MMP-8 AS A MARKER FOR IDENTIFYING INFECTIOUS DISEASE

Abstract

The invention relates to a method for the diagnosis, prognosis, risk assessment, risk stratification, monitoring, therapy guidance and/or therapy control of an infectious disease in a subject, wherein said method comprises providing a sample of said subject; determining a level of matrix metalloprotease-8 (MMP-8) or fragment(s) thereof in a sample of said subject, wherein said level of MMP-8 or fragment(s) thereof distinguishes between the presence and absence of an infectious disease in a patient with symptoms of a systemic inflammatory condition. In a preferred embodiment the invention relates to the determination of procalcitonin (PCT) and MMP-8 and their combined use to distinguish between the presence and absence of infectious disease in patients with symptoms of systemic inflammatory condition. The invention also relates to a computer-implemented method and a kit for conducting the method of the invention.

Claims

1. Method for the diagnosis, prognosis, risk assessment, risk stratification, monitoring, therapy guidance and/or therapy control of an infectious disease in a subject, wherein said method comprises: Providing a sample of said subject; determining a level of matrix metalloprotease-8 (MMP-8) or fragment(s) thereof in a sample of said subject, wherein said level of MMP-8 or fragment(s) thereof distinguishes between the presence and absence of an infectious disease in a patient with symptoms of a systemic inflammatory condition.

2. Method according to claim 1, additionally comprising determining a level of procalcitonin or fragment(s) thereof in a sample of the subject, wherein said level of procalcitonin or fragment(s) thereof and the level of MMP-8 or fragment(s) thereof differentiate between the presence and absence of an infectious disease in a patient with symptoms of a systemic inflammatory condition.

3. Method according to claim 1, wherein the determined level of MMP-8 or fragment(s) thereof is indicative of the presence of an infectious disease in a patient with symptoms of a systemic inflammatory condition, when said determined level(s) is above a reference level.

4. Method according to claim 1, wherein said reference level corresponds to MMP-8 levels in patients with a systemic inflammatory condition without an infection.

5. Method according to claim 1, wherein the infectious disease is sepsis, severe sepsis and/or septic shock.

6. Method according to claim 1, wherein the systemic inflammatory condition without an infection is Systemic Inflammatory Response Syndrome (SIRS) without infection by a pathogenic agent or microorganism.

7. Method according to claim 1, wherein determining a level of MMP-8 or fragment(s) thereof, and optionally additionally determining a level of procalcitonin or fragment(s) thereof, occurs within 72 hours; after symptoms of an infectious disease and/or systemic inflammatory condition occur in the patient.

8. Method according to claim 1, wherein the subject exhibiting symptoms of an infectious disease and/or of a systemic inflammation condition is a post-surgery patient.

9. Method according to claim 1, wherein the subject exhibiting symptoms of an infectious disease and/or of a systemic inflammation condition suffers from shortness of breath.

10. Method according to claim 1, wherein the level of MMP-8 or fragment(s) thereof is indicative of infectious disease severity.

11. Method according to claim 2, wherein the level of procalcitonin or fragment(s) thereof is indicative of infectious disease severity.

12. Method according to claim 1, wherein the sample is a blood sample or a sample derived from blood, preferably whole blood, plasma or serum.

13. Method according to claim 1, wherein the level of MMP-8 or fragment(s) thereof is determined by measuring MMP-8 gene expression.

14. Method according to claim 13, wherein measuring MMP-8 gene expression comprises a quantitative nucleic acid amplification reaction.

15. Method according to any claim 15, wherein the level of MMP-8 or fragment(s) thereof is determined by measuring MMP-8 protein.

16. Method according to claim 15, wherein measuring MMP-8 protein comprises carrying out an immunological assay, wherein said assay comprises: Bringing the sample of the subject into contact with a first anti-MMP-8 affinity reagent, coupled to a first fluorescent label, and a second anti-MMP-8 affinity reagent, coupled to a second fluorescent label, Allowing MMP-8 or fragment(s) thereof in said sample to bind to said first and second affinity reagents, thereby forming an antibody-MMP-8 complex, and Detecting the first and second fluorescent labels, wherein the fluorescent properties of one or more of said first and/or second fluorescent labels is altered upon binding of the first and second affinity reagents to MMP-8 in said sample.

