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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to an in vitro or ex vivo method for determining the risk of occurrence of a healthcare-associated infection in a patient, comprising a step for measuring the expression of TAP2, in a biological sample of said patient.
• the TAP2 gene encodes a membrane-associated protein of the ABC transporter superfamily (ATP-binding cassette), which is involved in the transport of peptides from the cytoplasm to the endoplasmic reticulum as part of the process of antigen presentation of molecules of class I.
• ABC transporter superfamily ATP-binding cassette
• mutations of the TAP2 gene have been associated with certain diseases, such as ankylosing spondylitis.
• the expression of the transcript of this gene has also been studied in septic patients, in which it has been shown that it makes it possible to identify patients with a particular endotype associated with the occurrence of death at 28 days (Scicluna et al. (2017), Lancet Respir Med 5(10):816-826).
• the subject of the present invention is an in vitro or ex vivo method for determining the risk of occurrence of a healthcare-associated infection in a patient, comprising a step for measuring the expression of the TAP2 gene, in a biological sample of said patient.
• the term "patient” designates an individual (human being), who has come into contact with a health professional, such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital, and more particularly the emergency department, the intensive care unit, an intensive care unit or a continuing care unit, or a medical structure for the elderly, of the EHPAD type).
• a health professional such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital, and more particularly the emergency department, the intensive care unit, an intensive care unit or a continuing care unit, or a medical structure for the elderly, of the EHPAD type).
• the patient may for example be an elderly person, as part of a vaccination protocol (in particular in an EHPAD or even at a general practitioner); an infection is said to be "associated with care", if it occurs during or after treatment (diagnostic, therapeutic, palliative, preventive, educational or operative) of a patient by a healthcare professional, and if it was not present at the start of treatment.
• Healthcare-associated infections include infections contracted within a healthcare facility (known as nosocomial infections) but also during care delivered outside this setting. When the infectious state at the start of treatment is not precisely known, a delay of at least 48 hours or a delay greater than the incubation period is commonly accepted to define an IAS.
• infectious disease occurs within 30 days of the operation or, if an implant, prosthesis or prosthetic material is placed, within one year of the operation.
• the infection can be of bacterial, fungal or even viral origin. It may also involve the reactivation of potentially pathogenic latent viruses, such as herpesviruses, for example CMV; "biological sample” refers to any sample from a patient, and may be of different natures, such as blood or its derivatives, sputum, urine, stool, skin, cerebrospinal fluid , bronchoalveolar lavage fluid, abdominal cavity puncture fluid, saliva, gastric secretions, semen, seminal fluid, tears, spinal cord, trigeminal nerve ganglion, adipose tissue, lymphoid tissue, placental tissue, gastrointestinal tract tissue, genital tract tissue, central nervous system tissue.
• this sample can be a biological fluid, such as a blood sample or a sample derived from blood, which can in particular be chosen from whole blood (as collected from the venous route, that is to say containing the white and red cells, platelets and plasma), plasma, serum, as well as any type(s) of cells extracted from blood, such as peripheral blood mononuclear cells (or PBMC, containing lymphocytes (B, T and NK cells), dendritic cells and monocytes), subpopulations of B cells, purified monocytes, or neutrophils; by “measurement of the expression of CD177” is meant the measurement of the expression of the CD177 gene, that is to say of the gene encoding the CD177 protein.
• the patient is a patient within a health establishment, preferably within a hospital, more preferably within the emergency department, the resuscitation department, in intensive care unit or continuing care unit; in a particularly preferred manner, the patient is a patient in a septic state (more particularly, in septic shock), a patient suffering from burns (more particularly, severe burns), a patient suffering from trauma (more particularly, severe trauma), or a patient undergoing surgery (more particularly, major surgery); and the method makes it possible to determine the risk of occurrence of a nosocomial infection in said patient.
• a septic state more particularly, in septic shock
• burns more particularly, severe burns
• trauma more particularly, severe trauma
• a patient undergoing surgery more particularly, major surgery
• a sepsis patient (or sepsis patient) is defined as a patient with at least one life-threatening organ failure caused by an inappropriate host response to an infection.
