 10023 (8309-15045) 11737 (8308-17880) 9840 (8303-11594) 0.446 D 3 11923 (10013-15182) 12307 (10872-15660) 11437 (9730-14929) 0.4284 Ang1 D 1 10768 (7530-13344) 11508 (7653-14140) 9243 (7328-13588) 0.5985 D 3 11880 (7988-14845) 11451 (8042-14203) 12053 (6878-16380) 0.8918 Ang2 D 1 2103 (1672-2862) 1711 (1591-2853) 2132 (1768-2870) 0.446 D 3 4517 (3014-6115) 3780 (2743-5046) 4803 (3655-7090) 0.238 Ang2/Ang1 D 1 0.18 (0.14-0.3425) 0.15 (0.125-0.3) 0.19 (0.14-0.49) 0.2688 D 3 0.39 (0.255-0.535) 0.325 (0.185-1.8475 0.4 (0.27-0.46) 0.4695 sTREM-1 D 3 − D 1 −2.995 (−36.47-18.83) −9.675 (−35.06-15.86) 1.655 (−38.85-42.96) 0.9203 % D 3 − D 1 −2.785 (−20.02-13.33) −7.09 (−19.01-17.1) 0.87 (−20.43-15.13) 0.9734 P-values are comparisons between the MV < 15 days subgroup and the MV ≥ 15 days and non-survivors (NS) subgroup. MV: mechanical ventilation. NS: non-survivors. D 1: day 1. D 3: day 3. WBC: white blood cells. Ly: lymphocytes. PMN: polymorphonuclear neutrophils. PLT: platelets. Hg: hemagglutinin. AST: aspartate aminotransferase TP: total proteins. ICAM-1: intercellular adhesion molecule 1. VCAM-1: vascular cell adhesion molecule 1. IL-6: interleukin 6. IL-8: interleukin-8. IL-10: interleukin-10. MCP-1: monocyte chemoattractant protein-1. RANTES: regulated on activation, normal T cell expressed and secreted. Ang1: angiopoietin-1. Ang2: angiopoietin-2. D3-D1 is the difference between sTREM-1 concentration at day 3 and day 1. % D3-D1 is the percentage of that difference compared to the value at D1.

[0343] The median duration of mechanical ventilation (MV) was 15 days in the study cohort (see Table 3 above). As indicated above, 11 patients had MV duration below 15 days, and 16 had at least 15 days of MV or died. As shown on FIGS. 3A-B, patients who required less than 15 days under MV had similar sTREM-1 levels at baseline and day 3 (150 pg/mL (IQR: 94,48-194.9) and 117.4 pg/mL (IQR:106.8-129.9), respectively) as compared to healthy volunteers (103.5 pg/mL (IQR:75.22-124.4)). Strikingly, patients requiring 15 days or more under MV and non-survivors had significantly higher sTREM-1 levels at baseline (176.8 pg/mL (IQR:147-195.9), p<0.0001) and at day 3 (163.4 pg/mL (IQR:142-263.7), p=0.0003) as compared to healthy volunteers (see FIGS. 3A-B), and had significantly higher sTREM-1 levels at day 3 as compared to patients who required less than 15 days incidence under MV (see FIG. 3B).

[0344] As shown in FIGS. 5A-B, sTREM-1 concentration at day 3 yielded a discriminative value in identifying patients with MV needs below 15 days from patients with MV needs above 15 days, with an area under the curve of 0.733 (95% confidence interval 0.4633-1.000) and a p-value of 0.0431. A sTREM-1 cut-off value of 186 pg/mL provided approximately 80% specificity and 90% sensitivity. Based on the sTREM-1 cut-off value of 186 pg/mL, analyses of the time to ICU exit and time to IMV free (i.e., time to extubation from IMV) were performed. For time to becoming IVM free (i.e., extubated from invasive mechanical ventilation), the hazard ratio (HR) and its respective 95% CI was 0.411 (0.155,1.087) at baseline (day 1) (see FIG. 6A), and 0.153 (0.034,0.685) at day 3 (see FIG. 6B). For time to ICU exit (i.e., exit from intensive care unit), the HR and its respective 95% CI was 0.374 (0.141,0.992) at baseline (day 1) (see FIG. 6C), and 0.151 (0.033, 0.681) at day 3 (see FIG. 6D).

[0345] The data thus show that COVID-19 patients displaying sTREM-1 levels at baseline and day 3 above 186 pg/mL had lower likelihood to be extubated before 15 days or to survive (baseline HR: 0.374 (95% CI 0.141-0.992), day3 HR: 0.151 (95% CI 0.033-0.681)).

Correlations Between sTREM-1 and Other Clinical Parameters

[0346] To further determine whether TREM-1 pathway activation was associated with complications in COVID-19, correlations between sTREM-1 and clinical parameters and features of COVID-19 were assessed at day 1 and day 3 (see Tables 5-7 below).

