WO2011036091A1 - Procédé pour détecter et différencier in vitro des états pathologiques - Google Patents

Procédé pour détecter et différencier in vitro des états pathologiques Download PDF

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WO2011036091A1
WO2011036091A1 PCT/EP2010/063659 EP2010063659W WO2011036091A1 WO 2011036091 A1 WO2011036091 A1 WO 2011036091A1 EP 2010063659 W EP2010063659 W EP 2010063659W WO 2011036091 A1 WO2011036091 A1 WO 2011036091A1
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sepsis
gene
infectious
polynucleotides
amplicon
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Eva Möller
Britta Wlotzka
Andriy Ruryk
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SIRS Lab GmbH
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SIRS Lab GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a method for the in vitro detection and / or early detection and / or differentiation and / or course monitoring of pathophysiological conditions according to claim 1, the use of
  • At least three polynucleotides for the formation of at least one multigene biomarker for the preparation of a multiplex assay as an aid for assessing whether a patient has a pathophysiological condition, and / or for determining the severity and / or early detection and / or for distinguishing and / or monitoring of pathophysiological conditions according to claim 5, a Verwenung according to claim 12; A primer for carrying out the method according to the invention as claimed in claim 16, and a kit for carrying out the method according to claim 17.
  • the present invention relates to the use of
  • Multigene biomarker for the preparation of a diagnostic aid in patients with certain pathophysiological conditions such as sepsis and sepsis-like conditions, with similar features as an in vitro diagnostic multivariate index assay (IVDMIA).
  • IVDMIA in vitro diagnostic multivariate index assay
  • Sepsis blood poisoning
  • blood poisoning is a life-threatening infection that affects the entire organism, is associated with high mortality, is becoming more prevalent and affects people of all ages.
  • the mortality rate of severe sepsis has not improved significantly in recent decades, with the last two breakthroughs in innovation since the introduction of blood culture (around 1880) being the introduction of antibiotics over 60 years ago and the onset of intensive care about 50 years ago.
  • novel diagnostic agents must be made available.
  • Sepsis is caused by infectious agents. As there is currently no specific therapy for sepsis, the success of the treatment largely depends on the successful control of the underlying infection and the quality of the intensive care treatment.
  • Antibiotic or antifungal (anti-fungal) substances are "calculated", that is to say suspected, and in 20-30% of cases, this choice is not correct.
  • Interpretation step to obtain a single patient-specific output value in the form of an index, score or classification. This value is for diagnostic statements, damage limitation, treatment or prevention of a disease.
  • Infection is associated with the uptake of pathogens, their multiplication in the organism and the associated triggering of pathophysiological and symptomatic reactions. In contrast, there are no disease symptoms of the host organism in a colonization.
  • Nonspecific defenses are endogenous, germ-damaging substances that are dissolved in the blood (humoral) as well as granulocytes and macrophages, which are limited in their ability to eliminate invaders, foreign bodies and cell debris.
  • the mode of action of the specific defense is to mark foreign bodies and pathogens with antibodies circulating in the blood in order to have them subsequently destroyed by T lymphocytes.
  • Multi-organ failure come. Depending on a pathogen the activator can excrete poisons, exotoxins which lead to partially violent reactions of a host response. Another possibility is that yourself
  • Influenza components so-called endotoxins, in the case of germ killing as a poison can affect.
  • Organism is called a local infection, such.
  • a local infection such.
  • the symptoms of a local infection are redness,
  • interventions on the gastrointestinal tract can lead to a fulminant spread of bacteria into the sterile abdominal cavity due to suture insufficiencies.
  • Infectious courses also play a role in the follow-up treatment after transplantations, thoracotomies, extremity and joint corrections and neurosurgical interventions.
  • the present invention relates to genes and / or fragments thereof and their use for generating multigene biomarkers which are specific to a condition and / or investigation.
  • the invention further relates to marker primers derived PCR primers and probes for hybridization or duplication methods.