• septic shock is meant a subtype of sepsis, in which hypotension persists, despite adequate vascular filling.
• the method according to the invention makes it possible to determine the risk of occurrence of a treatment-associated infection in a patient: - within 30 days from the day of the immuno-inflammatory attack, more preferably within 15 days from the day of the immuno-inflammatory attack (ie the trauma for patients with trauma, the burn for patients with burns, surgery for patients operated by surgery or the diagnosis of sepsis for patients in a septic state), i.e.
• the taking of the biological sample which may in particular have been performed during the 1 st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th , 11 th , 12 th , 13 th , 14 th or 15 th day from the immuno-inflammatory attack (the 1 st day corresponding here on the day of occurrence of the immuno-inflammatory attack); the taking of the biological sample which may in particular have been performed during the 1 st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th , 11 th , 12 th , the 13th, the 14th or 15th day from the immunoinflammatory aggression, preferably in 1, 2 nd, 3 rd, 4 th, 5 th, 6 th or the 7 th day from the immunoinflammatory aggression, preferably at the 3
• the method as described above further 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 coding for molecules involved in the innate immune system; and/or of at least one gene selected from the family of genes coding for cell cycle molecules; and/or at least one gene selected from the family of genes coding for cytokines; and/or at least one gene selected from the family of genes coding for anti-inflammatory cytokines; and/or at least one gene selected from the family of genes coding for pro-inflammatory cytokine receptors, located in chromosome 2, in the 2q11-2q12 region; and or at least one gene selected from the family of genes coding for pro-inflammatory cytokines; and/or at least one gene selected from the family of genes coding for molecules involved in the formation of the cytoskeleton; and/or at least one gene selected from the family of genes coding for molecules involved in the expression and/or transcription of genes; and/or at least one gene selected from at least one gene selected from the family of genes
• the molecules involved in the innate immune system are well known to those skilled in the art.
• the innate immune system includes all the cells and mechanisms that immediately and non-specifically defend the body against all types of infectious agents.
• Molecules involved in the innate immune system include anti-microbial peptides, the complement system, interferon l-alpha and l-beta, and acute phase proteins.
• genes coding for molecules involved in the innate immune system we can cite the following genes: GNLY, S100A9, C3AR1, ADGRE3, CD177, CX3CR1, IFIH1, OAS2, OAS3.
• Cell cycle molecules are also well known to those skilled in the art. They are involved in the regulation of at least one of the phases of the cell cycle, namely G1 phase, S phase, G2 phase and M phase. Thus, these may be the molecules ensuring the execution and transition of each phase of the cell cycle, such as cyclin-dependent kinases, or molecules monitoring the proper execution of each phase.
• genes coding for cell cycle molecules we can cite the CCNB1IP1, CDKN1A, CDKN1B, CDKN1C, CDKN2B, CDKN2C and CDKN2D genes.
• Cytokines are also molecules well known to those skilled in the art. These soluble molecules are synthesized by a large number of cells, in particular lymphocytes and macrophages, and are involved in the development and regulation of immune responses. There are different types of cytokines, including chemokines, interferons, interleukins (pro-inflammatory and anti-inflammatory cytokine), lymphokines and tumor necrosis factor (TNF). As examples of genes coding for cytokines, we can cite the following genes: IL15, IL2, MCP1 (CCL2), CXCL10. As examples of genes coding for anti-inflammatory cytokines, we can cite the genes following: IL10, IL1RN.
• genes encoding pro-inflammatory cytokine receptors located in chromosome 2, at the level of the 2q11-2q12 region, we can cite the following genes: IL18R1, IL1R2, IL1R1 and IL18RAP.
• genes encoding pro-inflammatory cytokines we can cite the following genes: IFNG, IL1B, IL17A, IL18, IL6, TNF.
• the molecules involved in the formation of the cytoskeleton are also well known to those skilled in the art. These include molecules involved in obtaining the protein filaments that make up the cytoskeleton, namely actin microfilaments, microtubules and intermediate filaments.
• genes coding for molecules involved in the formation of the cytoskeleton we can cite the following genes: ARL14EP, GSN.