[0347] Lymphocyte levels have been found to be predictor of prognosis in COVID-19 patients, and individuals who died of COVID-19 are demonstrated to have had expressively lower lymphocyte levels than survivors. Interestingly, at day 3, sTREM-1 showed a significant inverse correlation with lymphocyte counts in all COVID-19 patients (spearman r −0.6593 (−0.8530 to −0.3058), p-value=0.0012, see Table 5). This inverse correlation was lost when looking at less severely ill patients requiring less than 15 days under MV (see Table 6), but was still present in more severely ill patients (spearman r −0.5699 (−0.8425 to −0.06476), p-value 0.0291, see Table 7). sTREM-1 levels were also found to be correlated with bilirubin levels at day 3 (spearman r 0.5196 (0.07123 to 0.7933) p-value 0.0226, see Table 5), as well as with urea and creatine levels (see below Table 5, Table 6, and Table 7), different markers of liver and renal suffering or dysfunction. A positive correlation was also found between sTREM-1 and troponin levels in all patients (r=0.6559 p=0.0096, see Table 5), and this correlation was even more pronounced in the more severely ill patients (r=−0.8333, p=0.0154, see Table 7). This association between TREM-1 pathway activation and cardiac failure was confirmed by looking at the kinetics of sTREM-1 in patients presenting with troponins levels above 12 pg/mL: they showed higher levels of sTREM-1 from inclusion (day 1) to discharge (day 21), as compared to patients presenting with troponin levels below 12 pg/mL (see FIG. 4B).

[0348] Organ dysfunction, and in particular renal, liver and/or cardiac dysfunction, is a major complication in COVID-19 patients, associated with complicated and poor outcome. The data confirm that TREM-1 pathway engagement may drive inflammation and complications in COVID-19 patients.

[0349] No detectable levels of INF-γ, IL-1β, and VEGF, elevated levels of ICAM-1, E-selectin, P-selectin, VCAM-1, IL-6, IL-8, IL-10, MCP-1, RANTES, Ang1, and Ang2 were observed in the patients of the cohort (see Table 4 above). Unexpectedly, sTREM-1 was the only marker that showed a difference between the two groups of patients defined by either less than 15 days under MV, or by death or at least 15 days under MV. The difference was more pronounced, and statistically significant, at day 3.

