FR2941240A1 - Determining susceptibility of a patient for contracting nosocomial infection comprises collecting biological sample, extracting biological material, taking specific reagent from expression product of target gene S100A9 and determining - Google Patents

Abstract

Determining susceptibility of a patient for contracting nosocomial infection, comprises collecting a biological sample from the patient and extracting biological material from biological sample; taking at least one specific reagent from an expression product of the target gene S100A9; and determining the expression of the target gene S100A9. Independent claims are included for: (1) a kit for determining susceptibility of the patient for contracting nosocomial infection comprising one reagent specific for S100A9 gene expression product; and (2) use of at least one reagent for determining, in vitro, the susceptibility for contracting a nosocomial infection in a patient.

Description

The present invention relates to a method and kit for determining the susceptibility of a patient to acquiring a nosocomial infection.

Nosocomial infections constitute a real public health problem, generating consequences in a dramatic economic and human cost. In France, between 500,000 and 800,000 patients are estimated to have nosocomial infections each year. The occurrence of nosocomial infections is related to various factors, such as the medical techniques used, but also the susceptibility of the patient to contracting such infections.

Thus, the use of catheters, measurement of the central venous pressure, implantation of prostheses, perfusions, etc. are techniques favoring the emergence of nosocomial infections. Thus, the use of venous catheters is responsible for approximately 30 to 35% of nosocomial infections, the infection spreading from the tip of the catheter, in contact with the patient's skin, to the interior of the patient. venous circulation. In addition, hospitalized patients may have a deficient immune system, because of their pathology (diabetes, respiratory failure, immune pathologies, burns ...) or because of their general condition. Undernourished people, newborns or the elderly are more receptive to nosocomial infections. All nosocomial infections do not have the same character of gravity. A treatment is not always effective and efficient, depending on the location of the infection and the sensitivity of the infectious agent in question that can be resistant to a large number of antibiotics.

Therefore, the main measures to combat nosocomial infections remain preventive, including good hygiene of the hands of caregivers, patients and their entourage, septic isolation of patients likely to spread the infection, monitoring the use of antibiotics in the hospital ... It is also possible to isolate, as a preventive measure, subjects who are unusually susceptible to infections. Unfortunately, it is not always possible to determine when a patient is admitted, what may be his susceptibility to contracting a nosocomial infection.

The present invention proposes to solve the disadvantages of the state of the art by presenting a new reliable tool for determining the sensitivity of a patient to contract a nosocomial infection. Surprisingly, the inventors have demonstrated that such susceptibility can be determined by analyzing the expression of the S100A9 gene. The invention therefore presents an important advance in the field of the fight against nosocomial infections.