• the molecules involved in the expression and/or transcription of genes are well known to those skilled in the art. These molecules enable gene activity and define the extent to which a gene is expressed.
• genes coding for molecules involved in the expression and/or transcription of genes we can cite the following genes: CIITA, DYRK2, GATA3, MDC1, NFKB1, RORC, STAT4, TBX21, TDRD9.
• Growth factors are well known to those skilled in the art. These are the molecules that promote or inhibit cell growth, proliferation or differentiation. By way of example of a gene coding for growth factors, we can cite the CSF2 gene.
• Metabolism molecules are well known to those skilled in the art. These are the molecules involved in the catabolism or anabolism of cells. By way of examples of genes encoding metabolic molecules, we can cite the following genes: ALOX5, BPGM, TRAP1.
• Adaptive (or acquired) immunity is one of the mechanisms of action of the immune system.
• the adaptive immune system is made up of specialized cells and systemic processes that specifically eliminate each pathogen or prevent its development.
• adaptive immunity creates immunological memory after an initial response to a specific pathogen, then leading to an enhanced response in future encounters with that pathogen.
• genes coding for molecules involved in the adaptive immune system we can cite the following genes: CD40LG, CD3D, BT LA, CD274, CTL A4, ICOS, PDCD1, TNFSF4, CD74, FCGR1A, LILRB2.
• the molecules involved in signal transduction are also molecules well known to those skilled in the art.
• Transduction is the second step in the signaling cascade that follows an extracellular signal and precedes the internal or external cellular response.
• genes coding for molecules involved in signal transduction we can cite the following genes: FLT1, HAVCR2, IL7R, ZAP70.
• the molecules involved in the modulation of the acute phase are also well known to those skilled in the art.
• the acute phase is an innate defense of the body in response to inflammation, characterized by the stimulation or inhibition of the synthesis and/or secretion of blood proteins called acute phase proteins.
• acute phase proteins As an example of a gene coding for molecules involved in the modulation of the acute phase, we can cite the HP gene.
• the method as described previously comprises, in addition to the step of measuring the expression of TAP2, a step of measuring, in the biological sample of the patient, the expression: of at least one, two, three, four, five, six, seven, eight, nine gene(s) selected from the following genes: GNLY, S100A9, C3AR1, ADGRE3, CD177, CX3CR1, IFIH1, OAS2 and OAS3; and/or at least one, two, three, four, five, six, seven gene(s) selected from the following genes: CCNB1IP1, CDKN1A, CDKN1B, CDKN1C, CDKN2B, CDKN2C and CDKN2D; and/or at least one, two, three, four gene(s) selected from the following genes: IL15, IL2, MCP1(CCL2) and CXCL10; and/or at least one or two gene(s) selected from the following genes: IL10 and IL1RN;
• the method as described previously comprises, in addition to the step of measuring the expression of TAP2, a step of measuring, in the biological sample of the patient, the expression at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six gene(s) 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, CTL A4, CD74, IL7R, ZAP70 and HP.
• GNLY S100A9, C3AR1, ADGRE3, CD177, CX3CR1, OAS2, CCNB1IP
• the method as described previously comprises, in addition to the step of measuring the expression of TAP2, a step of measuring, in the biological sample of the patient, the expression at least one, two, three, four, five, six, seven, eight, nine, ten, eleven gene(s) selected from the following genes: S100A9, C3AR1, CD177, CX3CR1, IL1R2, IFNG, CIITA, CD3D, CTLA4, CD74 and HP.
• Table 3 List of preferred combinations of 1 to 11 other gene(s), the expression of which can be measured in combination with the expression of TAP2.
• the biological sample is a blood sample, preferably a whole blood sample or a sample derived from blood (eg PBMCs, which can be obtained by the Ficoll method, well known to those skilled in the art, or purified monocytes).
• PBMCs a sample derived from blood
• the measurement of the expression (or of the level of expression) of a gene consists in quantifying at least one expression product of the gene.
• the expression product of a gene within the meaning of the present invention, is any biological molecule resulting from the expression of said gene. More particularly, the gene expression product can be an RNA transcript.
