ASSESSMENT OF THE RISK OF COMPLICATION IN A PATIENT SUSPECTED OF HAVING AN INFECTION, HAVING A SOFA SCORE LOWER THAN TWO

Abstract

A method for the in vitro or ex vivo assessment of the risk of complications in a patient suspected of having an infection, having a SOFA score of less than two, including measuring the level of expression, in a biological sample obtained from said patient, of at least one expression product of the VEGFR2 gene.

Claims

1. A method for the assessment, in vitro or ex vivo, of the risk of complications in a patient suspected of having an infection, having a SOFA score of less than two, comprising measuring the level of expression of at least one expression product of the VEGFR2 gene in a biological sample obtained from the patient.

2. The method as claimed in claim 1, wherein the expression product of the gene is an RNA transcript.

3. The method as claimed in claim 1, wherein the expression product is a protein or a polypeptide.

4. The method as claimed in claim 1, comprising: measuring the quantity of at least one expression product of the VEGFR2 gene in the biological sample from the patient, comparing the quantity of the at least one expression product determined for the biological sample, or a value derived from this quantity with a predetermined reference value, and drawing a conclusion regarding the risk of complications from the result of the comparison.

5. The method as claimed in claim 1, comprising: measuring a first quantity of at least one expression product of the VEGFR2 gene in the patient's biological sample obtained by taking a first sample at time T1, measuring a second quantity of the at least one expression product of the VEGFR2 gene in the patient's biological sample obtained by taking a second sample at time T2, calculating the variation between the quantity of the at least one expression product of the VEGFR2 gene at T2 and the quantity of the at least one expression product of the VEGFR2 gene at T1, giving a value Δ, comparing the value Δ obtained in the preceding step with a reference value determined from two populations of patients suspected of having an infection, having a SOFA score of less than two, one developing complications and the other not, drawing a conclusion regarding the risk of complications from the result of the comparison.

6. The method as claimed in claim 1, also comprising measuring the level of expression of at least one expression product of the uPAR gene.

7. The method as claimed in claim 6, comprising: measuring the quantity of the at least one expression product of the VEGFR2 gene in the biological sample from the patient, measuring the quantity of the at least one expression product of the uPAR gene in the biological sample from the patient, comparing the quantity of the at least one expression product of the VEGFR2 gene determined for the biological sample or a value derived from this quantity with a predetermined reference value S.sub.VEGFR2; and comparing the quantity of the at least one expression product of the uPAR gene determined for the biological sample, or a value derived from this quantity with a predetermined reference value S.sub.uPAR; drawing a conclusion regarding the risk of complications from the result of the comparisons.

8. The method as claimed in claim 6, comprising: measuring a first quantity of the at least one expression product of the VEGFR2 gene in the patient's biological sample obtained by taking a first sample at time T1, measuring a second quantity of the at least one expression product of the VEGFR2 gene in the patient's biological sample obtained by taking a second sample at time T2, measuring a first quantity of the at least one expression product of the uPAR gene in the patient's biological sample obtained by taking a first sample at time T1, measuring a second quantity of the at least one expression product of the uPAR gene in the patient's biological sample obtained by taking a second sample at time T2, calculating the variation between the quantity of the at least one expression product of the VEGFR2 gene at T2 and the quantity of at least one expression product of the VEGFR2 gene at T1, giving a value Δ.sub.VEGFR2, calculating the variation between the quantity of the at least one expression product of the uPAR gene at T2 and the quantity of at least one expression product of the uPAR gene at T1, giving a value Δ.sub.uPAR, comparing the value Δ.sub.VEGFR2 with a reference value ΔS.sub.VEGFR2 determined from two populations of patients suspected of having an infection, having a SOFA score of less than two, one developing complications and the other not, comparing the value Δ.sub.uPAR with a reference value ΔS.sub.uPAR determined from two populations of patients suspected of having an infection, having a SOFA score of less than two, one developing complications and the other not, drawing a conclusion regarding the risk of complications from the result of the comparisons.

9. The method as claimed in claim 6, comprising: measuring the quantity of the at least one expression product of the VEGFR2 gene in the biological sample from the patient, measuring the quantity of the at least one expression product of the uPAR gene in the biological sample from the patient, calculating a combined score from the quantities determined in the preceding steps, comparing the combined score with a predetermined reference score, and drawing a conclusion regarding the risk of complications from the result of the comparison.