  • SIRS systemic inflammatory response syndrome
  • At least two of the following clinical criteria must be met: fever> 38 ° C or hypothermia ⁇ 36 ° C, leukocytosis> 12g / l or leukopenia ⁇ 4g / l or a left shift in the differential blood count, a heart rate of over 90 / min , a tachypnoea> 20 breaths / min or a PaCO 2 (partial pressure of carbon dioxide in the arterial blood) ⁇ 4.3 kPa.
  • This definition has a high sensitivity, but a low specificity. For intensive medical care, it is of little help, since, as a rule, every intensive care patient meets the SIRS criteria, at least for a short time.
  • Sepsis is defined as those clinical conditions in which the SIRS criteria are fulfilled and the cause of an infection is proven or at least very probable.
  • Infection is defined as a pathological process caused by invasion of pathogens or potentially pathogenic organisms into a normally sterile tissue. If the body fails to limit this infection to the site of origin, the pathogens or their toxins induce inflammation in the organs or tissues of the body remote from the site of infection. Immediate intensive care treatment, the targeted administration of antibiotics and the surgical rehabilitation of the infectious focus are needed to achieve recovery.
  • Severe sepsis is characterized by the additional occurrence of organ dysfunctions. Common organ dysfunctions include changes in the state of consciousness, oliguria, lactic acidosis or sepsis-induced hypotension with a systolic
  • Catecholamines the patient comes it is called a septic shock. This is detected in about 20% of all sepsis patients.
  • the first group includes score systems such as APACHE, SAPS and SIRS, which can stratify patients based on a variety of physiological indices. While some studies have demonstrated diagnostic potential for the APACHE II score, other studies have shown that APACHE II and SAPS II can not differentiate between sepsis and SIRS [Carrigan et al., 2004].
  • the second group contains protein markers derived from plasma and serum
  • IL-1 Ra MCP-1, MPIF-1, TNF-alpha, TNF-R1, MIG, BLC, HVEM, IL-10, IL-15, MCP-2, M-CSF, MIP-3b, MMP-9, PARC, ST-2; IL-6, SIL-2R, CD141, MMP-9, EGF, ENA-78, EOT, Gro-beta, IL-1b, leptin, MIF, MIP-1 a, OSM, protein C, P-selectin, and HCC4 (Molecular Staging, Inc., USA) or CD14 antigen, lipopolys
  • procalcitonin (PCT, BRAHMS) and C-reactive protein (CRP, Eli Lilly) appear to be the markers that best distinguish between infectious and non-infectious causes of SIRS.
  • Procalcitonin is a 1 16 amino acid peptide that plays a role in
  • CRP C-reactive protein
  • PCT is considered more suitable as a CRP to differentiate non-infectious versus infectious SIRS as well as bacterial versus viral infection [Simon et al., 2004].
  • biomarkers are currently being searched extensively by various scientific groups and commercial organizations, such as changes in blood cytokine concentrations caused by bacterial cell wall components such as lipopolysaccharides [Mathiak et al., 2003], or use of gene expression profiles in a blood sample to determine differences in survivors and non-survivors sepsis patients [Pachot et al., 2006].
  • Gene expression profiles or classifiers are for the determination of the severity of sepsis [WO 2004/087949], the distinction between a local or systemic infection [unpublished DE 10 2007 036 678.9], the identification of the source of infection [WO 2007/124820] or of
  • Gene expression signatures are useful for distinguishing between multiple etiologies and pathogen-associated signatures [Ramilo et al., 2007].
  • the currently available protein markers due to the insufficient specificity and sensitivity of the consensus criteria according to [Bone et al., 1992] the currently available protein markers, as well as due to the time required to detect the cause of the infection by blood culture, there is an urgent need for new methods that take into account the complexity of the disease.
  • MammaPrint The microarray-based, 70-gene signature called MammaPrint (Aqendia, NL) allows predicting the risk of recurrence and metastasis in women with breast cancer. It is being investigated whether the risk of developing distant metastases in the next few years could be considered high or low and that they would benefit from chemotherapy. The approval of this test by the FDA has led to the development of guidelines for a new class of diagnostic tests known as IVDMIA (in vitro diagnostic multivariate index assay). The MammaPrint signature is measured and calculated on a microarray in the manufacturer's laboratories.
