Methods for determining a patient's susceptibility of contracting a nosocomial infection and for establishing a prognosis of the progression of septic syndrome

Abstract

A method for determining a patient's susceptibility of contracting a nosocomial infection that includes obtaining a biological sample from the patient and extracting biological material from the biological sample; preparing a specific reagent of an expression product of at least one target gene selected from S100A9 and S100A8 target genes; and determining the expression of at least one of the target genes S100A9 and S100A8, where overexpression relative to a specified threshold value indicates susceptibility of contracting a nosocomial infection.

Claims

1. A method comprising: a) obtaining blood samples from a human patient with septic shock at multiple time points after septic shock onset, at least one of the time points being between 7 and 10 days after septic shock onset; b) extracting nucleic acids from the blood samples; and c) measuring an overexpression of the S100A9 gene relative to a predetermined threshold value in at least the blood sample(s) obtained between 7 and 10 days after septic shock onset, wherein the overexpression is measured via an amplification method comprising the use of a first primer consisting of SEQ ID NO: 1 and a second primer consisting of SEQ ID NO: 2.

2. The method as claimed in claim 1, wherein RNA is the nucleic acid extracted from the blood samples, and cDNA is generated from the RNA by an amplification method.

3. The method as claimed in claim 1, wherein the amplification method is a real-time PCR amplification method.

4. The method according to claim 1, wherein septic shock is characterized by an identified infectious site and persistent hypotension.

5. A method comprising: a) obtaining blood samples from a human patient with septic shock at multiple time points after septic shock onset, at least one of the time points being between 7 and 10 days after septic shock onset; b) extracting nucleic acids from the blood samples; and c) measuring an overexpression of the S100A8 gene relative to a predetermined threshold value in at least the blood sample(s) obtained between 7 and 10 days after septic shock onset, the threshold value being indicative of either a healthy subject or a non-surviving septic shock patient, wherein the overexpression is measured via an amplification method comprising the use of a first primer consisting of SEQ ID NO: 3 and a second primer consisting of SEQ ID NO: 4.

6. The method as claimed in claim 5, wherein RNA is the nucleic acid extracted from the blood samples, and cDNA is generated from the RNA by an amplification method.

7. The method as claimed in claim 5, wherein the amplification method is a real-time PCR amplification method.

8. The method according to claim 5, wherein septic shock is characterized by an identified infectious site and persistent hypotension.

Description

FIGURES

(1) FIG. 1 presents the correlation of the gene expression of S100A8 and S100A9 (n=86). It shows the strong correlation that exists between the gene expressions of the molecules S100A9 and S100A8 with a Spearman coefficient >0.90 (r=0.9134). The gene expressions of the molecules S100A9 and S100A8 possess similar capacity for discriminating patients who are likely to contract a nosocomial infection and for establishing a prognosis of the progression of septic syndrome.

(2) FIG. 2 illustrates the significant difference in expression of the S100A8 gene (p=0.0461) between the surviving and non-surviving patients for samples taken between D7 and D10 after the onset of septic shock. Legend: HV=healthy volunteers; S=patients surviving; NS=patients not surviving.

EXAMPLES

Example 1

Investigation of Expression of the S100A9 Gene

(3) Test Samples

(4) A group of 44 healthy volunteers (S) and a group of 148 patients with septic syndrome (SEP); sampled on D1, D2 or D3 after septic shock (H0 being injection of the vasopressor treatment), were used, for comparing the expression of the S100A9 gene in the peripheral blood. A group of 44 healthy patients (S) and a group of 148 patients developing septic shock (SEP) were identified.

(5) Extraction of the RNAs and Synthesis of the cDNAs

(6) For each patient, the biological sample was a blood sample, obtained regularly during the first 10 days following the onset of septic syndrome developed by the patients SEP. Sampling was also carried out by an identical protocol in healthy patients (S). These samples were collected directly in PAXgene™ Blood RNA tubes (PreAnalytiX, Frankin Lakes, USA).