Before going further, the following definitions are given to better understand the invention. Nosocomial infection means any infection contracted in a health facility 48 hours after admission and which was not present at the admission of the patient to that institution. The target gene / target gene S100A9 is the reference gene Genbank NM 002965.3. By expression product of the S100A9 gene is meant messenger RNA or an mRNA fragment, cDNA or cDNA fragment, protein or protein fragment. Biological sample means any material derived from the patient, which may contain biological material for detecting the expression of a gene or the corresponding protein, and may especially be a sample of blood, serum, saliva or tissue. or circulating cells of the patient. This biological sample is available by any type of sample known to those skilled in the art, such as in particular a blood test. By biological material is meant any material for detecting the expression of a gene, such as in particular a nucleic acid or a protein, or its coding sequence. The nucleic acid may in particular be an RNA (ribonucleic acid) such as an mRNA (messenger RNA). According to a preferred embodiment of the invention, the biological material is a nucleic acid, and even more preferably an mRNA (messenger RNA). The extraction of the biological material from the biological sample can be carried out by all the nucleic acid or protein extraction protocols well known to those skilled in the art. As an indication, the extraction of nucleic acids can in particular be carried out by: a step of lysis of the cells present in the biological sample, in order to release the nucleic acids contained in the protein and / or lipid envelopes of the microorganisms (as cellular debris that disrupts subsequent reactions). By way of example, it is possible to use the lysis methods as described in the Applicants' patent applications: o WO-A-00/05338 on magnetic and mechanical mixed lysis, o WO-A-99/53304 on electrical lysis, and WO-A-99/15321 on mechanical lysis. Those skilled in the art may use other well-known lysis methods, such as thermal or osmotic shocks or chemical lyses by chaotropic agents such as guanidium salts (US-A-5,234,809). A purification step, allowing separation between the nucleic acids and the other cellular constituents released in the lysis step. This step generally makes it possible to concentrate the nucleic acids. For example, magnetic particles optionally coated with oligonucleotides, by adsorption or covalence (see US Pat. are fixed on these magnetic particles, by a washing step. This nucleic acid purification step is particularly advantageous if it is desired to subsequently amplify said nucleic acids. A particularly interesting embodiment of these magnetic particles is described in the patent applications filed by the Applicant under the following references: WO-A-97/45202 and WO-A-99/35500. Another interesting example of a method for purifying nucleic acids is the use of silica either in the form of a column or in the form of inert particles (Boom R. et al., J. Clin Microbiol., 1990, No. 28 ( 3), pp. 495-503) or magnetic (Merck: MagPrep Silica, Promega: MagneSil'M Paramagnetic particles). Other widely used methods rely on columnar or paramagnetic particulate ion exchange resins (Whatman: DEAE-Magarose) (Levison PR et al., J. Chromatography, 1998, pp. 337-344). Another method that is very relevant but not exclusive to the invention is that of adsorption on a metal oxide support (Xtrana company: Xtra-BindTM matrix). Such an extraction step can be done in automata. The easyMAG is particularly suitable. By specific reagent is meant a reagent that reacts with the biological material in order to directly or indirectly highlight the expression of a target gene, which can be determined by the analysis of the mRNAs derived from this gene, or by the analysis of the protein encoded by this gene.

As an indication, when it is desired to determine the expression of a target gene by the analysis of the protein encoded by this gene, it is possible to use protein detection techniques with or without a ligand. Among the ligand detection techniques, it is possible to use, for example, an antibody specific for the protein encoded by this target gene. As an indication, when it is desired to determine the expression of a target gene by the analysis of messenger RNAs (mRNAs) transcribed from this gene, this specific reagent comprises at least one specific amplification primer of the DNA complementary to this mRNA (this is called specific amplification primer of a target gene). The DNA complementary to an mRNA can be obtained according to a protocol well known to those skilled in the art. As an indication, the total RNAs (including the ribosomal RNAs, the transfer RNAs, the mRNAs) are extracted from the biological sample. A reverse transcription reaction is then carried out using a reverse transcriptase enzyme which makes it possible to obtain, from an RNA fragment, a complementary DNA fragment (cDNA). The realization of such a step is well known to those skilled in the art. When it is more particularly desired to obtain the complementary DNAs of the messenger RNAs, this enzymatic step is carried out in the presence of nucleotide fragments comprising only thymine bases (polyT), which hybridize by complementarity on the polyA sequence of the different mRNAs in order to forming a polyT-polyA complex which then serves as a starting point for the reverse transcription reaction carried out by the enzyme reverse transcriptase. We then obtain different DNA complementary to the different messenger RNAs initially present in the biological sample. In the remainder of the description, the term "cDNA" designates a DNA complementary to a messenger RNA. By "amplification primer" is meant a nucleotide fragment comprising from 5 to 100 nucleotide units, preferably from 15 to 25 nucleotide units and having hybridization specificity under determined conditions for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction.

By enzymatic amplification reaction is meant a process generating multiple copies of a target nucleotide fragment using specific amplification primers by the action of at least one enzyme. Such amplification reactions are well known to those skilled in the art and may be mentioned in particular the following PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), RCR (Repair Chain Reaction), 3SR (Self Sustained Sequence Replication) ) with patent application WO-A-90/06995, NASBA (Nucleic Acid Sequence-Based Amplification), and TMA (Transcription Mediated Amplification) with US-A-5,399,491. We then speak of amplicons to designate the polynucleotides generated by an enzymatic amplification technique. Preferably, when the enzymatic amplification is a PCR, the specific reagent comprises at least 2 specific amplification primers in order to amplify a particular region of the DNA complementary to the mRNA derived from the target gene. When the enzymatic amplification is a PCR carried out after a reverse transcription reaction, it is called RT-PCR. By hybridization probe is meant a nucleotide fragment comprising from 5 to 100 nucleotide units, in particular from 6 to 35 nucleotide units, having hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide fragment. In the present invention, the target nucleotide fragment can be a nucleotide sequence included in a messenger RNA or a nucleotide sequence included in a complementary DNA obtained by reverse transcription of said messenger RNA.