17. Method according to claim 1, additionally comprising determination of one or more additional markers, wherein the one or more further marker is selected from the group consisting of Adrenomedullin (ADM), pro-Adrenomedullin, Histone H2A, Histone H2B, Histone H3, Histone H4, procalcitonin, calcitonin, Endothelin-1 (ET-1), Arginine Vasopressin (AVP), Atrial Natriuretic Peptide (ANP), Neutrophil Gelatinase-Associated Lipocalin (NGAL), Troponin, Brain Natriuretic Peptide (BNP), C-Reactive Protein (CRP), Pancreatic Stone Protein (PSP), Triggering Receptor Expressed on Myeloid Cells 1 (TREM1), Interleukin-6 (IL-6), Interleukin-1, Interleukin-24 (IL-24) other ILs, Presepsin (sCD14-ST), Lipopolysaccharide Binding Protein (LBP), Alpha-1-Antitrypsin, Matrix Metalloproteinase 2 (MMP2), Matrix Metalloproteinase 9 (MMP9), Matrix Metalloproteinase 7 (MMP9), soluble fms-like tyrosine kinase-1 (sFlt-1), Placental growth factor (PIGF), Chromogranin A, S100A protein, S100B protein and Tumor Necrosis Factor (TNF), Lipocalin-2 (LCN2), Olfactomedin 4 (OLFM4) and Proteinase 3 (PRTN3) or a fragment thereof.

18. Method according to claim 1, additionally comprising comparing the determined level of MMP-8 or fragment(s) thereof to a reference level, threshold value and/or a population average corresponding to MMP-8 levels in patients with systemic inflammatory condition without an infection, wherein said comparing is carried out in a computer processor using computer executable code.

19. Kit for carrying out the method of claim 1, comprising: detection reagents for determining the level of MMP-8 or fragment(s) thereof, and optionally additionally for determining the level of procalcitonin or fragment(s) thereof, in a sample from a subject, and reference data, such as a reference level, corresponding to MMP-8 levels, and optionally procalcitonin levels, in patients with systemic inflammatory condition without an infection, wherein said reference data is stored on a computer readable medium and/or employed in in the form of computer executable code configured for comparing the determined level of MMP-8 or fragment(s) thereof, and optionally additionally the determined level of procalcitonin or fragment(s) thereof, to said reference data.

20. Method according to claim 14, wherein the quantitative nucleic acid amplification reaction is real-time PCR (RT-PCR) or digital droplet PCR (ddPCR) employing detection of cDNA corresponding to MMP-8 encoding mRNA.

21. A method comprising: providing a reaction mixture comprising a complex between matrix metalloprotease-8 (MMP-8) or a fragment thereof and at least one reagent which binds to MMP-8 or a fragment thereof in a sample obtained from a subject, and wherein said subject has symptoms of a systemic inflammatory condition.

Description

FIGURES

[0218] FIG. 1: MMP-8 levels as assessed by droplet digital PCR using predesigned TaqMan Assay Primer/Probe Sets (FAM labelled MGB probes, Thermo Fisher/Scientific-Life Technologies, Waltham, Mass., USA). MMP-8 levels in different patient groups are shown, expressed as (copies/ng) log. Also shown in the table in FIG. 1c are AUROC and p values in SIRS and in sepsis patient groups for MMP-8, and MMP-8 and PCT.

[0219] FIG. 2: ROC curves for SIRS vs. Sepsis differentiation of PCT, MMP8 and the combination of both markers, when measured using ELISA. Combining PCT with MMP8 is also shown.

[0220] FIG. 3: ROC curves for Dyspnea vs. Sepsis differentiation of PCT, MMP8 measured by MagPix and the combination of both markers.