(7) After the blood sampling stage and in order to obtain complete lysis of the cells, the tubes were left at room temperature for 4 h and then stored at −20° C. until extraction of the biological material. More precisely, in this protocol, the total RNAs were extracted by means of PAXgene Blood RNA® kits (PreAnalytiX) following the manufacturer's recommendations. Briefly, the tubes were centrifuged (10 min, 3000 g) to obtain a pellet of nucleic acids. This pellet was washed and taken up in a buffer containing proteinase K required for digestion of the proteins (10 min at 55° C.). A repeat centrifugation (5 min, 19000 g) was carried out to remove the cellular debris, and ethanol was added in order to optimize the conditions for fixation of the nucleic acids. The total RNAs were specifically fixed on the columns PAXgene RNA spin column and, before elution of the latter, digestion of the contaminating DNA was carried out using the RNAse free DNAse Set™ (Qiagen Ltd, Crawley, UK).

(8) For each extraction, the quality of the total RNAs was verified by capillary electrophoresis using the RNA 6000 Nano Chip™ kit (Agilent technologies). The instrument used is the bioanalyzer 2100. A reverse transcription (RT) reaction was carried out in a final volume of 20 μl. The total. RNA (0.5 μg) was mixed with 1 μl of polyT at 50 μM and 1 μL of annealing buffer and was then incubated for 5 min at 65° C. After cooling in ice, the solution was mixed with 10 μl of 2× First Strand Reaction. Mix and 2 μL of SuperScript III/RNase™ out enzyme Mix RT (15 U/μl), all these products being obtained from the SuperScript First Strand Synthesis Super Mix™ RT-PCR system (Invitrogen). Reverse transcription was carried out for 50 min at 50° C. and then stopped by incubation for 5 min at 85° C. Finally, each solution of cDNA was diluted to 1/10 in DEPC water.

(9) Preparation of Standard Ranges for Quantification

(10) Starting from a pool of cDNA obtained from healthy subjects, amplification of the 153-179 region of the S100A9 target gene (GenBank No. NM_002965.3) was performed by PCR (Polymerase Chain Reaction), conducted to saturation, using the following primer pair:

(11) TABLE-US-00001 Sense primer: 5′-TCAAAGAGCTGGTGCGAAAA-3′ (SEQ ID NO: 1) Antisense primer: 5′-AACTCCTCGAAGCTCAGCTG-3′ (SEQ ID NO: 2)
Analysis of Expression of the mRNAs by Real-Time PCR

(12) The mRNAs of the S100A9 target gene retranscribed to cDNA, as described above, were quantified by real-time quantitative PCR using the LightCycler™ (Roche). The PCR reactions were carried out using the Fast-Start™ DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals). Each PCR was performed in a final volume of 20 μl containing 3.6 μl of water, 2.4 μl of MgCl.sub.2, 2 μl of SYBR Green mix (Fast-Start™ DNA master SYBR green dye+Taq DNA polymerase) and 10 of each primer (10 μM) and 10 μl of cDNA solution. The primers used are those described previously. After a stage of denaturation of 10 min at 95° C., amplification is carried out by 40 cycles of a “touch-down” PCR protocol (10 s at 95° C., 10 s of hybridization at 68-58° C., followed by extension of 16 s at 72° C.). At the end of each cycle, the fluorescence emitted by the SYBR Green dye was measured.

(13) To confirm the specificity of amplification, the PCR products were systematically submitted to analysis of the melting curve (LightCycler™—Roche). For this, the PCR products were treated at a temperature increasing from 58 to 98° C. with a rate of increase of 0.1° C./s. For each PCR product, a single peak was obtained during analysis of the curve, characterized by a specific melting temperature.

(14) The combination of primers required for quantification of the housekeeping gene CPB was supplied by Search-LC (Heidelberg, Germany; reference 488116).

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

(16) Five serial dilutions at 1/10 were performed in quadruplicate with each standard in order to generate a calibration curve expressing Cp as a function of the logarithm of the copy number. The standard dilutions were optimized so that the calibration curve covered the level of expression expected for the target gene and the housekeeping gene. The relative standard curves describing the effectiveness of PCR for the target gene and the housekeeping gene were generated and were used for performing quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

(17) Results Obtained

(18) The results obtained are presented in Table 1 below.

(19) TABLE-US-00002 TABLE 1 Lower Upper Patients N Median quartile quartile S 44 1.46 1.065 1.87 SEP 148 16.62 11.87 25.835

(20) The p_value of the Mann-Whitney test between the healthy subjects and the patients with septic shock is <0.0001.