10. A method for the treatment of a patient suspected of having an infection, having a SOFA score of less than two, comprising: identifying the patients presenting a risk of complications by carrying out a method as claimed in claim 1, and adapting the health care management of the patient identified in the preceding step in order to reduce the risk of complications.

11. A kit for measuring, in vitro or ex vivo, the level of expression of at least one expression product of the VEGFR2 gene and of at least one expression product of the uPAR gene in a patient suspected of having an infection, having a SOFA score of less than two, comprising at least one specific binding partner for the at least one expression product of the VEGFR2 gene and at least one specific binding partner for the at least one expression product of the uPAR gene.

12. A kit for measuring, in vitro or ex vivo, the level of expression of at least one expression product of the VEGFR2 gene and of at least one expression product of the uPAR gene in a biological sample, comprising: specific tools or reagents enabling the quantities of the at least one expression product of the VEGFR2 gene and of the at least one expression product of the uPAR gene to be measured in the biological sample, and a control sample which is a sample calibrated to contain the quantities of the at least one expression product of the VEGFR2 gene and of the at least one expression product of the uPAR gene which correspond to known quantities of the at least one expression product of the VEGFR2 gene, and of the at least one expression product of the uPAR gene.

13. The kit as claimed in claim 11, in which the specific binding partner for an expression product is (i) at least one hybridization probe and/or at least one amplification primer, or (ii) at least one antibody, or at least one antibody fragment, or at least one affinity protein, or at least one aptamer.

Description

[0294] The invention will be better understood with the aid of the following examples which are given by way of non-limiting illustration, as well as with the aid of FIGS. 1 to 4, in which:

[0295] FIG. 1 represents box and whisker plots (or box diagrams) which correspond to graphical representations of the levels of expression of sVEGFR2 and suPAR proteins in a sample taken upon admission to the emergency center (T0) as a function of the development or not of complications in the patients: A. Levels of expression for sVEGFR2 at T0; B. Levels of expression for suPAR at T0;

[0296] FIG. 2 represents box and whisker plots (or box diagrams) which correspond to graphical representations of the levels of expression of the proteins sVEGFR2 and suPAR in a sample taken six hours after the first sample (T6) as a function of the development or not of complications in patients: A. Levels of expression for sVEGFR2 at T6; B. Levels of expression for suPAR at T6;

[0297] FIG. 3 represents box and whisker plots (or box diagrams) which correspond to graphical representations of the variation in the level of expression of the marker sVEGFR2 between the first blood sample upon admission to the emergency center (T0) and the second blood sample six hours after the first sample (T6) as a function of the development or not of complications in patients.

[0298] FIG. 4 represents the apparent ROC curve for the association between the combination of sVEGFR2 and suPAR at T0 (first blood sample upon admission to the emergency center) and the probability of complications for patients during the 72 h following the first sample at T0.

EXAMPLES

Example 1

Obtaining and Preparing Blood Samples

[0299] This retrospective observational study was carried out on patients from 19 to 101 years of age (111 men, 122 women, median age: 53 years) newly admitted to the emergency center of 14 French and Belgian hospital centers between 2015 and 2017 for care in respect of a suspected infection. These patients were all hospitalized upon suspicion of an infection. The inclusion criteria were as follows: [0300] patients aged 18 years or over; [0301] patients having a single site of acute infection suspected or confirmed by the clinician on clinical or paraclinical signs; [0302] patients admitted to the emergency center presenting at least two criteria from the following: [0303] temperature higher than 38° C. or lower than 36° C.; [0304] heart rate more than 90 beats per minute; [0305] respiratory rate more than 20 breaths per minute or PaCO2<32 mmHg; [0306] number of leukocytes more than 12000/mm3 or less than 4000/mm3. [0307] patients having a SOFA score of less than 2; [0308] patients having symptoms for less than 72 h from their arrival in the emergency center; [0309] persons affiliated to a social security system or a beneficiary of a system of this type; and [0310] patients consenting to participation in the study.