[0221] FIG. 4: ROC curves for Dyspnea vs. Sepsis differentiation of PCT, MMP8 measured by ELISA and the combination of both markers. Combining PCT+MMP8 significantly increases the AUC and thereby the ability to differentiate dyspnea and sepsis patients.

EXAMPLES

Example 1

[0222] Patients with Sepsis Show Increased Levels of MMP-8 Over Control Groups with SIRS

[0223] Patients:

[0224] 158 adult patients (>18 years old) were assessed in the study. 57 uninfected SIRS patients were recruited as control group with no sepsis. In addition, 18 blood donors of similar age of the patients were recruited as healthy controls. 83 patients were diagnosed as septic patients (according to the definition proposed by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference), whereby of these 44 patients suffered a septic shock.

[0225] Written informed consent was obtained directly from all patients, or their legal representative, before enrolment. Scientific and ethical approval of the study protocol was obtained from the respective scientific committees for clinical research of the participant hospitals. Standard cultures in biological samples guided by the presumptive source of the septic insult were performed to assess the presence of bacterial and fungal infection. Potentially contaminant microorganisms were not considered.

[0226] ddPCR mRNA Extraction and Quality Evaluation:

[0227] A sample of 2.5 mL of blood was collected by using PaxGene (BD) venous blood vacuum collection tubes in the first 12 hours following diagnosis of sepsis or in the first 12 hours following surgery in the case of the surgical controls. Blood from healthy individuals was collected at the moment of donation. Total RNA was extracted from blood samples using the PAXgene Blood RNA System (PreAnalytix, Hombrechtikon, Switzerland). The evaluation of concentration and quality was performed by spectrometry (NanoDrop ND1000, NanoDrop Technologies, Wilminton, Delaware USA) and RNA Experion Bioanalyzer (BioRad, Calif. USA). Only samples with good quality and concentration were tested by ddPCR.

[0228] Quantification of Transcriptomic Response by ddPCR:

[0229] Gene expression was quantified by droplet digital PCR (ddPCR, Bio-Rad, Calif., USA) using predesigned TaqMane Assay Primer/Probe Sets, (FAM labelled MGB probes, Thermo Fisher/Scientific-Life Technologies, Waltham, Mass., USA): MMP8, matrix metallopeptidase 8 (neutrophil collagenase) (Reference Hs01029057 ml).

[0230] cDNA was generated from each sample on a Techne TC-512 thermal cycler (Bibby-Scientific, Staffordshire, OSA, UK) starting from 1000 ng of mRNA by using iScript Advanced cDNA Synthesis Kit (Biorad, Calif., USA, cat:1725038). The obtained volume of cDNA (20 ul) was further diluted (1/25), and 2.5 pl (5ng of total mRNA) were employed for quantification of target gene expression according to the manufacturer instructions.

[0231] Briefly, ddPCR was performed using the Bio-Rad QX200 Droplet Digital PCR system, ddPCR Supermix for Probes (No dUTP), and Bio-Rad standard reagents for droplet generation and reading. End-point PCR with 40 cycles was performed by using C1000 Touch Thermal Cycler (BioRad, Calif., USA) after splitting each sample into about 20000 droplets. Next, the droplet reader used at least 10000 droplets to determine the percentage of positive droplets and calculation of copy number of cDNA per ng of initial mRNA.

[0232] Statistical Analysis:

[0233] Differences in demographic and clinical characteristics between patient groups were assessed using the .sub.X.sup.2 test for categorical variables and the Mann Whitney U test for continuous variables. Differences in gene expression levels and expression ratios were assessed using Mann Whitney U test. The accuracy of individual genes and gene expression ratios for identifying the presence/absence of sepsis was studied by calculating areas under the receiver operating characteristic curve (AUROC).