(21) The expression of the marker S100A9 over the first 3 days of septic shock was significantly overexpressed relative to the healthy donors.

Example 2

Investigation of Expression of the S100A8 Gene

(22) Test Samples

(23) A group of 32 healthy volunteers (S) and a group of 86 patients with septic syndrome (SEP), sampled between D7 and D10 after septic shock (H0 being injection of the vasopressor treatment), were used in order to compare expression of the S100A8 gene in the peripheral blood. A group of 32 healthy patients (S) and a group of 86 patients developing septic shock (SEP) were identified.

(24) Extraction of the RNAs and Synthesis of the cDNAs

(25) For each patient, the biological sample is a blood sample, regularly obtained during the first 10 days following the onset of septic syndrome developed by the SEP patients. Sampling was also carried out according to an identical protocol in healthy patients (5). These samples were collected directly in PAXgene™ Blood RNA tubes (PreAnalytiX, Frankin Lakes, USA).

(26) After the stage of collecting blood samples and in order to obtain complete lysis of the cells, the tubes were left at room temperature for 4 h and then stored at −20° C. until extraction of the biological material. More precisely, in this protocol, the total RNAs were extracted by means of PAXgene Blood RNA® kits (PreAnalytiX) following the manufacturer's recommendations. Briefly, the tubes were centrifuged (10 min, 3000 g) to obtain a pellet of nucleic acids. This pellet was washed and taken up in a buffer containing proteinase K required for digestion of the proteins (10 min at 55° C.). A repeat centrifugation (5 min, 19000 g) was carried out to remove the cellular debris, and ethanol was added in order to optimize the conditions for fixation of the nucleic acids. The total RNAs were specifically fixed on the columns PAXgene™ RNA spin column and, before elution of the latter, digestion of the contaminating DNA was carried out using the RNAse free DNAse Set™ (Qiagen Ltd, Crawley, UK).

(27) For each extraction, the quality of the total RNAs was verified by capillary electrophoresis using the RNA 6000 Nano Chip™ kit (Agilent technologies). The instrument used is the bioanalyzer 2100. A reverse transcription (RT) reaction was carried out in a final volume of 20 μl. The total RNA (0.5 μg) was mixed with 1 μl of polyT at 50 μM and 1 μL of annealing buffer and was then incubated for 5 min at 65° C. After cooling in ice, the solution was mixed with 10 μl of 2× First Strand Reaction Mix and 2 μL of Superscript III/RNase™ out enzyme Mix RT (15 U/μl), all these products being obtained from the Superscript First Strand Synthesis Super Mix TM RT-PCR system (Invitrogen). Reverse transcription was carried out for 50 min at 50° C. and then stopped by incubation for 5 min at 85° C. Finally, each solution of cDNA was diluted to 1/10 in DEPC water.

(28) Preparation of Standard Ranges for Quantification

(29) Starting from a pool of cDNA obtained from healthy subjects, amplification of region 129-280 of the S100A8 target gene (GenBank No. NM_002964.3) was performed by PCR (Polymerase Chain Reaction), conducted to saturation, using the following primer pair:

(30) TABLE-US-00003 Sense primer: 5′-ATTTCCATGCCGTCTACAGG-3′ (SEQ ID NO: 3) Antisense primer: 5′-CACCAGAATGAGGAACTCCT-3′ (SEQ ID NO: 4)
Analysis of Expression of the mRNAs by Real-Time PCR

(31) The mRNAs of the S100A8 target gene retranscribed to cDNA, as described above, were quantified by real-time quantitative PCR using the LightCycler™ (Roche). The PCR reactions were carried out using the Fast-Start™ DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals). Each PCR was performed in a final volume of 20 μl containing 3.6 μl of water, 2.4 μl of MgCl.sub.2, 2 μl of SYBR Green mix (Fast-Start™ DNA master SYBR green dye+Taq DNA polymerase) and 10 of each primer (10 μM) and 10 μl of cDNA solution. The primers used are those described previously. After a stage of denaturation of 10 min at 95° C., amplification is carried out by 40 cycles of a “touch-down” PCR protocol (10 s at 95° C., 10 s of hybridization at 68-58° C., followed by extension of 16 s at 72° C.). At the end of each cycle, the fluorescence emitted by the SYBR Green dye was measured.