  • Oncotype DX multigene assav (Genomic Health, USA) is used to assess the likelihood of breast cancer recurrence in patients and to evaluate the response of patients to chemotherapy. 21 genes are summarized as "Recurrence-Score.” The measurement takes place in the rooms of the company, it is also the TaqMan- PCR technology used.
  • the test is based on 1 1 quantitative PCR assays (additional 9 controls and references) using the TaqMan
  • a common feature of these tests is that the addressed diagnostic question allows examination times until the result of several days is available. For diagnostic tests in the indication of sepsis, however, the information must be available within a single working day.
  • gene expression markers for the determination of a pathophysiological state, the amounts of the corresponding mRNA present in a sample, the gene expression levels, are always determined quantitatively.
  • the information determined by these gene expression levels is the respective over or under-expression of these mRNAs, which is determined experimentally with reference to a control state or with reference to control genes.
  • the detection of over or underexpression can be seen analogously to the determination of the concentration of a protein biomarker.
  • Patient selection was very heterogeneous. Patients were included in the study who had very different comorbidities, such as B to 1 1% to 16% had tumors or very different
  • therapeutic measures e.g., 27% to 64% vasopressor therapy, which greatly affected gene expression profiles.
  • Gene expression was reported by Feezor et al. also examined the plasma concentrations of some cytokines. The gene expression data did not necessarily correlate with the plasma concentrations. In gene expression, the amount of mRNA is measured. However, this is subject to posttranscriptional regulation prior to protein synthesis, from which the observed differences may result.
  • RNA samples isolated from peripheral blood mononuclear cells of ten patients each with E. coli and S. aureus infection, respectively.
  • the identification of the pathogens was done by blood culture. Based on the training dataset, 30 genes were identified, through which
  • the multigene biomarker optimal for a specific clinical problem [Simon et al., 2005].
  • pathophysiological condition such as sepsis and generalized infection
  • the object is characterized by the features of
  • a kit according to claim 17 solves the problem as well.
  • the present invention relates to a system comprising the following elements:
  • Amplification method or hybridization method • Using an algorithm to calculate the individual results of the
  • the present invention relates to a method for in vitro detection and / or early detection and / or differentiation and / or course observation and / or assessment of pathophysiological conditions selected from the group consisting of: SIRS, sepsis and their degrees of severity; sepsis-like states; septic shock; Bacteremia, infectious / non-infectious
  • Ivlultiorganversagen Early detection of these conditions; Focus control; Control of surgical remedial measures of the infection focus; Responder / non-responder for a given therapy; Therapy monitoring; Differentiation between infectious and non-infectious genesis in systemic reactions of the
  • Organism e.g. SIRS, sepsis, postoperative complications, chronic and / or acute organ dysfunction, shock reaction, inflammatory response and / or trauma; the method comprising the steps of: a) isolating sample nucleic acids from one of a patient
  • a preferred method is characterized in that the reference gene is selected from polynucleotides of the group consisting of R1, R2 and R3 and / or their isoforms and / or their gene loci and / or their transcripts and / or fragments having a length of at least 5 nucleotides thereof , wherein the reference genes are defined according to the following table:
  • Polynucleotide sequences Genloci, sense and / or antisense strands of pre-mRNA and / or mRNA, small RNA, especially scRNA, snoRNA, micro RNA, siRNA, dsRNA, ncRNA or transposable elements for the detection of
  • Another preferred embodiment lies in a method characterized in that in step b) the gene activity of 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 polynucleotides, or of all 13 polynucleotides is determined wherein the polynucleotides are selected from the group consisting of: M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and M17 and / or their isoforms and / or their gene loci and / or their transcripts and / or fragments of at least 5 nucleotides in length thereof, the polynucleotides being defined according to the following table: Transcript variant / ds' accession SEQ
  • the invention relates to the use of at least three polynucleotides selected from the group consisting of: M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and M17 and / or their isoforms and / or their gene loci and / or their transcripts and / or
  • Distinction between infectious and non-infectious genesis in systemic reactions of the organism e.g. SIRS, sepsis, postoperative complications, chronic and / or acute organ dysfunction, shock reaction,
  • a preferred embodiment of the present invention is a use in which the multigene biomarker is a combination of a plurality of polynucleotide, in particular gene sequences, by means of whose gene activities by means of a
  • Interpretation function is performed a classification and / or an index is formed.