• transcript we mean the RNAs, and in particular the messenger RNAs (mRNAs), resulting from the transcription of the gene. More precisely, the transcripts are the RNAs produced by the transcription of a gene followed by the post-transcriptional modifications of the pre-RNA forms.
• the measurement of the level of expression of one or more RNA transcripts of the same gene can be carried out.
• the expression of the gene(s) ie the expression of TAP2, and optionally of one or more other genes of interest, as previously described
• the expression of the gene(s) is measured at the RNA or mRNA transcribed level.
• the detection can be carried out by a direct method, by any method known to those skilled in the art making it possible to determine the presence of said transcript in the sample, or by indirect detection of the transcript after transformation of the latter into DNA, or after amplification of said transcript or after amplification of the DNA obtained after transformation of said transcript into DNA.
• Many methods exist for the detection of nucleic acids see for example Kricka et al., Clinical Chemistry, 1999, n° 45(4), p.453-458; Relier GH et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p.173-249).
• genes can in particular be measured by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably by quantitative RT-PCR or RT-qPCR (for example using FilmArray ® technology or the BiomarkTM platform from Fluidigm), by sequencing (from preferably by high-throughput sequencing) or by hybridization techniques (for example with hybridization microchips or by techniques of the NanoString ® nCounter ® type ).
• the measurement of the level of expression of a gene makes it possible in particular to determine the quantity of one or more transcripts present in the biological sample or to give a derived value therefrom.
• a value derived from the quantity can for example be the absolute concentration, calculated using a calibration curve obtained from successive dilutions of a solution of amplicons of known concentration. It can also correspond to the value of the standardized and calibrated quantity, such as the CNRQ.
• housekeeping genes include the DECRI, HPRT1, PPIB, RPLP0, PPIA, GLYR1, RANBP3, 18S, GAPDH and ACTB genes.
• the expression of the gene(s) of interest is normalized with respect to the expression of one or more housekeeping genes (or reference genes), as known to those skilled in the art; more preferably using one or more of the following housekeeping genes: DECRI (chromosomal location of the gene according to GRCh38/hg38: chr8:90, 001, 352-90, 053, 633), HPRT1 (chromosomal location of the gene according to GRCh38 /hg38: chrX:134, 452, 842-134, 520, 513) and PPIB (chromosomal location of the gene according to GRCh38/hg38: chrl5:64, 155, 812-64, 163, 205).
• DECRI chromosomal location of the gene according to GRCh38/hg38: chr8:90, 001, 352-90, 053, 633
• HPRT1 chromosomal location of the gene according to GRCh38 /hg38:
• the expression of the gene(s) of interest (preferably, the normalized expression) in the patient's biological sample is compared to a value of reference or to the expression of the same gene(s) of interest (preferably, the normalized expression) in a biological reference sample (these data being used for the calculation of the CNRQ, as mentioned above).
• the reference sample can be for example a sample from a volunteer (healthy individual), from a patient, or a mixture of samples from several volunteers (on the one hand) or from several patients (on the other hand ).
• the reference sample can also be a sample taken from a volunteer (or a mixture of samples taken from several volunteers) then treated ex vivo with an immune system stimulating agent (such as LPS or lipopolysaccharide).
• an immune system stimulating agent such as LPS or lipopolysaccharide.
• the reference sample can also be a mixture of untreated sample(s) and of samples treated ex vivo with an immune system stimulating agent.
• the method for determining the risk of occurrence of a healthcare-associated infection can further comprise the steps consisting in :
• the determination of a reference value is widely known to those skilled in the art in that it involves routine experimentation. This determination consists in particular in implementing an identical assay method, or at least comparable, to that implemented in the method of the invention, in a biological sample from a subject or a group of subjects considered ( s) as not being likely to suffer from a healthcare-associated infection. Also, said determination of a reference value may consist of the implementation of said assay method in biological samples of the two populations studied, and in the determination of the value of the test (quantity) making it possible to discriminate between these two populations, in the present case between that which will become complicated (risk of occurrence of a healthcare-associated infection) and that which will not become complicated (little or no risk of occurrence of a healthcare-associated infection).