(32) To confirm the specificity of amplification, the PCR products were systematically submitted to analysis of the melting curve (LightCycler™—Roche). For this, the PCR products were treated at a temperature increasing from 58 to 98° C. with a rate of increase of 0.1° C./s. For each PCR product, a single peak was obtained during analysis of the curve, characterized by a specific melting temperature.

(33) The combination of primers required for quantification of the housekeeping gene CPB was supplied by Search-LC (Heidelberg, Germany; reference 488116).

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

(35) Five serial dilutions at 1/10 were performed in quadruplicate with each standard in order to generate a calibration curve expressing Cp as a function of the logarithm of the copy number. The standard dilutions were optimized so that the calibration curve covered the level of expression expected for the target gene and the housekeeping gene. The relative standard curves describing the effectiveness of PCR for the target gene and the housekeeping gene were generated and were used for performing quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

(36) Results Obtained

(37) The results obtained are presented in Table 2 below.

(38) TABLE-US-00004 TABLE 2 Lower Upper Patients N Median quartile quartile S 32 12.9 8.5 20.4 SEP 86 183.5 96.6 438.5

(39) The p_value of the Mann-Whitney test between the healthy subjects and the patients with septic shock is <0.0001.

(40) The expression of the S100A8 marker on days D7 to D10 after septic shock was significantly overexpressed relative to the healthy donors.

Example 3

Investigation of the Survival Prognosis Value of S100A8 in Patients with Septic Shock

(41) Test Samples

(42) The study was conducted on a cohort of 86 patients who had developed septic shock, and had been admitted to a Surgical or Medical intensive care unit of the Lyon-Sud hospital center. Samples were collected from these patients on D7, D8, D9 or D10 after septic shock (H0 being the start, of vasopressor treatment).

(43) Extraction of the RNAs and Synthesis of the cDNAs

(44) For each patient, the biological sample is a blood sample, which was taken daily for the first 10 days following the onset of septic syndrome (patients SEP). These samples were collected directly in PAXgene™ Blood RNA tubes (PreAnalytiX, Frankin Lakes, USA).

(45) After the stage of collecting blood samples and in order to obtain complete lysis of the cells, the tubes were left at room temperature for 4 hours and then stored at −20° C. until extraction of the biological material. More precisely, in this protocol, the total RNAs were extracted by means of PAXgene Blood RNA® kits (PreAnalytiX) following the manufacturer's recommendations. Briefly, the tubes were centrifuged (10 min at 3000 g) to obtain a pellet of nucleic acids. This pellet was washed and taken up in a buffer containing proteinase K required for digestion of the proteins (10 min at 55° C.). A repeat centrifugation (5 min at 19000 g) was carried out to remove the cellular debris, and ethanol was added in order to optimize the conditions for fixation of the nucleic acids. The total RNAs were specifically fixed on columns PAXgene RNA Spin Column™ and, before elution of the latter, digestion of the contaminating DNA was carried out using the RNAse free DNAse Set™ (Qiagen Ltd, Crawley, UK).

(46) For each extraction, the quality of the total RNAs was verified by capillary electrophoresis using the RNA 6000 Nano Chip™ kit (Agilent technologies). The instrument used is the bioanalyzer 2100™. A reverse transcription (RT) reaction was carried out in a final volume of 20 μl. The total RNA (0.5 μg) was mixed with 1 μl of oligo (dT) 50 μM and 1 μL of annealing buffer and was then incubated for 5 min at 65° C. After cooling in ice, the solution was mixed with 10 μl of 2× First Strand Reaction Mix and 2 μL of SuperScript III/RNase out enzyme Mix RT (15 U/μl), all these products being obtained from the Superscript First Strand Synthesis Super Mix™ RT-PCR system (Invitrogen). Reverse transcription was carried out for 50 min at 50° C. and then stopped by incubation for 5 min at 85° C. Finally, each solution of cDNA was diluted to 1/10 in DEPC water.