Hybridization is understood to mean the process in 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 bonds. stable and specific hydrogens. 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. Hybridization is all the more specific as it is carried out with higher stringency. The 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 may also be a function of the parameters of the reaction, such as the concentration and 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 performed will depend primarily on the hybridization probes used. All 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. By detection is meant either a direct detection by a physical method or a detection method using a marker. Many detection methods exist for the detection of nucleic acids. [See, for example, Kricka et al., Clinical Chemistry, 1999, No. 45 (4), p.453-458 or Keller GH et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p.173-249]. By marker is meant a tracer capable of generating a signal. A nonlimiting list of these tracers includes the enzymes which produce a detectable signal for example by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, glucose-6-phosphate dehydrogenase; chromophores such as fluorescent, luminescent or coloring compounds; electron density groups detectable by electron microscopy or by their electrical properties such as conductivity, by amperometry or voltammetry methods, or by impedance measurements; groups detectable by optical methods such as diffraction, surface plasmon resonance, contact angle variation or by physical methods such as atomic force spectroscopy, tunneling effect, etc. ; Thus, the polynucleotide may be labeled during the enzymatic amplification step, for example using a labeled nucleotide triphosphate for the amplification reaction. The labeled nucleotide will be a deoxyribonucleotide in amplification systems generating a DNA, such as PCR, or a ribonucleotide in amplification techniques generating an RNA, such as TMA and NASBA techniques. The polynucleotide may also be labeled after the amplification step, for example by hybridizing a labeled probe according to the sandwich hybridization technique described in WO-A-91/19812.

For the purposes of the present invention, the hybridization probe may be a so-called capture probe. In this case, the target nucleotide fragment may be previously labeled with a marker. The so-called capture probe is immobilized or immobilizable on a solid support by any appropriate means, that is to say directly or indirectly, for example by covalence or adsorption. A hybridization reaction is then carried out between said detection probe and the labeled target nucleotide fragment.

The hybridization probe may also be a so-called detection probe. In this case, the hybridization probe can be labeled with a marker. A hybridization reaction is then carried out between said capture probe and the target nucleotide fragment.

Whether using a so-called capture probe or a so-called detection probe, the hybridization reaction can be carried out on a solid support that includes all the materials on which a nucleic acid can be immobilized. Synthetic materials or natural materials, optionally chemically modified, can be used as a solid support, in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and cellulose. nitrocellulose or dextran, polymers, copolymers, especially based on styrene-type monomers, natural fibers such as cotton, and synthetic fibers such as nylon; mineral materials such as silica, quartz, glasses, ceramics; latexes; magnetic particles; metal derivatives, gels etc. The solid support may be in the form of a microtiter plate, a membrane as described in application WOA-94/12670, a particle or a biochip. In the present invention, the determination of S100A9 gene expression can be analyzed by the expression of mRNAs that are transcribed at a given time. In this case, the biological material is a nucleic acid, and the specific reagent can be indifferently an amplification primer or a hybridization probe as defined above.

As an indication, the expression of a target gene can be determined in the following manner: 1) after extracting the total RNAs from a biological sample, a reverse transcription step, as described above, is carried out in order to obtain the different DNAs complementary to the different messenger RNA initially present in the biological sample (or cDNA) 2) the cDNAs are specifically amplified. In this case, the specific reagent used comprises at least one amplification primer specific for the target gene, as defined above. This step can be carried out in particular by a PCR type amplification reaction or by any other amplification technique. 3) the expression of the target gene is determined by quantifying the cDNAs. The cDNAs can be quantified in particular by the use of a quantization range obtained by an amplification reaction conducted until saturation. In order to take into account the variability of enzymatic efficiency that can be observed during the various steps (reverse transcription, PCR, etc.), the expression of the target gene of the different groups of patients can be standardized by simultaneously determining the expression of a so-called household gene, the expression of which is similar in the different groups of patients. By realizing a relationship between the expression of the target gene and the expression of the household gene, this corrects any variability between the different experiments. Those skilled in the art may refer in particular to the following publications: Bustin SA Journal of Molecular Endocrinology, 2002, 29: 23-39; Giulietti A Methods, 2001, 25: 386-401.