[0234] The optimal operating point (OOP) was the value for which the point on the curve had the minimum distance to the upper left corner (where sensitivity=1 and specificity=1). Categorical variables were further created using the OOPs as cut-offs. The ability of these categorical variables to diagnose the presence/absence of sepsis was further evaluated by using logistic regression analysis. Those variables yielding a p value <0.1 in the univariate regression analysis were included in the multivariate one. Logistic regression was employed also to evaluate potential associations between gene expression levels and risk of hospital mortality in sepsis patients. Napierian logarithm values of gene expression levels were employed for this analysis.

[0235] Clinical Characteristics of the Patients:

[0236] Elderly male predominated in both sepsis cases and SIRS, sepsis patients were slightly older. Profile of comorbidities was similar between sepsis cases and SIRS, but the former showed a higher prevalence of diabetes mellitus. Sepsis patients stayed longer at the hospital / ICU, presented with a higher degree of organ failure as assessed by the SOFA score. None of the patients of the SIRS control group died, but of sepsis patients did not survive to the disease. Most common sources of infection were the abdominal and respiratory ones. Sepsis patients showed a slightly higher frequency of positive cultures for Gramthan for Gram+bacteria. Fungal infection was present in 6.9% of the sepsis patients, and viral infection was uncommon. Sepsis patients showed higher levels of C reactive protein and procalcitonin (PCT), higher counts in blood of leukocytes, neutrophils, eosinophils and basophils. Both cases and controls had lymphocyte median levels close to lymphopenia (<1000 lymphocytes/mm3).

[0237] Variation of Gene Expression and Ratios Across Groups:

[0238] As is evident in FIG. 1b, the SIRS control group presented significantly higher concentrations of MMP8 (and PCT) than healthy controls, reflecting the existence of an active inflammatory process at the systemic level in SIRS patients with no infection. Of note, is that the data shown in FIG. 1 demonstrates significant differences in MMP-8 levels between SIRS patients (model for patients with systemic inflammation) compared to patients with infections (sepsis). Although SIRS controls show up-regulation of MMP-8 over healthy subjects, and show symptoms of systemic inflammation, the MMP-8 levels determined were significantly lower when compared to patients in which an infection (sepsis) was evident.

[0239] PCT levels were also determined in parallel using protein determination via an ELISA assay. As shown in the table in FIG. 1c, the combined analysis of PCT and MMP-8 leads to higher AUROC and more significant p values when compared to the measurement of MMP-8 alone.

[0240] Additional Data for Example 1:

[0241] Patients with Sepsis Show Increased Levels of MMP-8 Over Control Groups with SIRS, as Measured using MMP8 and PCT ELISA

[0242] Patients:

[0243] 56 adult patients (>18 years old) were assessed in the study. 17 uninfected SIRS patients were recruited as control group with no sepsis. 39 patients were diagnosed as septic patients (according to the definition proposed by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference).

[0244] Statistical Analysis:

[0245] The accuracy of individual protein concentration of PCT and MMP8 and combination of both for identifying the presence /absence of sepsis was studied by calculating areas under the receiver operating characteristic curve (AUROC). Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold/cutoff (table 2). The youden's index was calculated for each cutoff and the highest index depicts the cutoff with the best possible sensitivity/specificity pair. For example, if a clinician wants to use the MMP8 to rule out sepsis, a sensitivity of 100% is preferred, that would in this example correspond to a cutoff of 3.28 ng/mL. To rule-in sepsis, a specificity of 100% (cutoff 43.68 ng/mL) is preferred.

[0246] Absolute MMP8 concentrations may vary depending on differences in absolute quantities determined in different assay formats (see example 3), therefore fold changes were calculated to estimate the difference between sepsis and controls, independent of the test system.

[0247] Fold changes were estimated using an ANOVA model of log2-transformed biomarker values. The difference of means and a corresponding confidence interval of the log2-transformed values in the two groups was estimated using least square means. Then the inverse transformation 2.sup.difference of means gives an estimate of the fold change and the corresponding confidence limits because

[00001]$2^{{\log }_2\left(x\right)-{\log }_2\left(y\right)}=\frac{2^{{\log }_2\left(x\right)}}{2^{{\log }_2\left(y\right)}}=\frac{x}{y}.$

[0248] FIG. 2 shows a ROC curve for measurements of PCT and/or MMP8 using ELISA. As demonstrated in FIG. 2, MMP8 alone, PCT alone and MMP8 and PCT in combination show good specificity and sensitivity and demonstrate significant differences in MMP-8 and/or PCT levels between SIRS patients (model for patients with systemic inflammation) compared to patients with infections (sepsis).