(47) Preparation of Standard Ranges for Quantification

(48) Starting from a pool of cDNA obtained from healthy subjects, amplification of region 129-280 of the S100A8 target gene (GenBank No. NM_002964.3) was performed by PCR (Polymerase Chain Reaction), conducted to saturation, using the following primer pair:

(49) TABLE-US-00005 Sense primer: 5′-ATTTCCATGCCGTCTACAGG-3′ (SEQ ID NO: 3) Antisense primer: 5′-CACCAGAATGAGGAACTCCT-3′ (SEQ ID NO: 4)
Analysis of Expression of the mRNAs by Real-Time Quantitative PCR

(50) The mRNAs of the S100A8 target gene retranscribed to cDNA, as described above, were quantified by real-time quantitative PCR using the LightCycler™ 2.0 (Roche). The PCR reactions were carried out using the Fast-Start™ DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals). Each PCR was performed in a final volume of 20 μl containing 3.6 μl of water, 2.4 μl of MgCl.sub.2, 2 μl of SYBR Green mix (Fast-Start™ DNA master SYBR green dye+Taq DNA polymerase) and 10 of each primer (10 μM) and 10 μl of cDNA solution. The primers used are those described previously.

(51) After a stage of denaturation of 10 min at 95° C., amplification is carried out by 40 cycles of a “touch-down” PCR protocol (10 s at 95° C., 10 s of hybridization at 68-58° C. (0.5° C./cycle), followed by extension of 16 s at 72° C.). At the end of each cycle, the fluorescence emitted by the SYBR Green dye was measured.

(52) To confirm the specificity of amplification, the PCR products were systematically submitted to analysis of the melting curve (LightCycler™ 2.0—Roche). For this, the PCR products were treated at a temperature increasing from 58 to 98° C. with a rate of increase of 0.1° C./s. For each PCR product, a single peak was obtained during analysis of the curve, characterized by a specific melting temperature.

(53) The combination of primers required for quantification of the housekeeping gene CPB was supplied by Search-LC (Heidelberg, Germany; reference 488116).

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

(55) Five serial dilutions at 1/10 were performed in quadruplicate with each standard in order to generate a calibration curve expressing Cp as a function of the logarithm of the copy number. The standard dilutions were optimized so that the calibration curve covered the level of expression expected for the target gene and the housekeeping gene. The relative standard curves describing the effectiveness of PCR for the target gene and the housekeeping gene were generated and were used for performing quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

(56) Analysis of the Results

(57) To determine whether expression of the S100A8 gene is correlated with patient survival, a Mann Whitney test was performed. FIG. 2 illustrates the significant difference of expression of the S100A8 gene (p=0.0461) between the surviving and non-surviving patients for the samples taken between D7 and D10 after the onset of septic shock.

Example 4

Investigation of Expression of the S100A9 Gene for Determining Patients' Susceptibility of Contracting a Nosocomial Infection

(58) Test Samples

(59) The study was conducted on a cohort of 93 patients who had developed septic shock, and had been admitted to a Surgical or Medical intensive care unit of the Lyon-Sud hospital center. Samples were collected from these patients on D7, D8, D9 or D10 after septic shock (H0 being the start of vasopressor treatment).

(60) Among these patients who had developed septic shock, 27 patients contracted a nosocomial infection secondary to shock and 66 did not contract a nosocomial infection in the 53 days of observation of this cohort.

(61) Extraction of the RNAs and Synthesis of the cDNAs

(62) For each patient, the biological sample is a blood sample, which was regularly obtained during the first 10 days following the onset of septic syndrome (patients SEP). Samples were also taken from healthy subjects (S) according to an identical protocol. These samples were collected directly in PAXgene™ Blood RNA tubes (PreAnalytiX, Frankin Lakes, USA).

(63) After the stage of collecting blood samples and in order to obtain complete lysis of the cells, the tubes were left at room temperature for 4 hours and then stored at −20° C. until extraction of the biological material. More precisely, in this protocol, the total RNAs were extracted by means of PAXgene Blood RNA® kits (PreAnalytiX) following the manufacturer's recommendations. Briefly, the tubes were centrifuged (10 min at 3000 g) to obtain a pellet of nucleic acids. This pellet was washed and taken up in a buffer containing proteinase K required for digestion of the proteins (10 min at 55° C.). A repeat centrifugation (5 min at 19000 g) was carried out to remove the cellular debris, and ethanol was added in order to optimize the conditions for fixation of the nucleic acids. The total RNAs were specifically fixed on columns PAXgene RNA Spin Column™ and, before elution of the latter, digestion of the contaminating DNA was carried out using the RNAse free DNAse Set™ (Qiagen Ltd, Crawley, UK).

