METHOD FOR DETERMINING THE RISK OF INCIDENCE OF A CARE-ASSOCIATED INFECTION IN A PATIENT

Abstract

An in vitro or ex vivo method, based on the measurement of the expression of cytokine(s), from a patient's blood sample, incubated with a stimulus, for determining the risk of occurrence of a healthcare-associated infection in the patient, within seven days following the day on which the collection of the biological sample has been performed from the patient.

Claims

1. An in vitro or ex vivo method for determining the risk of occurrence of a healthcare-associated infection in a patient within seven days following the day on which the collection of the blood sample has been performed from the patient, comprising: a) a step of incubating a blood sample of the patient with a stimulus, the stimulus comprising a molecule selected from the group consisting of: a molecule capable of binding at least one type of antigen-presenting cell (APC), and at least one type of cell of the adaptive immunity, one or several molecule(s) allowing direct activation of T lymphocytes, the one or several molecule(s) being selected from antibodies and antibody analogs, and a molecule of the imidazoquinoline type; b) a step of measuring the expression, from the stimulated blood sample resulting from step a), of at least one cytokine, the cytokine being produced by innate immunity cells and/or by adaptive immunity cells.

2. The method according to claim 1, wherein the patient is a patient within a healthcare facility.

3. The method according to claim 1, wherein the patient is an adult patient, over the age of 18 years.

4. The method according to claim 1, wherein the blood sample is a whole blood sample.

5. The method according to claim 1, wherein the stimulus comprises a molecule capable of binding at least one type of antigen-presenting cell (APC), and at least one type of adaptive immunity cell, the molecule being a molecule of superantigen type.

6. The method according to claim 1, wherein the stimulus comprises a molecule selected from the superantigens produced by staphylococcal species and the superantigens produced by streptococcal species.

7. The method according to claim 1, wherein the stimulus comprises SEA (Staphylococcal Enterotoxin A), SEB (Staphylococcal Enterotoxin B) or SEC (Staphylococcal Enterotoxin C).

8. The method according to claim 1, wherein the stimulus comprises a molecule capable of binding at least one type of antigen-presenting cell (APC) and at least one type of adaptive immunity cell, the molecule being a molecule analogous to a superantigen.

9. The method according to claim 1, wherein the stimulus comprises one or several antibodies allowing a direct activation of the T lymphocytes, the one or several antibodies being selected from antibodies recognizing and activating a receptor on the surface of the T lymphocyte.

10. The method according to claim 1, wherein the stimulus comprises a molecule of the imidazoquinoline type.

11. The method according to claim 1, wherein in step b), the expression of at least one cytokine selected from the group consisting of GM-CSF, IFNγ, IL2, IL3, IL4, IL5, IL6, IL10, IL17 and TNFα (“adaptive immunity” list) is measured.

12. The method according to claim 1, wherein in step b), the expression of at least one cytokine selected from IFNγ and IL2 is measured.

13. The method according to claim 1, wherein in step b), the expression of at least one cytokine selected from the group consisting of CCL2 (MCP1), CCL3 (MIP1 alpha), CCL4 (MIP1 beta), CXCL8 (IL8), CXCL10 (IP10), IFNγ, IL1α, IL1β, IL1RA, IL3, IL6, IL10, IL18 and TNFα (“innate immunity” list).

14. The method according to claim 1, wherein in step b), the expression of at least two different cytokines, respectively selected from the “innate immunity” list and from the list “adaptive immunity”, is measured.

15. The method according to claim 14, wherein one of the at least two different cytokines is selected from IFNγ and IL2.

16. The method according to claim 1, wherein the expression of cytokine(s) is measured at the protein level.

17. The method according to claim 1, wherein the expression of cytokine(s) is measured by ELISA (Enzyme Linked ImmunoSorbent Assay), ELFA (Enzyme Linked Fluorescent Assay), RIA (radio immunoassays), ECL (Electrochemiluminescence) or mass spectrometry.

18. The method according to claim 1, wherein it comprises a step of measuring the expression, from a control blood sample without stimulation, of the same cytokine(s) than that/those measured from the stimulated blood sample.

19. The method according to claim 18, wherein it comprises a step of calculating the ratios of the expression of each cytokine in the stimulated blood sample, relative to the expression of the same cytokine in the control blood sample.

20. The method according to claim 1, wherein it further comprises a step of measuring, in a blood sample from the patient which has not been incubated with a stimulus, the concentration of IL10, the concentration of IL6, the number of molecules of HLA-DR per monocyte, the percentage of CD10.sup.low/CD16.sup.low neutrophils and/or the percentage of CD10.sup.high/CD16.sup.high neutrophils.