  • a use is particularly suitable, which is characterized in that the gene activities by enzymatic methods, in particular amplification method, preferably polymerase chain reaction (PCR), preferably real-time PCR, in particular probe-based methods such Taq Man, Scorpions, Molecular Beacons; and / or by means of hybridization methods, in particular those on microarrays; and / or direct mRNA detection, in particular sequencing or mass spectrometry; and / or isothermal amplification.
  • PCR polymerase chain reaction
  • probe-based methods such as Taq Man, Scorpions, Molecular Beacons
  • a further preferred embodiment of the present invention is a use, which is characterized in that from the individual specific gene activities an index is formed, which is a measure of the severity and / or the course of the pathophysiological state after appropriate calibration, preferably the index displayed on an easily interpretable scale.
  • the obtained gene activity data for the production of software for the description of at least one pathophysiological condition and / or an investigation question and / or as aids for diagnostic purposes and / or patient data management systems, in particular for use for lossstrat En and inclusion criterion for clinical trials.
  • RNA activity data such specific gene loci, sense and / or antisense strands of pre-mRNA and / or mRNA, small RNA, especially scRNA, snoRNA, microRNA, siRNA, dsRNA, ncRNA or transposable Elements, genes and / or
  • Gene fragments of at least 5 nucleotides in length which have a sequence homology of at least about 10%, in particular about 20%, preferably about 50%, particularly preferably about 80% to the polynucleotide sequences M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and
  • Another preferred embodiment of the present invention is a use characterized in that 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 polynucleotides, or all 13 polynucleotides are used, wherein the polynucleotides are selected from A group consisting of: M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and M17 and / or their isoforms and / or their gene loci and / or their transcripts and / or fragments a length of at least 5 nucleotides thereof, the polynucleotides being defined according to the following table:
  • the invention can also be carried out using at least one polynucleotide selected from the group consisting of: M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and M17 and / or their isoforms and / or their gene loci and / or their transcripts and / or fragments with a length of at least 5
  • pathophysiological condition is present, and / or to determine the severity and / or the course of a pathophysiological condition.
  • the pathophysiological condition is selected from the group consisting of: SIRS, sepsis and their severity; sepsis-like
  • Multi-organ failure Early detection of these conditions; Focus control; Control of surgical remedial measures of the infection focus; Responder / non-responder for a given therapy; Therapy monitoring; Differentiation between infectious and non-infectious genesis in systemic reactions of the
  • Organism e.g. SIRS, sepsis, postoperative complications, chronic and / or acute organ dysfunction, shock reaction, inflammatory response and / or trauma.
  • the sample nucleic acid is RNA, in particular total RNA or mRNA, or DNA, in particular cDNA.
  • the pathophysiological status in addition to at least one of the polynucleotides selected from the group consisting of: M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and M17 and / or their isoforms and / or their gene loci and / or their transcripts and / or fragments having a length of at least 5 nucleotides thereof, the polynucleotides being defined according to the following table:
  • M17 M17 NM_182491 28 to use at least one further marker which is selected from the group consisting of: procalcitonin (PCT), C-reactive protein (CRP);
  • primer pairs forward and reverse.