(64) For each extraction, the quality of the total RNAs was verified by capillary electrophoresis using the RNA 6000 Nano Chip™ kit (Agilent technologies). The instrument used is the bioanalyzer 2100™. A reverse transcription (RT) reaction was carried out in a final volume of 20 μl. The total RNA (0.5 μg) was mixed with 1 μl of polyT at 50 μM and 1 μl of oligo (dT) 50 μM and 1 μL of annealing buffer and was then incubated for 5 min at 65° C. After cooling in ice, the solution was mixed with 10 μl of 2× First Strand Reaction Mix and 2 μL of Superscript III/RNase out enzyme Mix RT (15 U/μl), all these products being obtained from the Superscript First Strand Synthesis Super Mix™ RT-PCR system (Invitrogen). Reverse transcription was carried out for 50 min at 50° C. and then stopped by incubation for 5 min, at 85° C. Finally, each solution of cDNA was diluted to 1/10 in DEPC water.

(65) Preparation of Standard Ranges for Quantification

(66) Starting from a pool of cDNA obtained from healthy subjects, amplification of the 153-179 region of the S100A9 target gene (GenBank No. NM_002965.3) was performed by PCR (Polymerase Chain Reaction), conducted to saturation, using the following primer pair:

(67) TABLE-US-00006 Sense primer: 5′-TCAAAGAGCTGGTGCGAAAA-3′ (SEQ ID NO: 1) Antisense primer: 5′-AACTCCTCGAAGCTCAGCTG-3′ (SEQ ID NO: 2)
Analysis of Expression of the mRNAs by Real-Time PCR

(68) The mRNAs of the S100A9 target gene retranscribed to cDNA, as described above, were quantified by real-time quantitative PCR using the LightCycler™ (Roche). The PCR reactions were carried out using the Fast-Start™ DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals). Each PCR was performed in a final volume of 20 μl containing 3.6 μl of water, 2.4 μl of MgCl.sub.2, 2 μl of SYBR Green mix (Fast-Start™ DNA master SYBR green dye+Taq DNA polymerase) and 10 of each primer (10 μM) and 10 μl of cDNA solution. The primers used are those described previously. After a stage of denaturation of 10 min at 95° C., amplification is carried out by 40 cycles of a “touch-down” PCR protocol (10 s at 95° C., 10 s of hybridization at 68-58° C., followed by extension of 16 s at 72° C.). At the end of each cycle, the fluorescence emitted by the SYBR Green dye was measured.

(69) To confirm the specificity of amplification, the PCR products were systematically submitted to analysis of the melting curve (LightCycler™—Roche). For this, the PCR products were treated at a temperature increasing from 58 to 98° C. with a rate of increase of 0.1° C./s. For each PCR product, a single peak was obtained during analysis of the curve, characterized by a specific melting temperature.

(70) The combination of primers required for quantification of the housekeeping gene CPB was supplied by Search-LC (Heidelberg, Germany; reference 488116).

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

(72) Five serial dilutions at 1/10 were performed in quadruplicate with each standard in order to generate a calibration curve expressing Cp as a function of the logarithm of the copy number. The standard dilutions were optimized so that the calibration curve covered the level of expression expected for the target gene and the housekeeping gene. The relative standard curves describing the effectiveness of PCR for the target gene and the housekeeping gene were generated and were used for performing quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

(73) Results of Univariate and Multivariate Analyses

(74) To determine the variables having an influence on occurrence of a nosocomial infection, a univariate analysis and then a multivariate analysis are performed. The univariate analysis is performed by effecting a logistic regression of the status attained or not attained by a nosocomial infection as a function of different variables and for the S100A9 marker in mRNA on days D7 to D10 after the onset of septic shock. Then a multivariate logistic regression is performed on the variables displaying a level of significance (p)<0.15 with the univariate analysis. The “backward” selection method (at the start, all the variables are taken into account in the model, then they are withdrawn one by one) is used with a significance threshold at 0.05. The results are presented in Table 3 below. The odds ratio (O.R.) and its 95% confidence interval are shown for each result.