The expression of a target gene can also be determined as follows: 1) after extracting the total RNAs from a biological sample, a reverse transcription step, as described above, is carried out in order to obtain the different DNAs complementary to the different messenger RNAs initially present in the biological sample (or cDNA) 2) the cDNAs are immobilized on a membrane 3) the expression of the target gene is determined by hybridizing the cDNAs to hybridization probes specific for the target gene previously marked. Such hybridization techniques are well known to those skilled in the art, and mention may in particular be made of the Northern blot technique. This hybridization reaction can be carried out after a step of specific amplification of the complementary DNAs of the messenger RNAs of a target gene when, in particular, the gene is weakly expressed. The expression of a target gene can also be analyzed by the expression of the proteins encoded by the target gene. In this case, the biological material is a protein and several detection techniques with or without ligand can be used. Mass spectrometry can be used as a ligand-free detection technique. The specific reagent may be an antibody specific for the protein encoded by the target gene for a ligand detection system.

The recombinant antibodies specific for the translated protein of the target gene can be obtained according to conventional methods known to those skilled in the art, from prokaryotic organisms, such as bacteria, or from eukaryotic organisms, such as yeasts, cells of mammals, plants, insects or animals, or by extracellular production systems. The monoclonal antibodies can be prepared according to conventional techniques known to those skilled in the art, such as the hybridoma technique, the general principle of which is recalled below. Firstly, an animal, usually a mouse (or cells cultured in the context of in vitro immunizations) is immunized with a target antigen of interest, whose B lymphocytes are then capable of producing antibodies against the said antigen. antigen. These antibody-producing lymphocytes are then fused with "immortal" myeloma cells (murine in the example) to give rise to hybridomas. From the heterogeneous mixture of cells thus obtained, the cells capable of producing a particular antibody and of multiplying indefinitely are then selected. Each hybridoma is multiplied in the clone form, each leading to the production of a monoclonal antibody whose recognition properties with respect to the antigen of interest can be tested, for example by ELISA, by immunoblotting in one or two-dimensional, in immunofluorescence, or with a biosensor. The monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique. For example, antibody fragments can be obtained by proteolysis. Thus, they can be obtained by enzymatic digestion, resulting in Fab-type fragments (papain treatment, Porter RR, 1959, Biochem J., 73: 119-126) or F (ab) '2 type (treatment pepsin, Nisonoff A. et al., 1960, Science, 132 1770-1771). They can also be prepared recombinantly (Skerra A., 1993, Curr Opin Immunol., 256-262). Another antibody fragment which is suitable for the purposes of the invention comprises a fragment Fv which is a dimer consisting of the non-covalent association of the light variable domain (VL) and the heavy variable domain (VH) of the Fab fragment, therefore of the association of two polypeptide chains. In order to improve the stability of the Fv fragment due to the dissociation of the two polypeptide chains, this Fv fragment can be modified by genetic engineering by inserting a suitable peptide link between the VL domain and the VH domain (Huston P. et al., 1988 Proc Natl.Acad Sci.USA 85: 5879-5883). We then speak of fragment scFv (single chain Fragment variable) because it consists of a single polypeptide chain. The use of a peptide bond preferably composed of 15 to 25 amino acids makes it possible to connect the C-terminal end of one domain to the N-terminus of the other domain, thus constituting a monomeric molecule with properties similar to those of the antibody in its complete form. The two orientations of the VL and VH domains are suitable (VL-link-VH and VH-link-VL) because they have identical functional properties. Of course, any fragment known to those skilled in the art and having the immunological characteristics defined above is suitable for the purposes of the invention. When the biological material is a protein resulting from the expression of a gene, one can determine the expression of the latter, by detecting and or quantifying said protein by Western Blot or ELISA, or any other method known to man of the art, such as a chemiluminescent method from biological material. ELISA (Enzyme Linked Immuno Sorbent Assay) is a solid enzyme immunoassay. This test is part of the more general EIA (Enzyme Immunoassays) in which the assay is coupled to an enzyme catalyzed reaction. The technique uses one or two antibodies. Antibody for the formation of immune complexes (antigen / antibody) is coupled to the enzyme and can generate the emission of a signal by a chromogenic or fluorogenic substrate. The Western Blot technique is a test for detecting a specific protein in a sample using an antibody specific for this protein comprising the following steps as described below. The first step is gel electrophoresis, which separates the proteins from the sample according to their size.