TABLE-US-00005 TABLE 5 Correlation table between sTREM-1 and clinical and inflammatory markers in all COVID-19 patients Correlation table All patients (n = 27) sTREM-1 with Spearman r (95% IC) p-value n WBC D 1 0.3662 (−0.05629 to 0.6774) 0.0784 24 D 3 −0.1364 (−0.5461 to 0.3261) 0.5556 21 Monocytes D 1 −0.04224 (−0.4483 to 0.3783) 0.8446 24 D 3 −0.2762 (−0.6406 to 0.1898) 0.2256 21 Lymphocytes D 1 −0.3484 (−0.6663 to 0.07656) 0.0952 24 D 3 −0.6593 (−0.8530 to −0.3058) 0.0012 21 ** Neutrophils D 1 0.3487 (−0.07624 to 0.6664) 0.0949 24 D 3 0.1494 (−0.3142 to 0.5554) 0.5182 21 Platelets D 1 −0.1391 (−0.5229 to 0.2916) 0.5167 24 D 3 −0.4208 (−0.7279 to 0.02698) 0.0575 21 Hemoglobin D 1 0.01174 (−0.4041 to 0.4236) 0.9566 24 D 3 0.1222 (−0.3389 to 0.5359) 0.5978 21 ALAT D 1 −0.08612 (−0.4828 to 0.3399) 0.6891 24 D 3 0.009785 (−0.4459 to 0.4615) 0.9673 20 ASAT D 1 −0.09702 (−0.4912 to 0.3302) 0.652 24 D 3 0.006769 (−0.4484 to 0.4591) 0.9774 20 Bilirubin D 1 0.3545 (−0.06964 to 0.6701) 0.0892 24 D 3 0.5196 (0.07123 to 0.7933) 0.0226 19 * Urea D 1 0.677 (0.3655 to 0.8521) 0.0003 24 *** D 3 0.5751 (0.2212 to 0.7952) 0.0026 25 ** Creatine D 1 0.7306 (0.4540 to 0.8788) <0.0001 24 **** D 3 0.7372 (0.4732 to 0.8797) <0.0001 25 **** Total Protein D 1 0.2311 (−0.2022 to 0.5887) 0.2773 24 D 3 0.003493 (−0.4110 to 0.4168) 0.9871 24 Lactate D 1 0.002182 (−0.4121 to 0.4157) 0.9919 24 D 3 0.1833 (−0.2401 to 0.5480) 0.3806 25 Troponin D 1 0.3926 (−0.02547 to 0.6938) 0.0577 24 D 3 0.6559 (0.2003 to 0.8783) 0.0096 15 ** ICAM-1 D 1 −0.193 (−0.5621 to 0.2401) 0.3661 24 D 3 −0.01615 (−0.4189 to 0.3919) 0.9389 25 E-selectin D 1 −0.213 (−0.5762 to 0.2203) 0.3175 24 D 3 0.3701 (−0.04169 to 0.6744) 0.0686 25 P-selectin D 1 −0.1652 (−0.5421 to 0.2670) 0.4404 24 D 3 −0.1377 (−0.5145 to 0.2836) 0.5116 25 VCAM-1 D 1 −0.02783 (−0.4367 to 0.3906) 0.8973 24 D 3 0.2169 (−0.2068 to 0.5721) 0.2976 25 IL-6 D 1 −0.02261 (−0.4325 to 0.3950) 0.9165 24 D 3 0.3462 (−0.06904 to 0.6591) 0.0901 25 IL-8 D 1 0.413 (−0.001068 to 0.7062) 0.0448 24 * D 3 0.5123 (0.1348 to 0.7599) 0.0088 25 ** IL-10 D 1 −0.03967 (−0.4463 to 0.3805) 0.854 24 D 3 0.3841 (−0.02541 to 0.6832) 0.058 25 MCP-1 D 1 0.3991 (−0.01773 to 0.6978) 0.0533 24 D 3 0.6508 (0.3332 to 0.8357) 0.0004 25 *** RANTES D 1 −0.2461 (−0.5990 to 0.1869) 0.2464 24 D 3 −0.3631 (−0.6699 to 0.04975) 0.0744 25 Ang1 D 1 0.1148 (−0.3141 to 0.5047) 0.5933 24 D 3 −0.2354 (−0.5850 to 0.1881) 0.2574 25 Ang2 D 1 0.1722 (−0.2603 to 0.5471) 0.4211 24 D 3 0.2592 (−0.1635 to 0.6015) 0.2108 25 Ang2/Ang1 D 1 0.09057 (−0.3360 to 0.4863) 0.6738 24 D 3 0.3882 (−0.02051 to 0.6858) 0.0551 25 WBC: white blood cells. ALAT: alanine aminotransferase. ASAT: aspartate aminotransferase. ICAM-1: intercellular adhesion molecule 1. VCAM-1: vascular cell adhesion molecule 1. IL-6: interleukin 6. IL-8: interleukin-8. IL-10: interleukin-10. MCP-1: monocyte chemoattractant protein-1. RANTES: regulated on activation, normal T cell expressed and secreted. Ang1: angiopoietin-1. Ang2: angiopoietin-2. * indicates statistical significance with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0005.

TABLE-US-00006 TABLE 6 Correlation table between sTREM-1 and clinical and inflammatory markers in COVID-19 patients with MV duration below 15 days Correlation table MV < 15 (n = 11) sTREM-1 with Spearman r (95% IC) p-value n WBC D 1 0.5667 0.1206 9 D 3 0.1581 0.6621 10 Monocytes D 1 −0.08333 0.8432 9 D 3 0.1337 0.7132 10 Lymphocytes D 1 −0.2167 0.5809 9 D 3 −0.1636 0.6567 10 Neutrophils D 1 0.5667 0.1206 9 D 3 0.3333 0.3487 10 Platelets D 1 −0.2167 0.5809 9 D 3 0.5152 0.1334 10 Hemoglobin D 1 −0.2833 0.463 9 D 3 0.1394 0.7072 10 ALAT D 1 0.06667 0.8801 9 D 3 0.3929 0.3956 7 ASAT D 1 0.08368 0.833 9 D 3 0.1071 0.8397 7 Bilirubin D 1 0.1368 0.7311 9 D 3 0.1518 0.8333 6 Urea D 1 0.4603 0.2149 9 D 3 0.3587 0.3067 10 Creatine D 1 0.7667 0.0214 9 * D 3 0.4303 0.2182 10 Total Protein D 1 −0.1681 0.6666 9 D 3 0.1519 0.6974 9 Lactate D 1 0.05858 0.886 9 D 3 −0.09847 0.7893 10 Troponin D 1 0.35 0.3586 9 D 3 0.1441 0.7603 7 ICAM-1 D 1 0.2167 0.5809 9 D 3 0.1152 0.7589 10 E-selectin D 1 −0.2333 0.5517 9 D 3 −0.6727 0.039 10 * P-selectin D 1 0.2167 0.5809 9 D 3 −0.1152 0.7589 10 VCAM-1 D 1 −0.45 0.2298 9 D 3 0.4545 0.1912 10 IL-6 D 1 0.2667 0.4933 9 D 3 0.1515 0.6821 10 IL-8 D 1 0.55 0.1328 9 D 3 0.2439 0.4936 10 IL-10 D 1 −0.03347 0.9377 9 D 3 0.1741 0.8 10 MCP-1 D 1 0.7167 0.0369 9 * D 3 0.2364 0.5135 10 RANTES D 1 −0.06667 0.8801 9 D 3 −0.7333 0.0202 10 * Ang1 D 1 0.2 0.6134 9 D 3 0.3455 0.3304 10 Ang2 D 1 0.3667 0.3363 9 D 3 0.09091 0.8113 10 Ang2/Ang1 D 1 0.2845 0.454 9 D 3 −0.103 0.785 10 WBC: white blood cells. ALAT: alanine aminotransferase. ASAT: aspartate aminotransferase. ICAM-1: intercellular adhesion molecule 1. VCAM-1: vascular cell adhesion molecule 1. IL-6: interleukin 6. IL-8: interleukin-8. IL-10: interleukin-10. MCP-1: monocyte chemoattractant protein-1. RANTES: regulated on activation, normal T cell expressed and secreted. Ang1: angiopoietin-1. Ang2: angiopoietin-2. * p < 0.05.