TABLE-US-00001 TABLE 1 Fold changes with 95% confidence interval (95% CI), comparing PCT or MMP8 levels in sepsis patients compared to SIRS patients. Model FoldChange 95% CI p-value PCT 15.55 [4.88-49.51] 0.00002 MMP8-ELISA 3.63 [1.84-7.16] 0.00036

TABLE-US-00002 TABLE 2 Sensitivity, specificity and youden index for all possible cutoffs calculated by ROC analyses. Optimal combination of sensitivity and specificity is represented by the highest youden index. To rule out sepsis, e.g. a sensitivity of 100% (cutoff 3.28 ng/mL) is preferred, to rule-in sepsis, e.g. a specifity of 100% (cutoff 43.68 ng/mL) is preferred. The highest combined values for sensitivity and specificity (highest youden index) is marked in bold type. Cutoff MMP 8 ELISA [ng/ml] Sensitivity Specificity Youden 3.28 1.000 0.059 0.059 3.55 0.974 0.059 0.033 3.82 0.949 0.059 0.008 4.26 0.949 0.118 0.066 4.29 0.949 0.176 0.125 4.43 0.949 0.235 0.184 6.15 0.923 0.235 0.158 6.16 0.923 0.294 0.217 6.61 0.897 0.294 0.192 7.08 0.897 0.353 0.250 7.40 0.872 0.353 0.225 8.34 0.872 0.412 0.284 8.90 0.872 0.471 0.342 9.73 0.846 0.471 0.317 10.07 0.821 0.471 0.291 10.86 0.821 0.529 0.350 11.24 0.795 0.529 0.324 11.49 0.769 0.529 0.299 13.46 0.744 0.529 0.273 14.26 0.744 0.588 0.332 19.36 0.744 0.647 0.391 19.55 0.744 0.706 0.449 21.19 0.718 0.706 0.424 22.46 0.718 0.765 0.483 22.58 0.692 0.765 0.457 22.65 0.692 0.824 0.516 24.67 0.692 0.882 0.575 25.36 0.667 0.882 0.549 25.75 0.641 0.882 0.523 27.33 0.615 0.882 0.498 27.99 0.590 0.882 0.472 29.27 0.564 0.882 0.446 29.37 0.538 0.882 0.421 29.78 0.513 0.882 0.395 31.24 0.487 0.882 0.370 36.68 0.487 0.941 0.428 43.60 0.462 0.941 0.403 43.68 0.462 1.000 0.462 43.72 0.436 1.000 0.436 54.24 0.410 1.000 0.410 62.60 0.385 1.000 0.385 72.96 0.359 1.000 0.359 75.80 0.333 1.000 0.333 84.40 0.308 1.000 0.308 91.60 0.282 1.000 0.282 92.76 0.256 1.000 0.256 100.52 0.231 1.000 0.231 125.04 0.205 1.000 0.205 136.20 0.179 1.000 0.179 148.84 0.154 1.000 0.154 172.28 0.128 1.000 0.128 209.20 0.103 1.000 0.103 243.16 0.077 1.000 0.077 320.00 0.051 1.000 0.051 320.00 0.051 1.000 0.051

Example 2

[0249] Patients with Sepsis Show Increased Levels of MMP-8 Over Control Groups of Patients having Undergone Surgery

[0250] Patients:

[0251] In a similar study to Example 1 above, 101 adult patients (>18 years old) admitted to the Surgery Service or to the Surgical ICU of the participant hospitals with a diagnosis of sepsis (according to the definition proposed by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference) were recruited. 53 uninfected surgical patients were recruited as control group with no sepsis. In addition, 16 blood donors of similar age of the patients were recruited as healthy controls.