The proteins in the gel are then transferred to a membrane (nitrocellulose, PVDF, etc.) by pressure or by application of an electric current, the proteins being attached to the membrane by means of hydrophobic and ionic interactions. After saturation of the nonspecific interaction sites, a first antibody specific for the protein to be studied (primary antibody) is incubated with the membrane. The membrane is then rinsed to remove unbound primary antibodies and incubated with so-called secondary antibodies, which will bind to the primary antibodies. This secondary antibody is usually linked to an enzyme that allows visual identification of the protein being studied on the membrane. The addition of a substrate labeled with the enzyme generates a colored reaction that is visible on the membrane.

Analysis of the expression of the S100A9 gene then makes it possible to have a tool for determining the susceptibility of a patient to contracting a nosocomial infection. For example, the expression of the target gene can be analyzed in a patient whose susceptibility is unknown, and by comparing with known mean expression values of the target gene of patients with high susceptibility to acquiring a nosocomial infection and Known average expression values of the target gene of patients with low susceptibility to acquiring a nosocomial infection, determine the susceptibility of the patient to acquiring a nosocomial infection, in order to offer him, for example, preventive isolation if necessary.

Thus, the invention relates to a method for determining the susceptibility of a patient to acquiring a nosocomial infection wherein: a. a biological sample of the patient is available and biological material is extracted from the biological sample b. at least one reagent specific for an expression product of the target gene S100A9 c is available. the expression of the target gene S100A9 is determined. According to a preferred embodiment of the invention, the biological sample is a blood sample.

According to a preferred embodiment of the invention, the biological material is a nucleic acid. According to a preferred embodiment of the invention, the total RNAs are extracted from the biological sample. According to a preferred embodiment of the invention, the specific reagent of S100A9 comprises at least one specific amplification primer of the target gene S100A9 or at least one specific hybridization probe of the target gene S100A9. According to another embodiment of the invention, the specific reagent according to the invention comprises at least one antibody specific for the expression product of the target gene S100A9. The invention preferably relates to the determination of the expression of the target genes by the analysis of the expression of the mRNAs.

The invention also relates to a kit for determining the susceptibility of a patient to acquiring a nosocomial infection comprising at least one reagent specific for an expression product of the S100A9 gene.

Preferably, the specific reagent of the expression product of the S100A9 gene comprises at least one hybridization probe specific for the target gene S100A9 or at least one primer specific for the target gene S100A9.

Preferably, the specific reagent of the expression product of the S100A9 gene comprises at least one antibody.

The invention also relates to the use of at least one specific reagent of an S100A9 gene expression product for determining the susceptibility of a patient to acquiring a nosocomial infection. Preferably, the specific reagent of the expression product of the S100A9 gene comprises at least one hybridization probe specific for the target gene S100A9 or at least one primer specific for the target gene S100A9. Preferably, the specific reagent of the gene expression product. S100A9 comprises at least one antibody.

