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

Abstract

An in vitro or ex vivo method for determining the risk of incidence of a healthcare-associated infection includes a step of measuring the expression of TAP2 in a biological sample from said patient.

Claims

1. An in vitro or ex vivo method for determining the risk of incidence of a healthcare-associated infection in a patient, comprising a step of measuring the expression of TAP2 in a biological sample from said patient.

2. The method as claimed in claim 1, wherein it additionally comprises a step of measuring, in the biological sample from the patient, the expression: of at least one gene selected from the family of genes encoding molecules involved in the innate immune system, and/or of at least one gene selected from the family of genes encoding molecules of the cell cycle, and/or of at least one gene selected from the family of genes encoding cytokines, and/or of at least one gene selected from the family of genes encoding anti-inflammatory cytokines, and/or of at least one gene selected from the family of genes encoding pro-inflammatory cytokine receptors, located on chromosome 2 in the the region 2q11-2q12, and/or of at least one gene selected from the family of genes encoding pro-inflammatory cytokines, and/or of at least one gene selected from the family of genes encoding molecules involved in cytoskeleton formation, and/or of at least one gene selected from the family of genes encoding molecules involved in gene expression and/or transcription, and/or of at least one gene selected from the family of genes encoding growth factors, and/or of at least one gene selected from the family of genes encoding molecules of the metabolism, and/or of at least one gene selected from the family of genes encoding molecules involved in the adaptive immune system, and/or of at least one gene selected from the family of genes encoding molecules involved in signal transduction, and/or of at least one gene selected from the family of genes encoding molecules involved in modulation of the acute phase.

3. The method as claimed in claim 1, wherein it comprises a step of measuring, in the biological sample from the patient, the expression: of at least one gene selected from the following genes: GNLY, S100A9, C3AR1, ADGRE3, CD177, CX3CR1, IFIH1, OAS2, OAS3, and/or of the CCNB1IP1 gene, and/or of at least one gene selected from the following genes: IL15, IL2, MCP1(CCL2), CXCL10, and/or of at least one gene selected from the following genes: IL10, IL1RN, and/or of at least one gene selected from the following genes: IL18R1, IL1R2, IL1R1 and IL18RAP, and/or of at least one gene selected from the following genes: IFNG, IL1B, IL17A, IL18, IL6, TNF, and/or of at least one gene selected from the following genes: ARL14EP, GSN, and/or of at least one gene selected from the following genes: CIITA, DYRK2, GATA3, MDC1, NFKB1, RORC, STAT4, TBX21, TDRD9, and/or of the CSF2 gene, and/or of at least one gene selected from the following genes: ALOX5, BPGM, TRAP1, and/or of at least one gene selected from the following genes: CD40LG, CD3D, BTLA, CD274, CTLA4, ICOS, PDCD1, TNFSF4, CD74, FCGR1A, LILRB2, and/or of at least one gene selected from the following genes: FLT1, HAVCR2, IL7R, ZAP70, and/or of the HP gene.

4. The method as claimed in claim 1, wherein it comprises a step of measuring, in the biological sample from the patient, the expression of at least one gene selected from the following genes: GNLY, S100A9, C3AR1, ADGRE3, CD177, CX3CR1, OAS2, CCNB1IP1, IL10, IL1RN, IL1R2, IFNG, TNF, ARL14EP, CIITA, GATA3, MDC1, TDRD9, BPGM, CD3D, CD274, CTLA4, CD74, IL7R, ZAP70, HP.

5. The method as claimed in claim 4, wherein it comprises a step of measuring, in the biological sample from the patient, the expression of at least one gene selected from the following genes: S100A9, C3AR1, CD177, CX3CR1, IL1R2, IFNG, CIITA, CD3D, CTLA4, CD74, HP.

6. The method as claimed in claim 1, wherein the biological sample is a blood sample.

7. The method as claimed in claim 1, wherein the expression is measured at the messenger RNA (mRNA) level.

8. The method as claimed in claim 1, wherein the expression is measured by RT-PCR.

9. The method as claimed in claim 1, wherein the expression is measured by sequencing.

10. The method as claimed in claim 1, wherein the expression is measured by hybridization.

11. The method as claimed in claim 7, wherein the expression is normalized in relation to the expression of one or more housekeeping genes.

12. A kit comprising means for amplifying and/or means for detecting the expression of TAP2 and of at least one other gene, selected from the genes of claim 2; said kit wherein all the amplification and/or detection means of said kit enable the detection and/or amplification of at most 100 biomarkers in total.

13. A method comprising using the in vitro or ex vivo: of means for amplifying and/or means for detecting the expression of TAP2, or of a kit comprising such amplification and/or detection means, for determining the risk of incidence of a healthcare-associated infection in a patient.