• the predetermined reference value used to compare the quantity measured in the context of the invention, will be determined from the same expression product(s) of the TAP2 gene as those or that quantified in the biological sample to be tested. .
• reference samples are advantageously of the same nature as that of the biological sample to be tested or at least of a compatible nature to constitute a reference when quantification of TAP2 expression product(s).
• the reference sample(s) used are preferably from people with the same characteristics or a majority of common characteristics, in particular of the same sex and/or of a similar or identical age and/or of the same ethnic origin, with those of the patient for whom the risk of complication is to be assessed.
• the reference sample(s) used are preferentially taken from patients in a septic state (more particularly, in septic shock), from patients suffering from burns (more particularly, from severe burns), from patients suffering from trauma (more particularly, from severe trauma ), or patients undergoing surgery (more particularly, major surgery).
• the determination of the reference value, or value of the test (quantity), making it possible to discriminate between these two populations, can in particular be calculated using the ROC curve (Receiver Operating Characteristic Curve), as illustrated in the embodiments below. -below.
• the patient when the value of the quantity of the TAP2 expression product is lower than the reference value, then the patient presents an increased risk of occurrence of an infection associated with care, preferably a nosocomial infection .
• the method for determining the risk of occurrence of a healthcare-associated infection also comprises a healthcare management step to reduce the risk of occurrence of a healthcare-associated infection.
• a patient identified as being at increased risk of developing a healthcare-associated infection can have appropriate health care management with the aim of reducing the risk of developing a healthcare-associated infection and, for example, in order to reduce the risk of developing sepsis, septic shock or the risk of death.
• Examples of care management include an immunomodulatory treatment adapted to the patient or a prophylactic antibiotic treatment, the two treatments can be combined and/or refer to a continuing care or resuscitation service in order to reduce the risk occurrence of a treatment-associated infection, for example reducing the risk of developing sepsis, septic shock or even the risk of death in the days following the measurement of the expression of the biomarker(s).
• the immunomodulatory treatment is an immunostimulatory treatment, if the individual is determined to have an immunosuppressed status, or an anti-inflammatory treatment, if the individual is determined to have an inflammatory status. .
• immunostimulant treatments which can be selected, mention may be made by way of examples of the group of interleukins, in particular IL-7, IL-15 or IL-3, growth factors, in particular GM-CSF, interferons, in particular IFNy, Toll agonists, antibodies, in particular anti-PDl, anti-PDLl, anti-LAG3, anti-TIM3, anti-IL-10 or anti-CTLA4 antibodies , transferrins and molecules that inhibit apoptosis, FLT3L, Thymosin al, adrenergic antagonists.
• interleukins in particular IL-7, IL-15 or IL-3
• growth factors in particular GM-CSF
• interferons in particular IFNy
• Toll agonists antibodies, in particular anti-PDl, anti-PDLl, anti-LAG3, anti-TIM3, anti-IL-10 or anti-CTLA4 antibodies , transferrins and molecules that inhibit apoptosis, FLT3L, Thymosin al, a
• anti-inflammatory treatments particular mention may be made of the group of glucocorticoids, cytostatic agents, molecules acting on immunophilins and cytokines, molecules blocking the IL-1 receptor and anti-TNF treatments.
• glucocorticoids particularly mention may be made of the group of glucocorticoids, cytostatic agents, molecules acting on immunophilins and cytokines, molecules blocking the IL-1 receptor and anti-TNF treatments.
• prophylactic antibiotic treatments to prevent pneumonia are described in particular in the Annales conses d'Anesthésie et de Réanimation (30; 2011; 168-190).
• a patient who does not present a risk of occurrence of a healthcare-associated infection can be quickly referred to a day hospital service, for example an infectiology service, rather than remaining in a service with close monitoring. which he won't need.