TABLE-US-00007 TABLE 7 Correlation table between sTREM-1 and clinical and inflammatory markers in most severe COVID-19 patients, non-survivors and patients with MV duration above 15 days Correlation table MV >= 15 and NS (n = 16) sTREM-1 with Spearman r (95% IC) p-value n WBC D 1 0.175 (−0.3848 to 0.6407) 0.532 15 D 3 −0.2214 (−0.6683 to 0.3429) 0.4266 15 Monocytes D 1 −0.03943 (−0.5525 to 0.4953) 0.8926 15 D 3 −0.3596 (−0.7438 to 0.2033) 0.1873 15 Lymphocytes D 1 −0.3714 (−0.7498 to 0.1901) 0.1735 15 D 3 −0.5699 (−0.8425 to −0.06476) 0.0291 15 * Neutrophils D 1 0.1321 (−0.4216 to 0.6141) 0.6389 15 D 3 −0.03571 (−0.5499 to 0.4981) 0.9031 15 Platelets D 1 −0.007143 (−0.5297 to 0.5193) 0.9847 15 D 3 −0.7286 (−0.9066 to −0.3303) 0.0029 15 ** Hemoglobin D 1 0.08929 (−0.4566 to 0.5863) 0.7532 15 D 3 −0.05357 (−0.5623 to 0.4845) 0.8525 15 ALAT D 1 −0.1357 (−0.6163 to 0.4186) 0.6297 15 D 3 0.4897 (−0.1021 to 0.8255) 0.0914 13 ASAT D 1 −0.2218 (−0.6685 to 0.3425) 0.4238 15 D 3 −0.07428 (−0.6123 to 0.5107) 0.8101 13 Bilirubin D 1 0.2853 (−0.2813 to 0.7044) 0.3008 15 D 3 0.233 (−0.3806 to 0.7042) 0.4404 13 Urea D 1 0.6512 (0.1924 to 0.8764) 0.0101 15 * D 3 0.6409 (0.1754 to 0.8722) 0.0118 15 * Creatine D 1 0.5165 (−0.01092 to 0.8191) 0.0508 15 D 3 0.7721 (0.4162 to 0.9229) 0.0011 15 ** Total Protein D 1 0.4269 (−0.1257 to 0.7773) 0.1133 15 D 3 −0.2471 (−0.6831 to 0.3187) 0.3718 15 Lactate D 1 −0.2009 (−0.6562 to 0.3617) 0.4696 15 D 3 0.2757 (−0.2909 to 0.6991) 0.3174 15 Troponin D 1 0.2395 (−0.3259 to 0.6787) 0.3868 15 D 3 0.8333 ( ) 0.0154 8 * ICAM-1 D 1 −0.2607 (−0.6908 to 0.3056) 0.3469 15 D 3 0.3843 (−0.1756 to 0.7563) 0.1573 15 E-selectin D 1 −0.3607 (−0.7444 to 0.2020) 0.187 15 D 3 0.01071 (−0.5167 to 0.5322) 0.9744 15 P-selectin D 1 −0.1679 (−0.6363 to 0.3911) 0.5492 15 D 3 0.35 (−0.2137 to 0.7389) 0.2012 15 VCAM-1 D 1 0.1464 (−0.4095 to 0.6231) 0.6024 15 D 3 0.1643 (−0.3942 to 0.6341) 0.558 15 IL-6 D 1 −0.1643 (−0.6341 to 0.3942) 0.558 15 D 3 0.625 (0.1495 to 0.8657) 0.0148 15 * IL-8 D 1 0.3643 (−0.1980 to 0.7462) 0.1824 15 D 3 0.6245 (0.1487 to 0.8655) 0.0152 15 * IL-10 D 1 −0.01261 (−0.5336 to 0.5153) 0.9664 15 D 3 −0.1268 (−0.6107 to 0.4260) 0.6571 15 MCP-1 D 1 0.1036 (−0.4451 to 0.5957) 0.7144 15 D 3 0.6357 (0.1669 to 0.8701) 0.0128 15 * RANTES D 1 −0.3071 (−0.7163 to 0.2591) 0.265 15 D 3 −0.2 (−0.6557 to 0.3625) 0.4738 15 Ang1 D 1 0.09643 (−0.4509 to 0.5910) 0.7337 15 D 3 −0.4321 (−0.7799 to 0.1194) 0.1094 15 Ang2 D 1 −0.01071 (−0.5322 to 0.5167) 0.9744 15 D 3 0.3357 (−0.2291 to 0.7314) 0.2212 15 Ang2/Ang1 D 1 −0.03757 (−0.5512 to 0.4967) 0.8952 15 D 3 0.6845 (0.2497 to 0.8896) 0.0061 15 ** WBC: white blood cells. ALAT: alanine aminotransferase. ASAT: aspartate aminotransferase. ICAM-1: intercellular adhesion molecule 1. VCAM-1: vascular cell adhesion molecule 1. IL-6: interleukin 6. IL-8: interleukin-8. IL-10: interleukin-10. MCP-1: monocyte chemoattractant protein-1. RANTES: regulated on activation, normal T cell expressed and secreted. Ang1: angiopoietin-1. Ang2: angiopoietin-2. * indicates statistical significance with * p < 0.05, and ** p < 0.01.