Example - Study of S100A9 gene expression to determine the susceptibility to contracting a nosocomial infection. Samples The study was carried out on a cohort of 93 patients who developed septic shock, and admitted to a surgical or medical resuscitation department of the Lyon-Sud hospital center. These patients were collected on day 7, day 8, day 9 or day 10 after septic shock (HO being the beginning of vasopressor treatment). Among these patients who developed septic shock, 27 patients contracted a nosocomial infection secondarily to shock and 66 did not contract a nosocomial infection within 53 days of observation of this cohort. RNA extraction and cDNA synthesis For each patient, the biological sample is a blood sample, which was regularly obtained during the first 10 days after the onset of septic syndrome (MS patients). Sampling was also performed according to the same protocol in healthy subjects (S). These samples were collected directly into PAXgeneTM Blood RNA tubes (PreAnalytiX, Frankin Lakes, USA). After the blood sampling step and in order to obtain a total lysis of the cells, the tubes were left at ambient temperature for 4 hours and then stored at -20 ° C. until the biological material was extracted. Specifically, in this protocol, total RNAs were extracted using PAXgene Blood RNA kits (PreAnalytiX) according to the manufacturer's recommendations. Briefly, the tubes were centrifuged (10 min at 3000g) to obtain a nucleic acid pellet. This pellet was washed and taken up in a buffer containing proteinase K necessary for the digestion of the proteins (10 min at 55 ° C.). Further centrifugation (5 min at 19000g) was performed to remove cell debris and ethanol was added to optimize the nucleic acid binding conditions. The total RNAs were specifically fixed on the PAXgene RNA spin columnTM columns and, prior to elution thereof, a digestion of the contaminating DNA was performed using the RNase free DNAse setTM (Qiagen Ltd, Crawley, UK). For each extraction, the quality of the total RNAs was verified by capillary electrophoresis using the Nana ChipTM 6000 RNA Kit (Agilent Technologies). The instrument used is the 2100TM bioanalyser. A reverse transcription reaction (RT) was performed in a final volume of 20 μl. Total RNA (0.5 μg) was mixed with 1 μl of 50 μM polyT and 1 μl of 50 μM oligo (dT) and 1 μL of annealing buffer and incubated for 5 min. at 65 ° C. After cooling in ice, the solution was mixed with 10 μl of 2x First Strand Reaction Mix and 2 μl of SuperScript III / RNase out Mix RT enzyme (15 U / μl), all these products being derived from SuperScript First Strand Synthesis Super MixTM RT-PCR system (Invitrogen). Reverse transcription was performed for 50 min. at 50 ° C and then stopped by incubation for 5 min. at 85 ° C. Finally, each cDNA solution was diluted 1: 10E in DEPC water. Quantification Standard Range Preparation From a cDNA pool from healthy subjects, amplification of the 153-179 region of the S100A9 target gene (GenBank # NM8002965.3) was performed by PCR (Polymerase Chain Reaction). ), conducted to saturation, using the following primer pair: sense primer: 5'-TCAAAGAGCTGGTGCGAAAA-3 '(SEQ ID NO: 1) Antisense primer: 5'-AACTCCTCGAAGCTCAGCTG-3' (SEQ ID NO 2) Real-time PCR mRNA expression analysis The cDNA-tagged S100A9 target gene mRNAs, as described above, were quantitated by quantitative PCR in real time using LightCyclerTM (Roche). PCR reactions were performed using Fast-StartTM DNA Master SYBR Green I realtime PCR kit (Roche Molecular Biochemicals). Each PCR was carried out in a final volume of 20 μl containing 3.6 μl of water, 2.4 μl of MgCl 2, 2 μl of SYBR Green mix (Fast start DNA master SYBR green + Taq DNA polymerase) and 1 μl of each. primer (10 μM) and 10 μl of cDNA solution. The primers used are those described above. After a denaturation step of 10 min. at 95 ° C, the amplification is carried out using 40 cycles of a "touch-down" PCR protocol (10 s at 95 ° C, 10 s of hybridization at 68-58 ° C, followed by an extension of 16 s at 72 ° C). At the end of each cycle, the fluorescence emitted by the SYBR Green was measured. To confirm the specificity of the amplification, the PCR products were systematically subjected to a fusion curve analysis (LightCyclerTM - Roche). For this, the PCR products were treated with an increasing temperature of 58 to 98 ° C with an increase of 0.1 ° C / s. For each PCR product, a single peak was obtained during the analysis of the curve, characterized by a specific melting temperature. The combination of primers necessary for the quantification of the CPB household gene was provided by Search-LC (Heidelberg, Germany, reference 488116).

The amount of target mRNA relative to the amount of mRNA of the cyclophilin B household gene was analyzed by the relative quantization technique with the LightCycler Relative Quantification Software ™ (Roche Molecular Biochemicals). The Second Derivative Maximum Method of the LightCyclerTM software (Roche) was used to automatically determine the Crossing Point (Cp) for each sample. The Cp value was defined as the number of cycles for which the fluorescence was significantly different from the background.