(75) TABLE-US-00007 TABLE 3 Univariate analysis (N = 93) Multivariate analysis (N = 93) OR 95% CI p OR 95% CI p Sex 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 on >51 1 — — admission ≤51 1.107 0.450-2.723 0.8246 SOFA on <10 1 — — — — — admission ≥10 3.036 1.132-8.144 0.0274 — — 0.1384 Co-morbidities 0 1 — — ≥1 1.021 0.414-2.514 0.9645 Type of Nosocomial 1 — — infection Community 1.144 0.467-2.803 0.7682 Site of Pulmonary 1 — — infection Abdominal 1.735 0.644-4.672 0.3268 Others 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: Odds Ratio CI: confidence interval P: significance threshold

(76) As can be seen from Table 3, multivariate analysis reveals that expression of the S100A9 marker ≥6.1 on days 7 to 10 after the onset of septic shock significantly increases the probability of occurrence of a nosocomial infection (odds ratio at 6.2).

Example 5

Investigation of Correlation of Expression of the S100A9 Gene and of Expression of the S100A8 Gene

(77) Test Samples

(78) The study was conducted on a cohort of 86 patients who had developed septic shock, and had been admitted to a Surgical or Medical intensive care unit of the Lyon-Sud hospital center. Samples were collected from these patients on D7, D8, D9 or D10 after septic shock (H0 being the start of vasopressor treatment).

(79) Extraction of the RNAs and Synthesis of the cDNAs

(80) For each patient, the biological sample is a blood sample, which was taken daily for the first 10 days following the onset of septic syndrome (patients SEP). These samples were collected directly in PAXgene™ Blood RNA tubes (PreAnalytiX, Frankin Lakes, USA).

(81) After the stage of collecting blood samples and in order to obtain complete lysis of the cells, the tubes were left at room temperature for 4 hours and then stored at −20° C. until extraction of the biological material. More precisely, in this protocol, the total RNAs were extracted by means of PAXgene Blood RNA® kits (PreAnalytiX) following the manufacturer's recommendations. Briefly, the tubes were centrifuged (10 min at 3000 g) to obtain a pellet of nucleic acids. This pellet was washed and taken up in a buffer containing proteinase K required for digestion of the proteins (10 min at 55° C.). A repeat centrifugation (5 min at 19000 g) was carried out to remove the cellular debris, and ethanol was added in order to optimize the conditions for fixation of the nucleic acids. The total RNAs were specifically fixed on columns PAXgene RNA Spin Column™ and, before elution of the latter, digestion of the contaminating DNA was carried out using the RNAse free DNAse Set™ (Qiagen Ltd, Crawley, UK).

(82) For each extraction, the quality of the total RNAs was verified by capillary electrophoresis using the RNA 6000 Nano Chip™ kit (Agilent technologies). The instrument used is the bioanalyzer 2100™. A reverse transcription (RT) reaction was carried out in a final volume of 20 μl. The total RNA (0.5 μg) was mixed with 1 μl of oligo (dT) 50 μM and 1 μL of annealing buffer and was then incubated for 5 min at 65° C. After cooling in ice, the solution was mixed with 10 μl of 2× First Strand Reaction Mix and 2 μL of SuperScript III/RNase out enzyme Mix RT (15 U/μl), all these products being obtained from the Superscript First Strand Synthesis Super MiX™ RT-PCR system (Invitrogen). Reverse transcription was carried out for 50 min at 50° C. and then stopped by incubation for 5 min at 85° C. Finally, each solution of cDNA was diluted to 1/10 in DEPC water.

(83) Preparation of Standard Ranges for Quantification

(84) Starting from a pool of cDNA obtained from healthy subjects, amplification of the 153-179 region of the S100A9 target gene (GenBank No. NM_002965.3) was performed by PCR (Polymerase Chain Reaction), conducted to saturation, using the following primer pair:

(85) TABLE-US-00008 Sense primer: 5′-TCAAAGAGCTGGTGCGAAAA-3′ (SEQ ID NO: 1) Antisense primer: 5′-AACTCCTCGAAGCTCAGCTG-3′ (SEQ ID NO: 2)

(86) Starting from a pool of cDNA obtained from healthy subjects, amplification of region 129-280 of the S100A8 target gene (GenBank No. NM_002964.3) was performed by PCR (Polymerase Chain Reaction), conducted to saturation, using the following primer pair:

(87) TABLE-US-00009 Sense primer: 5′-ATTTCCATGCCGTCTACAGG-3′ (SEQ ID NO: 3) Antisense primer: 5′-CACCAGAATGAGGAACTCCT-3′ (SEQ ID NO: 4)
Analysis of Expression of the mRNAs by Real-Time Quantitative PCR

(88) The mRNAs of the S100A9 and S100A8 target genes retranscribed to cDNA, as described above, were quantified by real-time quantitative PCR using the LightCycler™ 2.0 (Roche). The PCR reactions were carried out using the Fast-Start™ DNA Master SYBR Green I real-time PCR kit (Roche Molecular Biochemicals). Each PCR was performed in a final volume of 20 μl containing 3.6 μl of water, 2.4 μl of MgCl.sub.2, 2 μl of SYBR Green mix (Fast-Start™ DNA master SYBR green dye+Taq DNA polymerase) and 10 of each primer (10 μM) and 10 μl of cDNA solution. The primers used are those described previously.

(89) After a stage of denaturation of 10 min at 95° C., amplification is carried out by 40 cycles of a “touch-down” PCR protocol (10 s at 95° C., 10 s of hybridization at 68-58° C. (0.5° C./cycle), followed by extension of 16 s at 72° C.). At the end of each cycle, the fluorescence emitted by the SYBR Green dye was measured.

(90) To confirm the specificity of amplification, the PCR products were systematically submitted to analysis of the melting curve (LightCycler™ 2.0—Roche). For this, the PCR products were treated at a temperature increasing from 58 to 98° C. with a rate of increase of 0.1° C./s. For each PCR product, a single peak was obtained during analysis of the curve, characterized by a specific melting temperature.

(91) The combination of primers required for quantification of the housekeeping gene CPB was supplied by Search-LC (Heidelberg, Germany; reference 488116).

(92) The amount of mRNA targets relative to the amount of mRNA of the housekeeping gene cyclophilin B was analyzed by the relative quantification technique with the LightCycler Relative Quantification Software™ (Roche Molecular Biochemicals). The “Second Derivative Maximum Method” of the LightCycler™ software (Roche) was used for automatically determining the “Crossing Point” (Cp) for each sample. The value of Cp was defined as the number of cycles for which the fluorescence was significantly different from the background noise.

(93) Five serial dilutions at 1/10 were performed in quadruplicate with each standard in order to generate a calibration curve expressing Cp as a function of the logarithm of the copy number. The standard dilutions were optimized so that the calibration curves cover the level of expression expected for the target genes and the housekeeping gene. The relative standard curves describing the effectiveness of PCR for the target gene and the housekeeping gene were generated and were used for performing quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

(94) Analysis of the Results

(95) To determine whether the expression of the S100A9 gene is correlated with the expression of the S100A8 gene, a correlation analysis was performed. The results given in FIG. 1 show that there is a strong correlation between the gene expressions of these two molecules, with a Spearman coefficient >0.90. The gene expressions of the molecules S100A9 and S100A8 therefore possess a similar capacity for discriminating patients who are likely to contract a nosocomial infection and for establishing a prognosis of the progression of septic syndrome.

REFERENCES

(96) [1] M. M. Levy, M. P. Fink, J. C. Marshall, E. Abraham, D. Angus, D. Cook, J. Cohen, S. M. Opal, J. L. Vincent and G. Ramsay, 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference, Crit. Care Med. 31 (2003), pp. 1250-1256 [2] Boom R. et al., J. Clin. Microbiol., 1990, No. 28(3), p. 495-503 [4] Kricka et al., Clinical. Chemistry, 1999, No. 45(4), p. 453-458 [5] Keller G. H. et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p. 173-249 [6] Bustin S A Journal of molecular endocrinology, 2002, 29: 23-39 [7] Giulietti A Methods, 2001, 25: 386-401 [8] Porter R R, 1959, Biochem. J., 73: 119-126 [9] Nisonoff A. et al., 1960, Science, 132: 1770-1771 [10] Skerra A., 1993, Curr. Opin. Immunol., 5: 256-262 [11] Huston P. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5879-5883 [12] Fluss R., Faraggi D., Reiser B. (2005), “Estimation of the Youden index and its associated cutoff point”. Biometrical Journal, 47:458-72