  • Such particularly suitable primers are those listed in the following table:
  • amplicons can be used, for example, as probes for
  • the invention further relates to a kit for carrying out the method according to the invention, comprising at least one IVIigenigen biomarker comprising a plurality of polynucleotide sequences which are selected from the group consisting of: M2, M3, M4, M6, M7, M8, M9, M10, M12, M13, M15, M16 and M17 and / or their isoforms and / or their gene loci and / or their transcripts and / or fragments having a length of at least 5 nucleotides thereof, the polynucleotides being defined according to the following table:
  • the multigene biomarker is specific for a
  • pathophysiological condition of a patient includes such conditions selected from the group consisting of: SIRS, sepsis and theirs
  • a preferred kit is characterized in that the polynucleotide sequences also include gene loci, sense and / or antisense strands of pre-mRNA and / or mRNA, small RNA, in particular scRNA, snoRNA, microRNA, siRNA, dsRNA, ncRNA or transposable elements.
  • a further preferred kit is characterized in that it contains at least one reference gene which is selected from the group consisting of: R 1, R 2 and R 3 and / or their isoforms and / or their gene loci and / or their
  • index is formed from the individual specific gene activities, which, after appropriate calibration, is a measure of the severity and / or the course of the pathophysiological state, in particular sepsis or the sepsis-like state.
  • the index is preferably determined by statistical techniques such as supervised machine and static learning classification techniques such as (diagonal, linear, quadratic) discriminant analysis, super vector machines, generalized partial least squares, k-nearest neighbors, random forests, k-nearest neighbor determined.
  • supervised machine and static learning classification techniques such as (diagonal, linear, quadratic) discriminant analysis, super vector machines, generalized partial least squares, k-nearest neighbors, random forests, k-nearest neighbor determined.
  • the multigene biomarker is a combination of several elements
  • Polynucleotide in particular gene sequences, based on their gene activities by means of an interpretation function performed a classification and / or an index or score is formed.
  • amplification method preferably polymerase chain reaction (PCR), preferably real-time PCR; and / or by means of hybridization methods, in particular those on microarrays.
  • PCR polymerase chain reaction
  • pathophysiological status a course and / or therapy monitoring.
  • an index is formed from the individual determined gene activities, which, after appropriate calibration, is a measure of the severity and / or the course of the pathophysiological state, in particular sepsis or the sepsis-like state.
  • This score can be a quick diagnostic for the attending physician
  • the present invention as part of an integrated system (In Vitro Diagnostic Multivariate Index Assay, IVDMIA), allows for a potential infectious
  • Sequence pools used to determine states and / or to distinguish or answer defined investigation questions. Examples are in the following
  • the invention relates to polynucleotide sequences, to a method and furthermore to kits for generating multigene biomarkers which have features of an "in vitro diagnostic multivariate index assay” (IVDMIA) in one and / or several modules.
  • IVDMIA in vitro diagnostic multivariate index assay
  • RefSeq is a public database that includes nucleotide and protein sequences with their characteristics and bibliographic information.
  • NCBI National Library of Medicine
  • NCBI builds RefSeq from the sequence data of the GenBank archive database (2), a large public database of sequences posted and shared between GenBank in the United States, the EMBL Data Library in the United Kingdom, and the Japan DNA Database
  • the RefSeq collection is unique when it comes to delivering
  • Protein databases goes. The entries are not redundant with the aim of the represent different biological molecules that are characteristic of organism, strain or haplotype.
  • transcript variants it encodes alternative spliced transcripts for the same protein product (so-called transcript variants).
  • RefSeq database provides the critical foundation for sequence integration, genetic and functional information and is internationally recognized as the standard for genome annotation.
  • BLAST Sequence Search provides RefSeq information in multiple NCBI resources, including Entrez Nucleotide, Entrez Protein, Entrez Gene, Map Viewer, for FTP Download; or by networking with PubMed (Pruitt et al., 2007; The NCBI Handbook, 2002).
  • RefSeq statements can be identified by the unique Accession format, which includes the underscore (_).
  • Each entry has a comment that shows the status of the respective error correction as well as the assignment of the cooperating working group.