[0311] The clinical criteria for exclusion were as follows: [0312] patients arrived at the emergency center more than 12 hours ago; [0313] patients presenting septic shock upon arrival in the emergency center (organ failure and persistent hypotension despite adequate vascular fluid replacement (up to 20 ml/kg over 1 h) and/or the need to use catecholamines); [0314] patients having acute organ failure upon arrival in the emergency center of an origin other than septic; [0315] patients hospitalized in the week preceding inclusion; [0316] immunosuppressed patients (HIV, transplants, patients undergoing chemotherapy, patients receiving a treatment of >20 mg/jour of prednisolone or equivalent); [0317] patients with a known pathology from among non-infectious pathologies potentially associated with SIRS; [0318] patients with sepsis diagnosed in the 30 days before the date of ‘inclusion; [0319] patients who are already included in the study; [0320] persons refusing to sign the written consent form [0321] adult persons under legal protection; [0322] women who are pregnant, in labor or lactating; [0323] persons with a legal guardian; [0324] person deprived of liberty by a judicial or administrative decision and person hospitalized without consent under articles L.3212-1 and 3213-1 who do not fall within article L.1122-8 of the Public Health Code.

[0325] 233 patients were included in the study. All of these patients satisfied the following criteria: [0326] the first blood sample was taken at the latest in the first 12 hours following the patient's arrival in the emergency center (T0); [0327] the second blood sample was taken between 4 and 8 hours (T6±2 h) following taking the first blood sample (T0); [0328] the assessment of complications was observed by an adjudication committee in the 72 hours (T72) following taking the first blood sample (T0); and [0329] mortality was assessed 28 days following the patient's arrival in the emergency center.

[0330] Complications were determined by an adjudication committee composed of 3 physicians who were independent of the study. This committee determined the complications as a function of several criteria; in particular, the appearance of new organ failures (increase in SOFA score), death or the need to go into intensive care.

[0331] Of the 233 patients in this cohort, 36 patients (21%) developed complications in the 72 h following their admission (“COMPLICATIONS”) and 185 patients (79%) did not develop complications (“NO COMPLICATIONS»).

Example 2

Assay of the Soluble Form of the VEGF Receptor (sVEGFR2)

[0332] Human plasma was collected from patients described above in Example 1 at T0 and T6.

[0333] The protein sVEGFR2 was assayed with the aid of antibodies marketed by Bio-Techne® (Ac monoclonal anti Human VEGFR2 (KDR) ref: MAB3573, and Human VEGF R2/KDR/Flk-1 Antibody Antigen Affinity-purified Polyclonal Goat IgG ref: AF357) and an ELISA test using the automated instrument Vidas® (bioMérieux). To this end, the ELISA test was constructed using reagents from the cartridge of the Vidas® B.R.A.H.M.S. PCT™ kit (bioMérieux, Cat. No.30450) without using the antibodies and the control calibrators.

[0334] VIDAS® is an automated multi-parametric immunoanalyzer. It is a closed system for unitary tests offering great flexibility. This automated instrument is characterized by its robustness, its flexibility, its ease of use and is intended for small to medium sized laboratories. It can be used to carry out routine tests, confirmations and high value medical tests.

[0335] Detection was carried out using the ELFA (Enzyme Linked Fluorescent Assay) technique in serum or plasma. The principle of ELFA assay corresponds to a combination of immunoenzymatic reactions with detection of the end point using fluorescence. The enzyme used is the alkaline phosphatase that catalyzes the hydrolysis reaction of the substrate, 4-méthyl-ombelliferyl phosphate, to a product: 4-méthyl-ombellierone. The product emits at a wavelength of 450 nm after excitation at 370 nm. The results were automatically analyzed by VIDAS® and expressed as the relative fluorescence intensity or RFV (for “Relative Fluorescent Value”). This value for RFV was determined by subtracting the value for the background noise (BKG) from the gross value obtained.

[0336] The reagents were used as described in the notes, with the following modifications: [0337] 1. The cones were sensitized with MAB3573 monoclonal antibodies in a concentration of 2.5 μg/ml (indirect coating with a first anti-mouse Ac at 10 μg/ml then anti-sVEGFR2 Ac at 2.5 μg/ml); [0338] 2. The contents of the fourth well of the cartridge of the Vidas® B.R.A.H.M.S. PCT™ kit was replaced by 400 μl of revealing antibody (ref.: AF357), coupled to biotin, diluted to 1 μg/ml; [0339] 3. The plasma samples (200 μl) were used directly, pure; [0340] 4. The ELISA reaction was carried out with the aid of the automated Vidas® instrument and application of the Vidas® B.R.A.H.M.S. PCT™ kit protocol; [0341] 5. The results were obtained in the form of gross values after subtracting the background noise (reading for substrate before reaction). A calibration curve was produced by assaying a range of concentration for the marker in the form of recombinant protein (Recombinant Human VEGF R2/KDR/Flk-1 Fc Chimera. Bio-Techne® ref: 357-KD-050). The calibration curve was plotted by recording the concentration of marker along the abscissa and the signal read by Vidas® (RFV or Relative Fluorescence Value) up the ordinate. The concentration of marker present in the serum was calculated by recording the concentration corresponding to the RFV signal read by Vidas®.