Five 1 / 10th series dilutions were performed in quadruplicate with each standard to generate a calibration curve expressing the Cp as a function of the logarithm of the number of copies. The standard dilutions were optimized so that the calibration curve covers the level of expression expected for the target gene and the housekeeping gene. The standard relative curves describing the PCR efficiency for the target gene and the housekeeping gene were generated and used to perform quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

Results of univariate and multivariate analyzes In order to determine the variables influencing the occurrence of a nosocomial infection, a univariate analysis and then a multivariate analysis are performed. Univariate analysis is performed by logistic regression of status with or without nosocomial infection as a function of different variables and on the S100A9 marker in mRNA on days D7 to D10 after the onset of septic shock. Then a multivariate logistic regression is carried out on the variables presenting a level of significance (p) <0.15 with the univariate analysis.

The backward selection method (initially all the variables are taken into account in the model, then they are removed one by one) is used with a significance threshold of 0.05. The results are shown in Table 2 below. The odd ratio (O.R.) and its 95% confidence interval are given for each result.

21 Table 2 Univariate analysis (N = 93) Multivariate analysis (N = 93) OR 95% CI p OR 95% CI p Gender Male 1 - - Female 1.6 0.641-3.997 0.3143 Age (years)> 65 1 - - - - - <- 65 2.04 0.814-5.115 0.1285 - - 0.1186 SAPS II to> 51 1 - - admission <_ 51 1.107 0.450-2.723 0.8246 SOFA to <10 1 - - - - - admission> _ 10 3.036 1.132-8.144 0.0274 - - 0.1384 Co-morbidity 0 1 - -> _ 1 1.021 0.414-2.514 0.9645 Nosocomial Type 1 - - Community Infection 1.144 0.467-2.803 0.7682 Pulmonary Site 1 - - Abdominal Infection 1.735 0.644-4.672 0.3268 Other 1.172 0.299 -4.597 0.8540 S100A9 (mRNA) <6.1 1 - - 1 - - 6.1 6.27 1.718-22.891 0.0055 6.242 1.662-23.447 0.0067 OR: Odd Ratio CI: confidence interval P: threshold of significance As can be seen from Table 2, the analysis multivariate reveals that an expression of the marker S100A9? 6.1 on days 7 to 10 after the onset of septic shock significantly increase the probability of occurrence of a nosocomial infection (odd ratio to 6.2).

Claims (14)

REVENDICATIONS1. A method for determining the susceptibility of a patient to acquiring a nosocomial infection wherein: a. a biological sample of the patient is available and biological material is extracted from the biological sample b. at least one reagent specific for an expression product of the target gene S100A9 c is available. the expression of the target gene S100A9 is determined. The method of claim 1 wherein the biological sample is a blood sample. The method of claim 1 or 2 wherein the biological material is a nucleic acid. 4. Method according to any one of claims 1 to 3 wherein the specific reagent of the S100A9 gene expression product comprises at least one amplification primer specific for the target gene S100A9. 5. Method according to any one of claims 1 to 3 wherein the specific reagent of the S100A9 gene expression product comprises at least one hybridization probe specific for the target gene S100A9. The method of claim 1, wherein the specific reagent of the S100A9 gene expression product comprises at least one antibody. 7. Kit for determining the susceptibility of a patient to a nosocomial infection comprising at least one reagent specific for an S100A9 gene expression product. 8. Kit according to claim 7 wherein the specific reagent of the S100A9 gene expression product comprises at least one hybridization probe specific for the target gene S100A9. 9. Kit according to claim 7 wherein the specific reagent of the S100A9 gene expression product comprises at least one amplification primer specific for the target gene S100A9. 10. Kit according to claim 7, wherein the specific reagent of the expression product of the S100A9 gene comprises at least one antibody. 11. Use of at least one specific reagent of an S100A9 gene expression product to determine, in vitro, the susceptibility of a patient to acquiring a nosocomial infection. 12. Use according to claim 11, wherein the specific reagent of the S100A9 gene expression product comprises at least one hybridization probe specific for the target gene S100A9. The use according to claim 11, wherein the S100A9 specific reagent comprises at least one amplification primer specific for the S100A9 target gene expression product. The use of claim 11, wherein the S100A9 specific reagent comprises at least one antibody.