Description

EXAMPLE 1: MEASUREMENT OF THE EXPRESSION OF TAP2 MAKES IT POSSIBLE TO PREDICT THE RISK OF INCIDENCE OF A HEALTHCARE-ASSOCIATED INFECTION IN A PATIENT

[0078] Materials and Methods

[0079] A prospective, longitudinal, single-center observational clinical study was carried out at Hôpital Edouard Herriot (Lyon, France). The design of this clinical study was published in Rol et al (2017), BMJ Open 7(6): e015734. The clinical study was approved by the Agence Nationale de Sécurité du Medicament et des produits de sante (ANSM) [French Agency for the Safety of Drugs and Health Products] and the Comité de Protection des Personnes Sud-Est II [South-East II Independent Ethics Committee] in December 2015. Amendments to the protocol were made in July 2016 and 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), from severe injury (n=137) or hospitalized in a resuscitation unit or an intensive care unit following major surgery (n=109), and 175 healthy volunteers, were included between December 2015 and March 2018. [0080] Patients in a septic state/in septic shock: according to the first clinical protocol, only those patients in septic shock were included, on the basis of a suspected infected site, treatment with catecholamimes was started within the 48 h following admission to resuscitation, and treatment with catecholamines (noradrenalin)>0.25 μg/kg/min for at least 2 hours. Subsequently, 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). Patients in septic shock were therefore included on the basis of a suspected infected site, treatment with catecholamimes was started within the 48 h following admission to resuscitation, along with vasopressive therapy required to maintain arterial pressure of 65 mm Hg and lactate concentration of >2 mmol/1 (18 mg/dl), despite correction of hypovolemia. In 2017, the possibility of including patients in a septic state (according to the definition of Sepsis 3) was added, namely a suspected infected site and an increase in the SOFA score 2 points compared to the baseline SOFA in the 48 h following admission to resuscitation. For this population, day 1 corresponds to the day of diagnosis of sepsis or septic shock. [0081] Severe injury: in the first protocol, only those patients with a sever injury were included (Injury Severity Score (ISS)≥25). In August 2016, the possibility of also including less sever injuries (16<ISS<24) was added. For this population, day 1 corresponds to the day of admission to the resuscitation unit or to the intensive care unit (.sup.˜day of the injury). [0082] Major surgery: in the first protocol, only esogastrectomy, Bricker-type bladder resection, cephalic duodenopancreatectomy and abdominal aorta surgery by laparotomy were considered. Other types of surgery with a high risk of complications were added in January 2017: pancreatectomy (total or caudal), neuroendocrine tumors, hepatectomy (on the right-hand side), extended colectomy (laparotomy), abdoperineal resection, nephrectomy (laparotomy, PKD), ilio-femoral bypass (Scarpa). For this population, day 1 corresponds to the day of surgery. [0083] Severe burns: the patients were selected on the basis of a total burn surface area of greater than 30%. For this population, day 1 corresponds to the day of admission to the resuscitation unit or to the intensive care unit (.sup.˜day of the burn).

[0084] The exclusion criteria related essentially to factors which could have impacted the immune status and could have biased the results (for example: severe neutropenia, corticosteroid treatments, an onco-haematological disease, etc.). Each event leading to a suspected healthcare-associated infection, occurring within the hospital before D30, was reviewed independently by three physicians who were not involved in patient recruitment. Twenty-six percent of the patients developed at least one healthcare-associated infection before D30, or before leaving hospital.

[0085] Blood samples were collected in PAXgene® tubes (ref. 762165, PreAnalytiX GmbH Hombrechtikon Switzerland), once for healthy volunteers and several times for a sub-cohort of 242 patients (i.e. 82 patients in a septic state/in septic shock, 83 patients suffering from severe injuries, 61 patients hospitalized in a resuscitation unit or an intensive care unit after major surgery, and 16 patients suffering from severe burns), 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).

[0086] The expression level of TAP2 was measured in these samples by RT-qPCR. A volume of 100 μl of blood collected in the PAXgene® was directly injected into a FilmArray® pouch optimized for detecting a panel of genes involved in the host response, including TAP2, by nested PCR. The steps of extraction of the nucleic acids, reverse transcription and qPCR were carried out sequentially and automatically by the Filmarray® instrument, without external intervention. The cycle thresholds (Ct) determined by the instrument were normalized in relation to the expression of 3 reference genes (DECR1, HPRT1 and PPIB).

[0087] Regarding data analysis, associations between the expression of TAP2, measured at different times during the first week, and the incidence of a healthcare-associated infection before D30 from inclusion in the study were evaluated. The results were calculated in the form of Hazard Ratios expressed as the inter-quartile distance with the associated 95% confidence interval (HR IQR). Next, univariate logistic regression was implemented in order to predict the risk of incidence of a healthcare-associated infection before D15. The power of the values predicted by logistic regression to distinguish between healthcare-associated infection and lack of healthcare-associated infection was quantified by the area under the curve (AUC) of the ROC curve (Receiver Operating Characteristic), and 95% confidence intervals were estimated.