Example 3

Assay of the Soluble Form of the Receptor for uPA (suPAR) by ELISA

[0342] Human serum was collected at from patients described in Example 1 T0 and T6.

[0343] The suPAR blood counts were measured with the aid of frozen serum (samples stored at −80° C.). The samples were analyzed using the commercial CE/IVD marked ELISA suPARnostic® AUTO Flex kit, in accordance with the instructions of the manufacturer (Virogates, Birkeroed, Denmark). The suPARnostic® ELISA test is based on a simplified double monoclonal antibody sandwich ELISA assay in which serum samples and peroxidase-conjugated anti-suPAR are initially mixed then incubated in anti-suPAR pre-coated micro-wells. The recombinant suPAR standards of the kit were calibrated and enabled a calibration curve to be calculated. The concentrations of suPAR were determined in ng/ml of plasma. The test was validated for measuring suPAR levels between 0.6 and 22 ng/ml.

Example 5

Statistical Analyses

[0344] The statistical analyses were carried out using R software, version 3.4.0. The differences observed were considered to be significant for values of p, or p-values, of less than 0.05.

[0345] Association Between the Level of Expression of Markers sVEGFR2 and suPAR and the Appearance or Otherwise of Complications

[0346] The predictive capacity of measuring the level of expression of the markers was studied having regard to the appearance or not or complications in the patients in the 72 hours which followed taking the first blood sample at T0. The Wilcoxon-Mann-Whitney test was used to characterize this association.

[0347] The levels of expression of VEGFR2 and suPAR at T0 and T6 were measured as described above in blood samples from 233 patients suspected of having an infection, having a SOFA score of less than two. The results are presented in Table 5 and in FIGS. 1 and 2.

TABLE-US-00005 TABLE 5 Mann-Whitney (p-value) sVEGFR2 suPAR T0 <0.0001 0.02 T6 <0.001 0.00737

[0348] The results shown in FIGS. 1 and 2 provide, up the ordinate, the level of expression of sVEGFR2 (in pg/mL) and suPAR (in ng/mL) at T0 and T6, as a function of the development or not of complications in patients.

[0349] These results showed a significant association (p<0.05) between the level of expression of the markers sVEGFR2 and suPAR at T0 and at T6 and complications in the 72 hours following taking the first blood sample at T0. The sVEGFR2 protein exhibited better performances at T0 and T6 than suPAR.

[0350] The level of expression of the studied markers can thus be used to distinguish patients who will develop complications in the 72 hours which follow taking the first blood sample at T0 from those who will not develop complications.

[0351] More precisely, the patients in whom complications will appear had lower levels of expression of sVEGFR2 than patients who did not suffer from any complications. Concerning suPAR, the patients in whom complications will develop had higher levels of expression than patients who did not suffer from any complications.

[0352] Association Between the Variation in the Level of Expression of sVEGFR2 and the Development or Not of Complications

[0353] Apart from the association between the level of expression of sVEGFR2 and the development of complications, the association between the difference in the level of expression of sVEGFR2 measured in two successive samples taken between 4 and 8 hours and the appearance of complications in the 72 hours which followed taking the first sample was observed.

[0354] The level of expression of sVEGFR2 at T0 and T6 was measured as described above in the plasma samples from 233 patients suspected of having an infection, having a SOFA score of less than two. For each patient, the variation was calculated in accordance with the following formula:

[00003]$\Delta =\frac{\mathrm{sVEGFR}.Math..Math.2.Math..Math.\mathrm{at}.Math..Math.T.Math..Math.0-\mathrm{sVEGFR}.Math..Math.2.Math..Math.\mathrm{at}.Math..Math.T.Math..Math.6}{\mathrm{sVEGFR}.Math..Math.2.Math..Math.\mathrm{at}.Math..Math.T.Math..Math.0}$

[0355] The results are presented in FIG. 3 giving, up the ordinate, the result of a variation between T0 and T6 (Δ in accordance with the above formula), as a function of the development or not of complications in patients, patients for whom clinical monitoring had shown that their condition then deteriorated (“COMPLICATIONS”), and patients for whom clinical monitoring had shown that their condition was not going to deteriorate (“NO COMPLICATIONS”).