[0252] Sepsis patients were in need of urgent surgery in of the cases, while this was a rare event in controls. Kind of surgery was similar in both groups. Sepsis patients stayed longer at the hospital/ICU, presented with a higher degree of organ failure as assessed by the SOFA score. None of the patients of the surgical control group died, but of sepsis patients did not survive to the disease.

[0253] Methods for ddPCR, ELISA and statistical analyses were performed essentially as in Example 1.

[0254] Variation of Gene Expression and Ratios Across Groups:

[0255] As is evident from Table 3 below, surgical controls presented significantly higher concentrations of PCT and MMP8 than healthy controls, reflecting the existence of an active inflammatory process at the systemic level in post-operated patients with no infection. Of note, is that the data shown below in table 3 demonstrates significant differences in both MMP-8 and PCT levels between surgical controls ((1); model for patients with systemic inflammation) compared to patients with infections ((2); sepsis). Although surgical controls show up-regulation of MMP-8 over healthy subjects, and show symptoms of systemic inflammation, the MMP-8 levels determined were significantly lower when compared to patients in which an infection (sepsis) was evident.

TABLE-US-00003 TABLE 3 Comparison of gene expression levels, procalcitonin levels and ratios between groups. Healthy Surgical Sepsis Sepsis Controls Controls SOFA < 8 SOFA 8 p p p P p p (0) (1) (2) (3) (0 vs 1) (0 vs 2) (0 vs 3) (1 vs 2) (1 vs 3) (2 vs 3) MMP8 23 125 856 5116 <0.001 <0.001 < 0.001 <0.001 <0.001 <0.001 [68] [302] [4169] [12636] PCT 0.03 0.50 1.63 12.09 <0.001 <0.001 < 0.001 <0.001 <0.001 <0.001 [0.02] [0.97] [5.25] [50.46] (MMP8 + N. A 0.3 0.8 1.0 N. A N. A N. A <0.001 <0.001 <0.001 PCT) * [0.4] [0.3] [0.1] Table 3: Comparison of gene expression levels, procalcitonin levels and ratios between groups. Results for gene expression levels are provided as cDNA copies/ng total mRNA. Results for PCT are provided as ng/mL. The values shown above for each data row relate to the measured mean values for each marker in each patient group (either cDNA copies/ng total mRNA or ng/mL). The values shown in square brackets relate to the median interquartile range (IQR). * Results from a probabilistic function built using logistic regression combining MMP8 and PCT levels to distinguish sepsis from no sepsis patients. Log values of PCT and MMP8 were used for building this function, since neither MMP8 not PCT values followed a normal distribution. N.A: not applicable.

[0256] Further analysis of MMP-8 protein levels via immunoassays (e. g. Luminex) confirms the results above.

Example 3

[0257] Patients with Sepsis Show Increased Levels of MMP-8 Over Control Groups with Dyspnea Patients:

[0258] 68 adult patients (>18 years old) were assessed in the study. 21 patients with dyspnea were recruited as control group. 22 patients with sepsis and 25 patients with severe sepsis were recruited (according to the definition proposed by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference).

[0259] Statistical Analysis:

[0260] As described for example 1.

[0261] Results:

[0262] MMP8 levels were also determined in parallel using protein determination via an ELISA and a Luminex MAGPIX assay. As shown in FIGS. 3 and 4, and Tables 4, 5 and 6 below, the combined analysis of PCT and MMP-8 leads to higher AUROC and more significant p values when compared to the measurement of MMP-8 alone.