Example 2

Materials and Methods

Study Design and Participants

[0350] The retrospective study presented below was conducted on a validation cohort of 192 patients with COVID-19 (both ICU and non-ICU) admitted to the Radboud University Medical Centre (Radboudumc), Nijmegen, the Netherlands. The validation cohort consists of patients with a PCR-proven or clinically diagnosed SARS-CoV-2 infection admitted to the Radboudumc between 6 Mar. 6th 2020 and Apr. 15.sup.th 2020. Clinical diagnosis of COVID-19 infection was defined based on signs and symptoms, specific computed tomography (CT) findings according to the Dutch COVID-19 Reporting and Data System (CO-RADS) classification, and final consensus of clinical experts. 95% (183/192) of the COVID-19-diagnosed patients had a positive PCR result at the time of diagnosis. The study protocol was approved by the local ethics committee (CMO 2020 6344 and CMO 2016 2963). All patients or legal representatives were informed about the study details and could decline to participate.

[0351] Ethylenediaminetetraacetic acid (EDTA) plasma was collected at the first routine blood withdrawal for laboratory testing after COVID-19 diagnosis in the hospital (i.e., baseline). For analysis, patients were stratified into groups based on disease severity and mortality. Disease severity was defined based on the need for ICU admission during hospital stay: severe illness was defined as patients needing ICU admission, and moderate illness was defined as patients without the need for ICU admission during hospital stay. patients were admitted to a non-ICU ward at the time of sampling, but needed ICU admission later during their hospital stay. These patients were classified in the severe illness group.

Data Collection

[0352] Clinical data and laboratory results were collected from the electronic patient files (EPIC, EPIC Systems Corporation, Verona, Wis., USA) and recorded in electronic Case Report Forms (Castor EDC, Amsterdam, the Netherlands). Values of white blood cell (WBC) counts, lymphocyte counts, C-reactive protein (CRP), ferritin and D-dimer were collected at the day of plasma sampling. Clinical outcomes (ICU admission, hospital length of stay, ICU length of stay, incidence of thromboembolic events (TEE), and mortality) were recorded until hospital discharge.

Measurements and Assays

[0353] Venous blood was collected in EDTA tubes and thereafter centrifuged at 2954 g (3800 rpm) at room temperature for 10 minutes. Plasma was collected and aliquoted before storage at −80° C. for further analysis. Concentrations of IL-6 were measured using enzyme-linked immunosorbent assays (ELISA, Quantikine, R&D systems) according to the manufacturer's instructions, with a lower detection limit of 16 pg/mL. Plasma sTREM-1 levels were measured using an analytically validated ELISA assay according to regulatory requirements (EMA 2011) using a commercially available research use only ELISA assay (Human TREM-1 Quantikine® ELISA kit, R&D Systems, reference DTRM10C). This method was validated with lower and upper limit of quantification of 34.2 and 2070 pg/mL, respectively. For routine analysis, each analytical run contained 3 levels of quality control (QC) sample (Low, Mid and High, with QC.Low=93 pg/mL, QC.Mid=728 pg/mL, and QC.High=1610 pg/mL), and each run was accepted if standard curve and QC samples are within acceptance criteria. The analytical performances and acceptance criteria of this method are summarized in Table 1 above (see Example 1).