• the invention also relates to a kit comprising means for amplifying (eg primers) and/or means for detecting (eg probes) the expression of TAP2 and of one or more other gene(s) , as indicated previously, in all the embodiments (and, in a particularly preferred manner, one, two, three, four, five, six, seven, eight, nine, ten or eleven genes, selected from the group consisting of: CD74 , CIITA, IFNG, IL1R2, C3AR1, CD177, HP, CX3R1, S100A9, CTLA4 and CD3D); said kit being characterized in that all of the amplification and/or detection means of said kit allow the detection and/or amplification of at most 100, 90, 80, 70, 60, 50, 40,
• biomarker is meant an objectively measurable biological characteristic which represents an indicator of normal or pathological biological processes or of pharmacological response to a therapeutic intervention. This biomarker can in particular be detectable at the mRNA level. More particularly, the biomarker can be an endogenous biomarker or loci (such as a gene or a HERV / Human Endogenous RetroVirus, which is found in the chromosomal material of an individual) or an exogenous biomarker (such as a virus).
• endogenous biomarker or loci such as a gene or a HERV / Human Endogenous RetroVirus, which is found in the chromosomal material of an individual
• exogenous biomarker such as a virus
• said kit can for example also comprise means for amplifying and/or detecting one or more housekeeping genes (preferably selected from the list consisting of: DECRI, HPRT1 and PPIB).
• the kit can also comprise positive control means making it possible to qualify the quality of the extraction of the RNA, the quality of any process of amplification and/or hybridization.
• the kit according to the invention comprises means for amplifying (eg primers) and/or means for detecting (eg probes) the expression of TAP2 and of at least one or more other genes selected from the following genes: GNLY, S100A9, C3AR1, ADGRE3, CD177, CX3CR1, IFIH1, OAS2, OAS3, CCNB1IP1, CDKN1A, CDKN1B, CDKN1C, CDKN2B, CDKN2C, CDKN2D, IL15, IL2, MCP1(CCL2), CXCL10, IL10 , IL1RN, IL18R1, IL1R2, IL1R1, IL18RAP, IFNG, IL1B, IL17A, IL18, IL6, TNF, ARL14EP, GSN, CIITA, DYRK2, GATA3, MDC1, NFKB1, RORC, STAT4, TBX21, TDRD9, C
• primer or “amplification primer” is understood to mean a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably of 15 to 30 nucleotides, and possessing a specificity of hybridization with a target nucleotide sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence.
• primers consisting of two primers.
• probe or “hybridization probe” is understood to mean a nucleotide fragment typically consisting of 5 to 100 nucleotides, preferably of 15 to 90 nucleotides, even more preferably of 15 to 35 nucleotides, possessing a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence.
• the probe also comprises a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence.
• the target nucleotide sequence can be a nucleotide sequence included in a messenger RNA (mRNA) or a nucleotide sequence included in a complementary DNA (cDNA) obtained by reverse transcription of said mRNA.
• mRNA messenger RNA
• cDNA complementary DNA
• probes are preferably used, each preferably having a capacity to hybridize specifically with a different biomarker.
• hybridization is meant the process during which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, having sufficiently complementary sequences, are capable of forming a double strand with stable and specific hydrogen bonds.
• a nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner.
• the hybridization conditions are determined by the stringency, that is to say the rigor of the operating conditions.
• the hybridization is all the more specific as it is carried out at higher stringency.
• Stringency is defined in particular according to the base composition of a probe/target duplex, as well as by the degree of mismatch between two nucleic acids.
• the stringency can also be a function of the reaction parameters, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and/or the hybridization temperature.
• the stringency of the conditions under which a hybridization reaction must be carried out will mainly depend on the hybridization probes used. All of these data are well known and the appropriate conditions can be determined by those skilled in the art. In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70°C, in particular between 35 and 65°C in a saline solution at a concentration of about 0 .5 to 1 M. A step of detecting the hybridization reaction is then carried out.
• enzyme amplification reaction is meant a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme.
• PCR Polymerase Chain Reaction
• LCR Liigase Chain Reaction
• RCR Repair Chain Reaction
• 3SR Self Sustained Sequence Replication
• NASBA Nucleic Acid Sequence-Based Amplification
• TMA Transcription Mediated Amplification
• US-A-5,399,491 and LAMP (Loop mediated isothermal amplification) with patent US6410278.
