Process for determining the susceptibility to nosocomial infections

Abstract

The invention relates to a process for determining the susceptibility to nosocomical infections in a patient, comprising the measurement of the expression of sCD127 in a biological sample.

Claims

1. A process for determining the susceptibility of a patient with septic shock to nosocomial infections, comprising the following steps: taking a first biological sample from said patient up to 48h after an onset of septic shock; taking a second biological sample from said patient at least 24h after the first biological sample is taken, and up to 10 days after the onset of septic shock; and measuring the expression of soluble CD127 in the first biological sample and in the second biological sample using anti-soluble CD127 antibodies, whereby a concentration of soluble CD127 in the first biological sample and a concentration of soluble CD127 in the second biological sample is obtained; wherein an increased susceptibility to nosocomial infections in the patient is indicated when the concentration of soluble CD127 in the second biological sample is not significantly reduced compared to the concentration of soluble CD127 in the first biological sample.

2. The process according to claim 1, wherein the expression of soluble CD127 in the second biological sample is measured within 10 days after septic shock, or at day 10 after septic shock.

3. The process according to claim 1, wherein the expression of soluble CD127 in the second biological sample is measured within or at about 7 days after septic shock.

4. The process according to claim 1, wherein the second biological sample is taken from said patient within 48h of taking the first biological sample, or at 48h after the first biological sample is taken.

5. The process according to claim 1, wherein the second biological sample is a plasma sample or a serum sample.

6. The process according to claim 1, comprising measuring the expression of soluble CD127 using an ELISA technique.

7. The process according to claim 1, comprising measuring the expression of soluble CD127 using monoclonal antibodies.

8. The process according to claim 1, wherein the increased susceptibility to nosocomial infections in the patient is indicated when the expression of soluble CD127 measured in the second biological sample is not reduced by more than 25%, compared with the expression of soluble CD127 measured in the first biological sample.

9. The process according to claim 1, wherein the increased susceptibility to nosocomial infections in the patient is indicated when the expression of soluble CD127 measured in the second biological sample is not reduced by more than 20%, compared with the expression of soluble CD127 measured in the first biological sample.

10. The process according to claim 1, wherein the expression of soluble CD127 in the second biological sample is measured within 4 days, or at day 4 after septic shock.

11. The process according to claim 1, wherein the expression of soluble CD127 in the second biological sample is measured within 3 days, or at day 3, after septic shock.

Description

(1) Various other characteristics will become apparent from the description given below with reference to the appended Figures which, as non-limiting examples, illustrate embodiments of the subject of the invention and in which:

(2) FIG. 1 shows the IL-7 plasma concentration in 35 patients in septic shock at days 1-2 (1-2) and 3-4 (3-4) and in 30 healthy volunteers “HV” (A), and the comparison of these results between “NS” and “S” patient groups (FIG. 1B) and “NI” and “No NI” patient groups (FIG. 1C).

(3) ** p<0.005 vs. “HV”—Mann Whitney U-test;

(4) FIG. 2 gives the expression of CD127 in T CD4.sup.+ cells (FIG. 2A) and T CD8.sup.+ cells (FIG. 2B) in 35 patients with septic shock at days 1-2 and 3-5 and in 30 healthy volunteers “HV”.

(5) * p<0.05 vs. “HV”—Mann Whitney U-test; §p<0.05—trend in time within one same group of patients—Wilcoxon paired t-test.

(6) FIG. 3 gives the sCD127 plasma concentration in 35 patients with septic shock at days 1-2 and 3-4, and in 30 healthy volunteers (FIG. 3A) and the comparison of these results between the “NS” or “S” patient groups (FIG. 3B) and “NI” or “No NI” patient groups (FIG. 3C).

(7) # p<0.05 ## p<0.005 vs. “No NI”—Mann Whitney U-test; §p<0.05 §§p<0.005—trend in time within one same group of patients—Wilcoxon paired t-test.

(8) FIG. 4 shows the expression of the HLA-DR marker in 35 patients with septic shock at days 1-2 and 3-4, and in 30 healthy volunteers as per patient groups “NI” or “No NI”.

METHODS

(9) Measurement of IL-7 Plasma Concentration

(10) The IL-7 plasma concentration was measured with a kit using the LUMINEX™ technique marketed by Bio-Rad (Bio-Plex Pro Cytokine, Chemokine and Growth Factor Assays: BioPlex Pro Reagent kit, Bio-Rad #171-304070 and SinglePlex IL-7) following the supplier's directions.