21. A method comprising applying the means for detecting the expression of at least one cytokine as defined in claim 1, the detection means being antibodies, or of a kit comprising such detection means as defined in claim 1, to determine the risk of occurrence of a healthcare-associated infection in a patient, within seven days following the day on which the collection of the blood sample, from which the expression of cytokine(s) is measured, has been performed from the patient.

Description

FIGURES

[0048] FIG. 1: Effect of different stimulus (SEB, SEC and a bifunctional conjugate ‘aHLADR/aCD3’ including anti-HLA-DR and anti-CD3 monoclonal antibodies grafted onto BSA) on the secretion of cytokines IL6, IL10 and CXCL10. The incubation of 100 μL (panel 1) or 300 μL (panel 2) of whole blood with the stimulus was carried out at 37° C. for 24 h (panel 1) or 16 h (panel 2), before measuring the quantity of secreted cytokines. “NS”: statistically non-significant.

[0049] The present invention is illustrated without limitation by the following examples.

EXAMPLE 1

Materials and Methods

[0050] A prospective, longitudinal and monocentric observational clinical study has been carried out at the Edouard Herriot Hospital (Lyon, France). The design of this clinical study has been published in Rol et al. (2017), BMJ Open 7(6): e015734. The clinical study was approved by the National Agency for the Safety of Medicines and Health Products (ANSM) in November 2015 and the South-East II Personal Protection Committee in December 2015. Amendments to the protocol were made in July 2016, then in January 2017. In brief, a total of 377 patients, in a septic state (n=35) or in septic shock (n=72), suffering from severe burns (n=24), severe trauma (n=137) or hospitalized in a resuscitation unit or intensive care unit after major surgery (n=109), and 175 healthy volunteers have been included between December 2015 and March 2018. [0051] Patients in septic state/in septic shock: according to the first clinical protocol, only patients in septic shock have been included, on the basis of a suspicion of an infectious focus, a start of treatment with catecholamines within 48 hours following admission to the resuscitation unit and of treatment with catecholamines (noradrenaline)>0.25 μg/kg/min for at least 2 hours. Then, the eligibility criteria were modified in August 2016, following the publication of a new definition of septic shock, Sepsis 3 (Singer et al. (2016), JAMA 810-801: (8)315). The patients in septic shock have therefore been included on the basis of a suspicion of an infectious focus, a start of treatment with catecholamines within 48 hours following admission to resuscitation unit and of vasopressor therapy necessary to maintain blood pressure 65 mm Hg and lactate concentration>2 mmol/L (18 mg/dL), despite the correction of hypovolaemia. In 2017, the possibility was added to include patients in a sepsis state (according to the Sepsis 3 definition), namely the suspicion of an infectious focus and the increase in the SOFA score 2 points compared to the basic SOFA within 48 hours following admission to the resuscitation unit. For this population, day 1 corresponds to the day of diagnosis of sepsis or septic shock; [0052] Severe trauma: in the first protocol, only patients with severe trauma have been included (Injury Severity Score (ISS)≥25). In August 2016, the possibility was added to also include less severe injuries (16<ISS<24). For this population, day 1 corresponds to the day of admission to the resuscitation unit or intensive care unit (˜trauma day); [0053] Major surgery: in the first protocol, only esogastrectomy, Bricker-type bladder resection, cephalic duodenopancreatectomy and surgery of the abdominal aorta by laparotomy have been considered. Other types of surgery with a high risk of complication were added in January 2017: (total or caudal) pancreatectomy, neuroendocrine tumors, hepatectomy (on the right side), extended colectomy (laparotomy), abdoperineal resection, nephrectomy (laparotomy, PKD), ilio-femoral bypass (Scarpa). For this population, day 1 corresponds to the day of surgery; [0054] Severe burns: the patients have been selected on the basis of a total surface area of burns greater than 30%. For this population, day 1 corresponds to the day of admission to the resuscitation unit or intensive care unit (˜day of the burn).

[0055] The exclusion criteria have mainly related to factors that could have impacted the immune status and biased the results (for example: severe neutropenia, corticosteroid treatments, onco-haematological pathology, etc.). Each event leading to a suspected healthcare-associated infection occurring in the hospital before day 30 has been independently reviewed by three physicians not involved in the recruitment of the patients. Twenty-six percent of the patients have developed at least one healthcare-associated infection before day 30, or before leaving the hospital.