[0356] These results show that the variation between T0 and T6 of the level of expression of sVEGFR2 is differentially associated (p-value=0.01) with patient complications in the 72 hours which follow the first sample.

[0357] More precisely, the patients who saw their condition deteriorate presented a variation in the level of expression of sVEGFR2 between T0 and T6 which was higher than for patients who will not suffer any complications.

[0358] Association Between the Level of Expression of sVEGFR2 and suPAR and the Probability of Complications

[0359] The association of the variables sVEGFR2 and suPAR with the status of the patient (in the present case «COMPLICATIONS″) was tested by means of a logistic regression. The strength of the association was estimated by calculating Odd Ratios (ORs), which is the ratio of the probability that the patient will develop at least one complication to the probability that the patient will not develop any complications.

[0360] For each quantitative variable: level of expression of sVEGFR2 and suPAR, the Odd Ratio was interpreted as follows: [0361] OR=1: no association [0362] OR<1: an increase from the first to the third quartile is associated with a decrease in the risk of complications [0363] OR>1: an increase from the first to the third quartile is associated with an increase in the risk of complications

[0364] The IQR is the interquartile range. The IQR is a measure of dispersion that is obtained by taking the difference between the third and the first quartile.

[0365] IQR.OR was measured for the blood samples from 233 patients suspected of having an infection, having a SOFA score of less than two.

[0366] The logistic regression model was also produced in order to analyze the performances of the ratio of the levels of expression of sVEGFR2 and suPAR. The objective was to demonstrate that the ratio of the markers is significantly associated with the risk of complications in the 72 hours which follow taking the first sample.

[0367] The results are given in Table 6 below.

TABLE-US-00006 TABLE 6 Markers IQR.OR p-value sVEGFR2 0.36 (1.7-4.8) 1.19 .Math. 10.sup.−4 suPAR 1.49 (1.1-2.1) 2.09 .Math. 10.sup.−2 sVEGFR2/suPAR 1.74 (1.3-2.4) 5.13 .Math. 10.sup.−4

[0368] The value of IQR.OR for the marker sVEGFR2 over the studied population was 0.36 with a p-value equal to 0.0001. Thus, patients with a low level of expression of sVEGFR2 (first quartile) had a significantly higher probability of complications (2.76 times) than patients with a high level of expression (third quartile).

[0369] The value of IQR.OR for the suPAR marker over the studied population was 1.49 with a p-value equal to 0.02. Thus, patients with a high level of expression of suPAR (third quartile) had a significantly higher probability of complications (1.49 times) than patients with a low level of expression (first quartile).

[0370] The value of IQR.OR for the ratio between sVEGFR2 and suPAR was 1.74 with a p-value equal to 0.0005. Thus, patients with an expression ratio between sVEGFR2 and suPAR (third quartile) had a significantly higher probability of complications (1.74 times) than patients with a low expression ratio (first quartile).

[0371] Analysis of the Predictive Performance of sVEGFR2 and suPAR Alone and in Combination

[0372] In order to analyze the predictive performance (sensibility/specificity) of the sVEGFR2 and suPAR markers alone and in combination at T0, the areas under the curve (AUC: Area Under Curve) were calculated; in addition, the maximum specificity, the maximum negative predictive value (NPV) and the maximum positive predictive value were assessed for a minimum imposed sensitivity of 0.90. The results are shown in Table 5 and in FIG. 4.

TABLE-US-00007 TABLE 5 Min imposed sensitivity = 0.90 Max NPV PPV Marker at T0 AUC 95% CI specificity Max max sVEGFR2 0.70 0.614-0.783 0.17 0.87 0.22 suPAR 0.61 0.516-0.701 0.18 0.87 0.22 sVEGFR2 + 0.72 0.641-0.8  0.31 0.92 0.25 sUPAR

[0373] The markers exhibited very good overall performances at T0. However, sVEGFR2 had better overall performances at T0 (AUC=0.70). In addition, the results show that the combination of sVEGFR2 and suPAR markers can be used to increase the overall performances of the prognostic test. (cf. FIG. 4).

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