  • the RefSeq specification either contains the essentially unchanged, initially valid copy of the original GenBank entries or corrected as well as additional ones
  • the main objective is to avoid known mutations, sequencing errors, cloning artifacts and erroneous annotations. RefSeq sequences that are affected by such types of errors are corrected. Sequences are validated by checking that the genomic sequence corresponding to the annotated mRNA actually matches the mRNA sequence indication and whether coding regions
  • Another important task is to expand the collection by adding previously unknown underrepresented genes and / or alternative splice products, as well as provide additional annotation of sequence properties representing mature peptide products and their functional domains and / or rare biological phenomena such as e.g. non-AUG initiation sites of transcription or selenoproteins, highlight (The NCBI handbook 2002).
  • the quality check is done on a regular basis to be questionable
  • NCBI handbook 2002 Localization, potential cloning errors (such as chimeras) and aligning the data with other NCBI resources, including homolog gene, map viewer and GenBank related sequences (The NCBI handbook 2002).
  • the quality assurance processes involve the registration of database attributes to document that
  • the decision to use the marker population named in the present application based on its RefSeq identity for purposes of the present invention was made based on the properties of the RefSeq database described above.
  • the properties of this database concerning the generation, quality, maintenance and updates of the biological sequences, as well as the presence of functional information at the nucleic acid level, equally for alternative splice variants, were the decisive factors.
  • Multi-organ failure is the failure of two or more vital organ systems occurring simultaneously or in rapid succession.
  • Multi-organ dysfunction syndrome precedes MOV as initial organ failure [Zeni et al., 1997].
  • Multi-organ failure if two or more organs show functional disorders simultaneously or consecutively, whereby chronic persistent organ failure is ruled out.
  • the prognosis of MOV is closely related to the number of involved organ systems. Mortality is 22% in the first 24 hours of an organ failure and 41% after 7 days. When three organ systems fail, mortality increases to 80% on the first day and to 100% on the first day [Knaus et al., 1985].
  • MODS and MOV can be both infectiologic and non-infectiological.
  • Fever of unclear origin Fever of unknow origin (FUO) is clinically defined as a fever in which the temperature is higher than 38.8 ° C for more than 3 weeks, with no evidence of a 1-week follow-up there is a clear diagnosis of the cause.
  • FUO Fever of unknow origin
  • four classes of FUO have been described: FUO classic, nosocomial, immunodeficient or HIV-related origin [Roth and Basello, 2003].
  • FUO was also called "a more well-known disease with one
  • Examination Question A clinically relevant question that is important for the treatment of a patient, for example: prediction of disease progression, therapy monitoring, focus of infection, chances of survival, predisposition, etc.
  • a systemic infection is an infection in which the pathogens have spread through the bloodstream throughout the organism.
  • SIRS Systemic Inflammatory Response Syndrome, according to Bone [Bone et al., 1992] and Levy [Levy et al., 2003] a generalized, inflammatory,
  • Biological fluids within the meaning of the invention are understood to be all body fluids of mammals, including humans.
  • a gene is a section of the deoxyribonucleic acid (DNA) that contains the basic information needed to produce a biologically active ribonucleic acid (RNA) as well as regulatory elements that activate or inactivate this production.
  • genes are also understood as meaning all derived DNA sequences, partial sequences and synthetic analogs (for example peptido-nucleic acids (PNA)). The determination of gene expression on RNA Level-related description of the invention thus expressly none
  • Genlocus is the position of a gene in the genome. If the genome consists of several chromosomes, the position is within the
  • Chromosome meant on which the gene is located. Different manifestations or variants of this gene are referred to as alleles, all located at the same site on the chromosome, namely the gene locus.
  • the term "gene locus” includes, on the one hand, the pure genetic information for a specific gene product and, on the other hand, all regulatory DNA segments as well as any additional DNA sequences that are in any functional relationship with the gene at the gene locus in the immediate vicinity (1 Kb) but outside the 5 'and / or 3' end of a gene locus
  • the specification of the gene locus is made by the accession number and / or RefSeq ID of the RNA main product, which is from this locus descended.