[0056] Heparinized whole blood samples were collected several times for the patients, i.e. 3-4 times in the first week (on days 1 or 2: D1/2, on days 3 or 4: D3/4 and on days 5, 6 or 7: D5/7), then 3 times at later times (around D14, D28 and D60). These samples were dispensed into preheated TruCulture tubes (Myriad Rbm, Austin, Tex., USA), containing either medium alone (“control sample”) or medium with SEB (100 ng/mL) (“stimulated sample”). These tubes were then inserted into a dry block incubator and maintained at 37° C. for 24 hours. After incubation, the concentrations of IFNγ or IL2 were measured by an ELISA assay (References: SPCKB-CS-000292 and SPCKB-CS-000955, Biotechne, respectively), using the nanofluidic platform ELLA (ProteinSimple, San José, Calif., USA), as recommended by the supplier.

[0057] Regarding the data analysis, the association between cytokine secretion and the risk of occurrence of a healthcare-associated infection was assessed for different time intervals of infection occurrence (i.e. time between sample collection and the first occurrence of an infection). The different time periods considered were: healthcare-associated infection within 4 days and within 7 days after sample collection, regardless of when the sample was collected. For each patient who has developed a healthcare-associated infection (i.e. “case patients”), the sample considered corresponds to the closest sample collection (with a minimum delay of 24 hours) before the occurrence of the first episode of healthcare-associated infection. For the patients who did not develop a healthcare-associated infection (i.e. “control patients”), a matching method was used to select, for each case patient, a control patient with the same sample collection day, and close SOFA and Charlson scores. Finally, a single control was selected for each unique case. Univariate logistic regressions were implemented. The analysis was made for all types of patients combined. The association between cytokine secretion and the occurrence of healthcare-associated infection was estimated in the form of Odds Ratios expressed as inter-quartile distance (OR IQR) with the 95% confidence interval associated therewith.

Results

[0058] It has been observed that a lower concentration of IL2 or IFNγ after stimulation with SEB was associated with a higher risk of occurrence of a healthcare-associated infection within 4 or 7 days following the day of sample collection. (Table 1).

TABLE-US-00001 TABLE 1 Association between the measurement of IFNγ or IL2 after stimulation by SEB and the risk of occurrence of a healthcare- associated infection within 4 or 7 days following the day of sample collection. The Odd Ratios (OR IQR) are expressed as inter-quartile distance with the associated 95% confidence interval (CI). Number of days after sample collection until first Measured occurrence of healthcare- Cytokine associated infection OR IQR (IC) P IFNγ 4 days 0.65 (0.44-0.88) 0.014 7 days 0.63 (0.43-0.86) 0.009 IL2 4 days 0.51 (0.30-0.82) 0.007 7 days 0.65 (0.42-0.96) 0.038

EXAMPLE 2

[0059] In this example, the inventors compared the effect of three stimulus (SEB, SEC and a bifunctional conjugate ‘aHLADR/aCD3’ including anti-HLA-DR and anti-CD3 monoclonal antibodies grafted onto BSA) on the secretion of different cytokines (IL6, IL10 and CXCL10).

Materials and Methods

[0060] Whole blood samples (100 μL or 300 μL) from healthy volunteers (n=5 for each condition) were incubated at 37° C. for 16 h or 24 h in the presence of the stimulus at a concentration of approximately 1.4 10.sup.−8 M (i.e. 0.0004 g/L of SEB (TruCulture®, Myriad, ref 782-001124), 0.0004 g/L of SEC (Toxin Technology, Inc., ref CT111-SEC1) or 0.0094 g/L of aHLADR/aCD3 conjugate (Ultra-LEAF Purified anti-human HLA-DR-NA.41 (Ozyme), ref BLE307666 and Ultra-LEAF Purified anti-human CD3-NA.41 (Ozyme), ref BLE317347)) in RPMI. After incubation, the concentrations (in pg/mL) of the cytokines (IL6, IL10, CXCL10—ProteinSimple) were measured using the ELLA nanofluidic platform (ProteinSimple). Comparisons between different stimulus were made using a non-parametric test t (Wilcoxon signed rank test on paired samples). A value of p≤0.05 was considered statistically significant (two-sided test).

Results

[0061] FIG. 1 shows the results obtained with the different stimulus. Panel 1 illustrates that there was no statistically significant difference in the level of IL6, IL10 and CXCL10 secretion between a stimulation by SEB and a stimulation by SEC. Similarly, panel 2 shows that there is no statistically significant difference in the level of secretion of these cytokines between a stimulation by SEC and a stimulation by the aHLADR/aCD3 conjugate. These three stimulus therefore induce the secretion of similar amounts of cytokines in blood samples.