• RT-PCR reverse transcription
• mRNA messenger RNA
• cDNA complementary DNA
• a subject of the invention is also the use: of means for amplifying (eg primers) and/or means for detecting (eg probes) the expression of TAP2, and optionally also the expression of one or more other gene(s), as indicated previously, in all the embodiments (and, in a particularly preferred manner, one, two, three, four, five, six, seven, eight, nine, ten or eleven genes, selected from the group consisting of: CD74, CIITA, IFNG, IL1R2, C3AR1, CD177, HP, CX3R1, S100A9, CTLA4 and CD3D); or of a kit comprising such amplification and/or detection means, and preferably all of the amplification and/or detection means of said kit allow the detection and/or amplification of at most 100 , 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 , 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 biomarkers, in total, and optionally said kit
• Example 1 The measurement of the expression of TAP2 makes it possible to predict the risk of occurrence of a healthcare-associated infection in a patient
• - Patients in a septic state/in septic shock according to the first clinical protocol. only patients in septic shock were included, on the basis of a suspicion of an infectious focus, a start of treatment with catecholamines within 48 hours of admission to intensive care and treatment with catecholamines (noradrenaline) > 0.25 pg /kg/min for at least 2 hours.
• day 1 corresponds to the day of diagnosis of sepsis or septic shock
• pancreatectomy total or caudal
• neuroendocrine tumors to the right side
• hepatectomy on the right side
• extended colectomy laparotomy
• abdoperineal resection nephrectomy (laparotomy, PKD)
• ilio-femoral bypass Scarpa
• day 1 corresponds to day admission to the intensive care unit or intensive care unit ( ⁇ day of the burn).
• the exclusion criteria essentially 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 suspicion of healthcare-associated infection occurring in the hospital before D30 was independently reviewed by three physicians not involved in patient recruitment. Twenty-six percent of patients developed at least one healthcare-associated infection before D30, or before discharge from hospital.
• TAP2 The expression level of TAP2 was measured in these samples by RT-qPCR.
• a volume of 100 pL of blood collected in the PAXgene ® tubes was directly injected into a FilmArray ® bag optimized to detect a panel of genes involved in the host response, including TAP2, by nested PCR.
• the nucleic acid extraction, reverse-transcription and qPCR steps were carried out sequentially and automatically by the Filmarray ® instrument without external intervention.
• the threshold cycles (or Ct) determined by the instrument were normalized with respect to the expression of 3 reference genes (DECRI, HPRT1 and PPIB).
• the association between the expression of TAP2 and the occurrence of a healthcare-associated infection was evaluated for different time intervals of onset of the infection (ie time between sample collection and the 1 st 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.
• the sample considered corresponds to the closest sample taken before the occurrence of the first episode of healthcare-associated infection.
• the prediction models showed that the expression of TAP2 at the mRNA level, measured at D3/4 or at D5/7 from inclusion in the cohort, made it possible to predict the occurrence of an infection associated care before D15 from inclusion in the cohort (Table 4).
• Prediction models also showed that expression of TAP2 at the mRNA level predicted the occurrence of healthcare-associated infection within 4 days or within 7 days of sample collection (Table 5). .
• results obtained show that the measurement of the expression of TAP2 alone makes it possible to predict the occurrence of infection(s) associated with care within 15 days from the immuno-inflammatory attack, within 4 days of sample collection or within 7 days of sample collection.
• 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 predictive performance of the risk of occurrence of an infection associated with care
• the expression level of TAP2 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 predictive performance (compared to the measurement of the expression of TAP2 alone) of the risk of occurrence of a healthcare-associated infection, whether before D15 from inclusion in the cohort (Table 6) or within 4 days or within 7 days of sample collection (Table 7).
• TAP2 in combination with one or more other biomarker(s) (multivariate analysis), measured on D3/4 or D5/7 from inclusion in the cohort, for the prediction of the occurrence of an infection associated with care before D15 from inclusion in the cohort, in patients in a septic state/in septic shock, with severe trauma, or hospitalized after major surgery.
• biomarker(s) multivariate analysis

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