[0088] Next, the association between the expression of TAP2 and the incidence of a healthcare-associated infection was evaluated for different time intervals of incidence of the infection (i.e. periods between taking the sample and the 1st incidence of an infection). The different periods considered were: a healthcare-associated infection in the 4 days and in the 7 days following the sample being taken, regardless of when the sample was taken. For each patient who developed a healthcare-associated infection, the sample considered corresponds to the sample taken closest to the incidence of the first episode of healthcare-associated infection.

[0089] For patients who did not develop a healthcare-associated infection (i.e. control patients), a matching method was used to select, for each case, a control patient whose sample was taken on the same day and with close SOFA and Charlson scores. Finally, a single control was selected for each unique case. Univariate logistic regressions were implemented. The power of the values predicted by logistic regression to distinguish between healthcare-associated infection and lack of healthcare-associated infection was quantified by the area under the curve (AUC) of the ROC curve, and 95% confidence intervals were estimated.

[0090] Results

[0091] A reduction in the expression of TAP2 at the mRNA level, measured on D5/7 from inclusion in the cohort, was associated with a greater risk of incidence of a healthcare-associated infection before D30 in the overall patient population (univariate analysis: HR IQR=0.39 [0.24-0.63], p=0.0002). This association was always significant after adjustment with the SOFA and Charlson score:multivariate analysis: HR IQR=0.40 [0.24-0.67], p=0.0005).

[0092] Moreover, the prediction models showed that the expression of TAP2 at the mRNA level, measured on D3/4 or D5.7 from inclusion in the cohort, made it possible to predict the incidence of a healthcare-associated infection before D15 from inclusion in the cohort (table 4).

TABLE-US-00004 TABLE 4 Performance (AUC and 95% confidence intervals, with AUC.2.5 and AUC.97.5 limits) of the measurement of the expression of TAP2, measured on D 3/4 or D 5/7 from inclusion in the cohort, in predicting the incidence of a healthcare-associated infection before D 15 from inclusion in the cohort in patients in a septic state/in septic shock, suffering from severe injuries, or hospitalized following major surgery. Day on which sample was taken AUC AUC.2.5 AUC.97.5 D 3/4 0.587 0.468 0.706 D 5/7 0.732 0.638 0.825

[0093] The prediction models also showed that the expression of TAP2 at the mRNA level made it possible to predict the incidence of a healthcare-associated infection in the 4 days or 7 days following the sample being taken (table 5).

TABLE-US-00005 TABLE 5 Performance (AUC and 95% confidence intervals, with AUC.2.5 and AUC.97.5 limits) of the measurement of the expression of TAP2 in predicting the incidence of a healthcare-associated infection in the 4 days or in the 7 days following the sample being taken. Maximum time interval between the sample being taken and the potential incidence of first healthcare-associated infection AUC AUC.2.5 AUC.97.5 4 days 0.637 0.432 0.842 7 days 0.67 0.488 0.852

[0094] Thus, the results obtained show that the measurement of the expression of TAP2 alone makes it possible to predict the incidence of healthcare-associated infection(s) in the 15 days starting from the immuno-inflammatory attack, in the 4 days following the sample being taken or in the 7 days following the sample being taken.

EXAMPLE 2: THE MEASUREMENT OF THE EXPRESSION OF ONE OR MORE OTHER GENE(S) IN COMBINATION WITH THE MEASUREMENT OF THE EXPRESSION OF TAP2 MAKES IT POSSIBLE TO IMPROVE THE PERFORMANCE IN PREDICTING THE RISK OF INCIDENCE OF A HEALTHCARE-ASSOCIATED INFECTION

[0095] In this example, the expression level of TAP2 and also that of other genes was measured by RT-qPCR. Multivariate logistic regressions (combination of the measurement of the expression of TAP2 and of one or more other gene(s)) were then carried out. The measurement of the expression of one or more of these other genes, in addition to the measurement of the expression of TAP2, makes it possible to improve the performance (compared to the measurement of the expression of TAP2 alone) in predicting the risk of incidence of a healthcare-associated infection, whether before D15 starting from inclusion in the cohort (table 6) or in the 4 days or in the 7 days following the sample being taken (table 7).

TABLE-US-00006 Lengthy table referenced here US20230220477A1-20230713-T00001 Please refer to the end of the specification for access instructions.

TABLE-US-00007 Lengthy table referenced here US20230220477A1-20230713-T00002 Please refer to the end of the specification for access instructions.

TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).