Statistical Analysis

[0354] The obtained data were analyzed using SPSS version 25.0 (IBM Corp., Armonk, N.Y., USA), GraphPad Prism version 8.0 (GraphPad Software, Inc., San Diego, Calif., USA), and MedCalc version 19.6.4 (MedCalc Software Ltd, Ostend, Belgium). Differences between groups were assessed by Mann-Whitney U tests for continuous variables and by Fisher's exact tests for discrete variables. Correlations between sTREM-1 concentration and inflammatory parameters, and sTREM-1 concentration and duration of hospital stay were assessed by Spearman's rank correlation tests. Receiver operating characteristic (ROC) analyses were performed to assess the prognostic performance of several biomarkers by calculating the area under the curve (AUC). The optimal cut-off values for the biomarkers were defined based on the maximal Youden's J index and used to assess differences in survival during hospital stay for high versus low biomarker concentrations by Kaplan-Meier survival analysis. Hazard ratios were based on the log-rank test. A p-value <0.05 (two-tailed) was considered statistically significant.

Results

[0355] Elevated sTREM-1 Concentrations in Patients with Severe COVID-19

[0356] 218 patients diagnosed with COVID-19 and admitted to the Radboudumc were assessed for study inclusion (FIG. 7). Of these, 11 patients refused to participate. Moreover, 14 other patients were excluded because no plasma sample was available from first routine blood withdrawal after COVID-19 diagnosis (baseline), and one patient was excluded due to a measurement error. The final study population (n=192) was divided in groups based on disease severity (moderate illness (n=119) and severe illness (n=73)) and outcome (survivors (n=166) and non-survivors (n=26)). 72 patients out of the 73 patients admitted in ICU (i.e., severely ill patients) required invasive mechanical ventilation. 109 patients out of the 119 patients who were moderately ill (i.e., no ICU admission) received oxygen therapy: 8 patients received non-invasive ventilation and 101 patients received supplemental oxygen by nasal cannula or mask. Table 8 below shows the characteristics of the COVID-19 patients, divided in groups according to disease severity (i.e., moderate illness/severe illness). Table 9 below shows the characteristics of COVID-19 patients, divided in groups of according to outcome (i.e., survivors/non-survivors).

TABLE-US-00008 TABLE 8 Characteristics of the patients included in the validation cohort (all patients and patients divided in groups according to disease severity) All patients Moderate illness Severe illness P- (n = 192) (n = 119) (n = 73) value.sup.a Age (years) 65 (54-72) 66 (53-73) 64 (57-71) 0.285 Gender (n, %) Male 133 (69) 79 (66) 54 (74) 0.334 Female 59 (31) 40 (34) 19 (26) BMI (kg/m.sup.2) 26.4 (24.0-29.0) 26.0 (23.7-28.9) 26.8 (24.7-29.3) 0.250 Comorbidity (n, %) Diabetes mellitus 38 (20) 22 (19) 16 (22) 0.580 Cardiovascular disease 102 (53) 64 (54) 38 (52) 0.882 Pulmonary disease 39 (20) 31 (26) 8 (11) 0.016 Malignancy 48 (25) 34 (29) 14 (19) 0.171 Days of illness at day of sampling (days) 11 (8-15) 10 (8-14) 13 (9-16) 0.022 ICU admission (n, %).sup.b 73 (38) NA NA NA Length of hospital stay (days) 9 (6-24) 7 (5-9) 31 (19-45) <0.001 Mortality (n, %) 26 (14) 10 (8) 16 (22) 0.010 sTREM-1 (pg/mL) 208 (151-292) 195 (139-283) 235 (176-319) 0.017 CRP (mg/L) 109 (60-173) 81 (41-120) 175 (128-291) <0.001 Ferritin (μg/L) 1058 (543-1879) 822 (399-1461) 1694 (935-2554) <0.001 IL-6 (pg/mL).sup.c 72 (28-118) 43 (23-82) 144 (79-405) <0.001 Data are presented as median (IQR) or n (%). BMI: body mass index. CRP: C-reactive protein. IL-6: interleukin-6. .sup.aModerate illness versus severe illness (Mann-Whitney U test), .sup.bICU during total hospital admission, .sup.cMeasured in 151 of the 192 patients.