TABLE-US-00004 TABLE 4 .sup.2 test statistics for PCT, two different MMP8 assays, and the PCT + MMP8 combinations. Addition of MMP8 to PCT shows a significant add-on to diagnose sepsis, independent of the MMP8 assay type. LR added LR added added p N Events Chi2 DF p value C Index Chi2 DF value PCT 68 47 23.16 1 0.00000 0.837 MMP8- 68 47 19.45 1 0.00001 0.819 Luminex MMP8-ELISA 68 47 36.30 1 0.00000 0.903 PCT + MMP8- 68 47 33.04 2 0.00000 0.905 9.88 1 0.00167 Luminex PCT + MMP8- 68 47 44.82 2 0.00000 0.933 21.66 1 0.00000 ELISA DF = degree of freedom

[0263] With respect to mean fold changes, MMP-8 and PCT levels were compared in patient groups with sepsis, dyspnea and severe sepsis, in order to determine whether mean fold change values between the population groups could be determined.

TABLE-US-00005 TABLE 5 Fold changes with 95% confidence interval (95% CI). Fold Contrast change 95% CI p value PCT Sepsis - Dyspnea 3.53 [1.09-11.39] 0.03238 PCT Severe Sepsis - 16.99 [5.45-53.00] 0.00000 Dyspnea MMP8- Sepsis - Dyspnea 4.27 [1.75-10.43] 0.00068 Luminex MMP8- Severe Sepsis - 4.48 [1.88-10.65] 0.00029 Luminex Dyspnea MMP8- Sepsis - Dyspnea 2.75 [1.59-4.73] 0.00010 ELISA MMP8- Severe Sepsis - 5.77 [3.40-9.78] 0.00000 ELISA Dyspnea

[0264] With respect to mean fold changes, MMP-8 and PCT levels were compared in patients groups with sepsis, dyspnea and severe sepsis, in order to determine whether mean fold change values between the population groups could be determined. The analysis showed [0265] 2.75 to 4.27-fold increased MMP-8 in Sepsis vs. Dyspnea [0266] 3.53-fold increased PCT in Sepsis vs. Dyspnea [0267] 4.48 to 5.77-fold increased MMP-8 in severe sepsis vs. Dyspnea [0268] 16.99-fold increased PCT in severe sepsis vs. Dyspnea

[0269] As is evident from the above, patients with dyspnea (shortness of breath; a control group for patients with a systemic inflammatory condition but without an infection) showed reduced levels in both MMP-8 and PCT compared to patients with sepsis (patients with an infection).