(11) Measurement of Cell Expression of the IL-7 Receptor (CD127)

(12) In brief, 50 μl of whole blood were incubated in the presence of 5 μl of anti-CD4 antibody conjugated with Phycoerythin-TexasRed (ECD) (Beckman Coulter #6604727) or 5 μl of ECD-conjugated anti-CD8 antibody (Beckman Coulter #737659) and 10 μl of anti-CD127 antibody conjugated with phycoerythrin (PE) (Beckman Coulter #IM1980U) for 15 minutes at ambient temperature and in the dark. The red blood cells were then lysed by hypotonic lysis and the cells fixed by automatic lysis on TQ-Prep automated system (Beckman Coulter). The membrane expression of CD127 on the surface of the T CD4+ or CD8+ lymphocytes was measured by flow cytometry.

(13) Assay of the Soluble Form of the IL-7 Receptor (sCD127) by ELISA

(14) Coating

(15) A coating buffer was prepared to contain 0.8 g of Na.sub.2CO.sub.3, 1.4 g NaHCO.sub.3, 0.1 g NaN.sub.3 in 500 ml of water (pH 9.6).

(16) 100 μL of capture antibody (Ab) (Mouse Monoclonal Antibody Anti-CD127, human, R34.34, Beckman Coulter®) diluted in coating buffer were deposited per well of an assay plate ([Ab]=8 μg/mL). The plate was then covered for incubation at 4° C. overnight.

(17) The content of the wells was then aspirated and the wells washed 3 times with at least 300 μL of 0.05% PBS-TWEEN20® non-ionic detergent wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer.

(18) Blocking

(19) Non-specific fixing was blocked using 150 μL of blocking buffer per well (10% foetal bovine serum (FBS)/0.05% PBS-TWEEN20® non-ionic detergent), and the plate incubated for 1 h at 37° C.

(20) The content of the wells was again aspirated and the wells washed 3 times with at least 300 μL of 0.05% PBS-TWEEN20® non-ionic detergent wash buffer. All the liquid was carefully removed at each wash. After the last wash, the plate was turned over onto absorbent paper to remove all traces of buffer.

(21) Samples and Standards

(22) A standard range was obtained with recombinant human IL-7Rα/CD127 Fc chimera (R&D Systems—Catalog Number: 306-IR) diluted in PBS containing 5% FCS diluting buffer, as described in Table 1 below and in accordance with C. Janot-Sardet et al. Journal of Immunological Methods, 2010, 28, 115-123.

(23) TABLE-US-00001 TABLE 1 rh IL-7Rα/CD127 Fc chimera [c] (ng/mL) 500 250 125 62.5 31.25 15.7 7.85 0 Diluent (μL) 0 100 100 100 100 100 100 100 500 ng/mL 100 100 Successive dilutions 0 solution (μL)

(24) 100 μL of sample or standard (extemporaneously-reconstituted solution of CD127 Fc chimera aliquoted at concentrations of 60 ng/ml and 10 ng/ml) were added to each well and the plate incubated for 1 h at 37° C.

(25) The content of the wells was again aspirated and the wells washed 3 times with at least 300 μL of 0.05% PBS-TWEEN20® non-ionic detergent wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer.

(26) Detection Antibody

(27) 100 μL of detection antibody (biotinylated polyclonal anti-CD127 goat antibody reconstituted with 1 mL of 1% TBS-BSA, R&D Systems®) diluted in PBS/5% FBS, were added to each well ([Ab]=200 ng/mL), and the plate then incubated for 1 h at 37° C.

(28) The content of the wells was again aspirated and the wells washed 3 times with at least 300 μL of 0.05% de PBS-TWEEN20® non-ionic detergent wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer.

(29) Detection

(30) 100 μL of Streptavidin-HRP were added to each well ([Streptavidin-HRP]=8 μL/mL). The plate was covered to be incubated for 30 min at ambient temperature.

(31) The content of the wells was again aspirated and the wells washed 3 times with at least 300 μL of 0.05% PBS-TWEEN20® non-ionic detergent wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer. At this washing step the wells were impregnated with the wash buffer 1 to 2 min before aspiration.

(32) The two bottles of TMB colorimetric substrate solution (3,3′,5,5′-tetramethylbenzidine, bioMérieux #XX7LF1UC) were volume/volume mixed. 100 μL of this substrate solution were deposited in each well. The plate was covered to be incubated 30 min at ambient temperature.

(33) Finally, plate read-off was performed by absorbance measurement at 450 nm.