TABLE-US-00009 TABLE 9 Characteristics of the patients included in the validation cohort (all patients and patients divided in groups according to outcome) All patients Survivors Non-survivors P- (n = 192) (n = 166) (n = 26) value.sup.a Age (years) 65 (54-72) 64 (53-71) 73 (69-75) <0.001 Gender (n, %) Male 133 (69) 115 (69) 18 (69) 1.000 Female 59 (31) 51 (31) 8 (31) BMI (kg/m.sup.2) 26.4 (24.0-29.0) 26.6 (23.9-29.3) 25.8 (24.0-28.4) 0.651 Comorbidity (n, %) Diabetes mellitus 38 (20) 33 (20) 5 (19) 1.000 Cardiovascular disease 102 (53) 84 (51) 18 (69) 0.092 Pulmonary disease 39 (20) 31 (19) 8 (31) 0.188 Malignancy 48 (25) 41 (25) 7 (27) 0.810 Days of illness at day of sampling (days) 11 (8-15) 11 (8-15) 11 (8-17) 0.882 ICU admission (n, %).sup.b 73 (38) 57 (34) 16 (62) 0.010 Length of hospital stay (days) 9 (6-24) 9 (6-24) 15 (6-15) 0.339 Mortality (n, %) 26 (14) NA NA NA sTREM-1 (pg/mL) 208 (151-292) 199 (142-278) 326 (207-445) <0.001 CRP (mg/L) 109 (60-173) 103 (56-172) 139 (83-210) 0.154 Ferritin (μg/L) 1058 (543-1879) 996 (491-1864) 1270 (717-1962) 0.237 IL-6 (pg/mL).sup.c 72 (28-118) 67 (26-104) 188 (70-480) <0.001 Data are presented as median (IQR) or n (%). BMI: body mass index. CRP: C-reactive protein. IL-6: interleukin-6. .sup.aSurvivors versus non-survivors (Mann Whitney U test), .sup.bICU during total hospital admission, .sup.cMeasured in 151 of the 192 patients.

[0357] As compared to moderately ill patients (i.e., patients only admitted to the clinical ward), severely ill patients had higher concentrations of inflammatory parameters (CRP, D-dimer and IL-6), a longer hospital stay (31 days versus 7 days, p<0.001), and a higher mortality rate (22% versus 8%, p=0.010). As compared to the patients who survived, non-survivors were older (73 years vs. 64 years, p<0.001) and were more frequently admitted to the ICU (62% vs. 34%, p=0.010). No other differences in gender, BMI, comorbidities were observed between the groups.

As shown on FIG. 8A, higher sTREM-1 plasma concentrations were observed in severely ill patients as compared to patients with moderate illness (235 pg/mL (IQR 176-319) vs. 195 pg/mL (IQR 139-283), respectively, p=0.017). Similarly, as shown on FIG. 8B, non-survivors had higher sTREM-1 plasma concentrations compared to survivors (326 pg/mL (IQR 207-445) vs. 199 pg/mL (IQR 142-278), respectively, p<0.001).
The relationship between sTREM-1 and disease severity or mortality was separately assessed in these subgroups, indicating the strongest difference in sTREM-1 concentrations in survivors vs. non-survivors (FIGS. 9A-D).
Furthermore, patients who developed thromboembolic events (n=28, 14%) presented with increased sTREM-1 concentrations compared to patients without this complication (FIG. 8C, p=0.044). Thromboembolic events encompassed pulmonary embolisms (n=26), cerebrovascular accidents (n=2) and deep venous thrombosis (n=1). Additionally, sTREM-1 levels were weakly positively correlated with the total duration of hospital stay and ICU stay (r=0.22, p=0.002 and r=0.26, p=0.030, respectively, FIGS. 10A-B), but no correlation was found with duration of symptoms at time of baseline sample collection (r=0.09, p=0.211; FIG. 10C). These latter findings suggest that sTREM-1 may be persistently increased during the disease course in severely ill patients.

[0358] Positive correlations were observed for sTREM-1 concentration and white blood cell (WBC) count, lymphocyte count, C-reactive protein (CRP), ferritin, D-dimer and IL-6 (see FIGS. 11A-F, respectively). These findings indicate that sTREM-1 concentrations are directly associated with a systemic inflammatory response, although they may be independent of standard laboratory inflammatory markers such as CRP or ferritin.

Discriminatory Power of sTREM-1 on Mortality

[0359] FIG. 12 presents the ROC-curve for discrimination between survivors and non-survivors based on sTREM-1 concentrations (FIG. 12A); the ROC-curves for CRP (FIG. 12B), ferritin (FIG. 12C) and IL-6 (FIG. 12D) are presented for comparison. The characteristics of the tests conducted for each biomarker are provided in Table 10 below.

TABLE-US-00010 TABLE 10 Test characteristics Cut-off Youden’s J Sensitivity Specificity PPV NPV sTREM-1 >315 pg/ml 0.41 57.7% 83.7% 35.7% 92.7% CRP >69 mg/mL 0.21 86.4% 34.4% 17.1% 94.2% Ferritin >665 μg/mL 0.23 86.4% 36.2% 17.5% 94.4% IL-6 >237 pg/ml 0.42 50.0% 91.7% 48.6% 92.1% AUC: area under the curve. CRP: C-reactive protein. IL-6: interleukin-6. PPV: positive prediction value. NPV: negative prediction value.