TABLE-US-00006 TABLE 6 Sensitivity, specificity and youden index for all possible cutoffs calculated by ROC analyses. Optimal combination of sensitivity and specificity is represented by the highest youden index (bold type). For example, to rule out sepsis a sensitivity of 100% is preferred, to rule-in sepsis a specifity of 100% is preferred. MMP8-Luminex MMP8-ELISA [pg/mL] [pg/mL] Cutoff Sensitivity Specificity Youden Cutoff Sensitivity Specificity Youden 32.98 0.957 0.095 0.053 8892.00 1.000 0.048 0.048 34.39 0.936 0.095 0.031 9292.00 1.000 0.095 0.095 34.39 0.936 0.095 0.031 9360.00 1.000 0.143 0.143 42.04 0.915 0.143 0.058 10364.00 1.000 0.190 0.190 42.89 0.915 0.190 0.105 11568.00 1.000 0.238 0.238 45.73 0.915 0.238 0.153 13160.00 1.000 0.286 0.286 50.02 0.915 0.286 0.201 15716.00 1.000 0.333 0.333 62.92 0.915 0.333 0.248 15812.00 0.979 0.333 0.312 63.08 0.915 0.381 0.296 16500.00 0.957 0.333 0.291 68.72 0.915 0.429 0.343 16832.00 0.957 0.381 0.338 69.41 0.894 0.429 0.322 17572.00 0.957 0.429 0.386 69.43 0.872 0.429 0.301 17836.00 0.957 0.476 0.434 70.15 0.851 0.429 0.280 20804.00 0.957 0.524 0.481 74.48 0.830 0.429 0.258 22448.00 0.936 0.524 0.460 79.13 0.830 0.476 0.306 22592.00 0.936 0.571 0.508 83.21 0.809 0.476 0.285 23220.00 0.915 0.571 0.486 88.10 0.787 0.476 0.263 23548.00 0.915 0.619 0.534 91.91 0.787 0.524 0.311 24660.00 0.894 0.619 0.513 96.29 0.787 0.571 0.359 25488.00 0.894 0.667 0.560 96.61 0.787 0.619 0.406 25544.00 0.872 0.667 0.539 97.89 0.787 0.667 0.454 27300.00 0.872 0.714 0.587 98.86 0.787 0.714 0.502 28148.00 0.872 0.762 0.634 102.87 0.787 0.762 0.549 28612.00 0.851 0.762 0.613 106.54 0.766 0.762 0.528 28980.00 0.830 0.762 0.592 118.94 0.766 0.810 0.575 29792.00 0.830 0.810 0.639 138.72 0.766 0.857 0.623 31148.00 0.809 0.810 0.618 162.31 0.745 0.857 0.602 31724.00 0.787 0.810 0.597 167.16 0.723 0.857 0.581 33424.00 0.766 0.810 0.575 173.12 0.702 0.857 0.559 38052.00 0.766 0.857 0.623 192.24 0.681 0.857 0.538 38920.00 0.766 0.905 0.671 207.95 0.681 0.905 0.586 42280.00 0.745 0.905 0.649 211.60 0.681 0.952 0.633 48080.00 0.723 0.905 0.628 217.71 0.660 0.952 0.612 48960.00 0.723 0.952 0.676 220.35 0.638 0.952 0.591 53480.00 0.702 0.952 0.655 254.52 0.617 0.952 0.569 55480.00 0.681 0.952 0.633 258.28 0.596 0.952 0.548 56400.00 0.660 0.952 0.612 280.89 0.574 0.952 0.527 59640.00 0.638 0.952 0.591 302.83 0.553 0.952 0.506 63120.00 0.617 0.952 0.569 318.25 0.532 0.952 0.484 68560.00 0.596 0.952 0.548 353.50 0.511 0.952 0.463 72040.00 0.574 0.952 0.527 399.94 0.489 0.952 0.442 72480.00 0.553 0.952 0.506 481.98 0.468 0.952 0.420 72640.00 0.553 1.000 0.553 530.84 0.447 0.952 0.399 73680.00 0.532 1.000 0.532 538.81 0.426 0.952 0.378 77640.00 0.511 1.000 0.511 568.13 0.404 0.952 0.357 78480.00 0.489 1.000 0.489 580.18 0.383 0.952 0.335 83160.00 0.468 1.000 0.468 685.58 0.383 1.000 0.383 85280.00 0.447 1.000 0.447 728.74 0.362 1.000 0.362 89800.00 0.426 1.000 0.426 769.54 0.340 1.000 0.340 91320.00 0.404 1.000 0.404 835.26 0.319 1.000 0.319 92200.00 0.383 1.000 0.383 837.23 0.298 1.000 0.298 110840.00 0.362 1.000 0.362 855.38 0.277 1.000 0.277 112520.00 0.340 1.000 0.340 880.72 0.255 1.000 0.255 121280.00 0.319 1.000 0.319 957.64 0.234 1.000 0.234 127760.00 0.298 1.000 0.298 969.63 0.213 1.000 0.213 133680.00 0.277 1.000 0.277 1013.76 0.191 1.000 0.191 173480.00 0.255 1.000 0.255 1120.33 0.170 1.000 0.170 176800.00 0.234 1.000 0.234 1173.56 0.149 1.000 0.149 180120.00 0.213 1.000 0.213 1206.70 0.128 1.000 0.128 184320.00 0.191 1.000 0.191 1346.59 0.106 1.000 0.106 185560.00 0.170 1.000 0.170 1430.56 0.085 1.000 0.085 214120.00 0.149 1.000 0.149 1855.90 0.064 1.000 0.064 221240.00 0.128 1.000 0.128 4161.58 0.043 1.000 0.043 246960.00 0.106 1.000 0.106 6459.76 0.021 1.000 0.021 258880.00 0.085 1.000 0.085