(34) Measurement of the Monocyte Expression of HLA-DR

(35) Measurement was performed by the flow cytometry technique, using direct staining with EDTA whole blood.

(36) The two following antibodies were incubated with 50 μL of whole blood (30 minutes at ambient temperature and in the dark): 5 μL of fluorescein-conjugated anti-CD14 antibody (Beckman Coulter Immunotech—Ref.: IM0645), allowing identification of the monocytes among the white blood cells); 10 μL of phycoerythrin-conjugated anti-HLA-DR antibody (Becton Dickinson—Ref.: 347401), allowing quantification of HLA-DR expression on the surface of the cells.

(37) The red blood cells were then removed: lysis by adding 1 ml of lysis solution (marketed by Becton Dickinson under reference 349202), 1:10 dilution, 15 minutes at ambient temperature and in the dark.

(38) The cells were then analysed on a flow cytometer (FC500—Beckman Coulter).

(39) Results are expressed as a % of positive cells, the positivity threshold being defined by isotype control (Mouse IgG2a PE Becton Dickinson—Ref.: 349053).

(40) Results

(41) Plasma samples were taken from 35 patients in septic shock at days 1-2 (D1-2) and 3-4 (D3-4) after septic shock and then stored (retrospective cohort). Different parameters or markers were measured such as IL-7 and sCD127 plasma concentrations, and CD127 expression on T CD4.sup.+ cells, and HLA-DR expression on the monocytes. At 28 days after admission into intensive care for septic shock, 13 patients did not survive (“NS”) i.e. 37% whereas 22 patients did survive (“S”) out of 35 patients. 6 patients contracted a nosocomial infection (“NI”) i.e. 17%, whereas 29 patients remained free of any nosocomial occurrence (“No NI”).

(42) The same measurements were performed in 30 healthy volunteers (HV).

(43) The results obtained were compared using a Mann Whitney U-test in these different populations of volunteers or patients, and are grouped together in appended FIGS. 1 to 4.

(44) These results show that the IL-7 plasma concentration is significantly reduced (at D1-2), but not significantly at D3-4 in patients who develop secondary nosocomial infection compared with patients who did not suffer any nosocomial infection occurrence (FIG. 1C), whereas it remained unchanged between the surviving patients “S” and non-surviving patients “NS”.

(45) In addition, the cell expression of the IL-7 receptor (CD127) was maintained after septic shock (FIGS. 2A and 2B), and without any difference between the surviving patients “S” and non-surviving patients “NS” or the patients who did “NI” or did not “No NI” develop nosocomial infections (results not shown).

(46) The slight increase in the plasma concentration of the soluble form of the I-7 receptor, or sCD127, observed at the onset of septic shock is not statistically significant and returns to “normal” values over time (FIG. 3A).

(47) On the other hand, whereas the plasma concentration of sCD127 remained unchanged between the surviving patients “S” and non-surviving patients “NS” (FIG. 3B), a marked increase in the plasma concentration of sCD127 was observed in “NI” patients who developed a nosocomial infection compared with the “No NI” patients who did not contact a nosocomial infection (FIG. 3C).

(48) Also it was observed that the plasma concentration of sCD127 does not change over time (between D1-2 and D3-4) in “NI” patients who developed a nosocomial infection, contrary to the “No NI” patients who did not contract a nosocomial infection (FIG. 3C).

(49) By way of comparison, study of the expression of the prior art marker HLA-DR on the same cohort of patients did not allow evidencing of the reduction in expression of this marker between the “NI” patients who suffered a nosocomial infection and the “No NI” who did not contract a nosocomial infection (FIG. 4), contrary to what is clearly established in the prior art.

(50) This difference, probably related to the size of the cohort studied herein which is smaller compared with the cohorts analysed in prior art studies, allows evidencing of the pertinence of the sCD127 marker compared with HLA-DR. Despite the size of the cohort studied herein the results show that the study of the expression of sCD127 according to the invention is informative with regard to the susceptibility of patients to nosocomial infections.

(51) It follows that all these results show that measurement of sCD127 expression is a useful immunological marker to determine the susceptibility of a patient to nosocomial infections, and in particular patients hospitalized in intensive care and/or having sustained an insult (surgery, burns, trauma, . . . ) generating a systemic inflammatory response (SIRS), and in particular patients with sepsis, particularly severe sepsis, and preferably patients in septic shock.

(52) The invention is not limited to the examples described and illustrated since various modifications can be made thereto without departing from the scope of the invention.