[0360] The AUC for sTREM-1 was 0.73 (95% CI 0.62-0.83) indicating moderate discrimination between survivors and non-survivors. The discriminatory power of sTREM-1 was similar to that of IL-6 (AUC=0.77, 95% CI 0.65-0.88; p=0.887), but performed better than that of CRP (AUC=0.59, 95% CI 0.47-0.72; p=0.132) and ferritin (AUC=0.58, 95% CI 0.46-0.70; p=0.078), although not significantly different. Next, survival analysis for sTREM-1 indicated that patients with sTREM-1 plasma concentrations of more than 315 pg/mL had an increased risk for in-hospital mortality (hazard ratio=3.3, 95% CI 1.4-7.8) (FIG. 13A). Hazard ratios of mortality for high circulating concentrations of CRP (FIG. 13B), ferritin (FIG. 13C), and IL-6 (FIG. 13D) were 1.2 (95% CI 0.4-3.7), 1.9 (95% CI 0.7-5.1), and 2.5 (95% CI 0.9-6.9), respectively.

Combining the use of sTREM-1 and IL-6 did not improve the discrimination between survivors and non-survivors. The AUC for the combination of sTREM-1 and IL-6 was 0.79, not statistically different from that of IL-6 alone (p=0.562). Similarly, combining the use of sTREM-1 and IL-6 in a survival analysis (see FIG. 13E) did not show a significant difference as compared to either of the two biomarkers alone. Nevertheless, a significant difference was observed in patients presenting with sTREM-1>315 pg/mL and IL-6>237 pg/mL, compared to patients presenting with sTREM-1<315 pg/mL and IL-6<237 pg/mL (HR: 4.5, p=0.005).

[0361] The data presented above in Example 1 and Example 2 demonstrate that sTREM-1 concentrations are significantly increased in patients with COVID-19, and show that they are correlated with severity of the disease and with mortality (see FIGS. 1-2 and 8). Discrimination between patients requiring <15 days under mechanical ventilation and patients >15 days under mechanical ventilation (see FIG. 5), and between survivors and non-survivors was thus possible based on sTREM-1 plasma concentrations (see FIGS. 12A and 13A). Accordingly, patients presenting with high sTREM-1 at entry to hospital or at entry to ICU have a lower likelihood to be extubated before 15 days or to survive (see FIG. 6). The data also demonstrate that elevated sTREM-1 concentrations are significantly correlated with the presence of a complication, such as cardiovascular failure (see FIG. 4B) and thromboembolic events, i.e., thrombotic complications (see FIG. 8C).

Example 3

Materials and Methods

[0362] sTREM-1 was quantified in 109 plasma samples collected from patients recruited in the study NCT03158948 by ELISA (Human TREM-1 Quantikine® ELISA kit, R&D Systems, reference DTRM10C) and by ECLIA (Elecsys from Roche Diagnostics). The ECLIA method comprises an incubation of the samples with a first sTREM-1-specific antibody which is biotinylated and a second sTREM-1-specific antibody which is labeled with a ruthenium complex to form a sandwich complex. After the addition of streptavidin-coated microparticles, a second incubation allows the binding of the sandwich complex to the microparticles, thus forming a solid phase. The reaction mixture is then aspirated into the measuring cell of an analyzer (Cobas analyzer from Roche Diagnostics), where the microparticles are magnetically captured onto the surface of an electrode. Unbound substances are removed. The application of a voltage to the electrode excites the ruthenium complex and induces a chemiluminescent emission which is measured by a photomultiplier. Results are determined via a calibration curve. The ELISA method comprises a first incubation of the samples in a plate (96-well plate) with wells pre-coated with a capture sTREM-1-specific antibody. After several washes of the wells, the samples are incubated with a detection sTREM-1-specific antibody which is coupled to horseradish peroxidase (HRP). After several washes, a colorimetric reagent is added, followed by a stop solution 30 minutes later. The optical density (OD) is measured in each well within 30 minutes, using a microplate reader set to 450 nm, and wavelength correction with OD 540 nm. Results are determined via a calibration curve

Results

[0363] The correlation between the sTREM-1 plasma concentrations measured with the Elecsys sTREM-1 ECLIA assay and those measured with the Quantikine sTREM-1 ELISA assay was investigated. Results show a linear relation between the sTREM-1 plasma concentrations obtained with the two methods. As shown on FIG. 14, the measurements are highly correlated (Pearson's r 0.962, Spearman's rho 0.962, Kendall's tau 0.858). The regression analysis using a weighted Deming regression yields an intercept at 50.0 (−6.41; 106) pg/ml and a slope of 2.89 (2.74; 3.04).