  • Gene activity is the measure of the ability of a gene to be transcribed and / or to form translation products.
  • Gene Expression The process of forming a gene product and / or expression of a genotype into a phenotype.
  • Multigenbiomarker Combination of several gene sequences whose gene activities form a combined overall result (for example a classification and / or an index) by means of an interpretation function. This result is specific to a condition and / or an investigation question.
  • Hybridization Conditions Physical and chemical parameters well known to those skilled in the art that may affect the establishment of a thermodynamic equilibrium of free and bound molecules. In the interest of optimal hybridization conditions, the duration of contact of the probe and sample molecules, cation concentration in the hybridization buffer, Temperature, volume and concentrations and ratios of the hybridizing molecules are coordinated.
  • Amplification conditions Constant or cyclic reaction conditions that allow the amplification of the starting material in the form of nucleic acids.
  • the reaction mixture are the individual components (deoxyribonucleotides) for the resulting nucleic acids, as well as short oligonucleotides, which can attach to complementary regions in the starting material, as well as a nucleic acid synthesis enzyme, called polymerase.
  • the cation concentrations, pH, volume and the duration and temperature of the individual reaction steps which are well known to the person skilled in the art are of importance for the course of the amplification.
  • Primer is an oligonucleotide which can be used as a starting point for nucleic acid-replicating enzymes, such as, for example, B. the DNA polymerase is used. Primers may consist of both DNA and RNA (primer 3, see, e.g., http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi of MIT).
  • a probe is a
  • Nucleic acid fragment (DNA or RNA) having a molecular tag (for example fluorescent markers, in particular Scorpion ®, molecular beacons, Minor Groove Binding- probes, TaqMan ® probes, isotopic label, etc.) can be provided and for sequence-specific detection of target DNA - And / or target RNA molecules is used.
  • a molecular tag for example fluorescent markers, in particular Scorpion ®, molecular beacons, Minor Groove Binding- probes, TaqMan ® probes, isotopic label, etc.
  • PCR is the abbreviation for the term "polymerase chain reaction.”
  • the polymerase chain reaction is a method to amplify DNA in vitro outside a living organism using a DNA-dependent DNA polymerase used according to the present invention to short parts - up to about 3,000
  • Base pairs - to amplify a DNA strand of interest It may be a gene or even a part of a gene or non-coding DNA sequences. It is well known to the skilled person that a A number of PCR methods are known in the art, all of which are encompassed by the term “PCR.” This applies in particular to “real-time PCR” (see also the explanations below).
  • PCR Primer Typically, a PCR requires two primers to detect the starting point of DNA synthesis on the two single strands of DNA
  • primers are well known to those skilled in the art, for example, from the website "Primer3", see, e.g., http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi of MIT.
  • RNA transcript For the purposes of the present application, a transcript is understood to mean any RNA product which is produced on the basis of a DNA template.
  • Small RNA Small RNAs in general. Representatives of this group are in particular, but not exclusively:
  • scRNA small cytoplasmic RNA
  • snRNA small nuclear RNA
  • RNAs small non-protein codinq RNAs, which include the so-called small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs (dsRNAs), which increase gene expression on many levels, including chromatin architecture, RNA editing, RNA stability, translation, and possibly also transcription and splicing.
  • these RNAs are multiply processed from the introns and exons of longer primary transcripts, including protein-coding transcripts.
  • ncRNA non-protein-coding RNAs
  • snoRNAs Small nucleolar RNAs
  • snoRNAs Pseudouridylation regulated by two large snoRNA families called box C / D snoRNAs on the one hand and box H / ACA snoRNAs on the other.
  • Such snoRNAs have a length of about 60 to 300 nucleotides.
  • miRNAs miRNAs
  • siRNAs short interfering RNAs
  • miRNAs are derived from endogenous short hairpin precursor structures and usually use other loci with similar - but not identical - sequences as the target of translational repression.
  • siRNAs arise from longer double-stranded RNAs or long hairpins, often of exogenous origin. They usually target homologous sequences at the same locus or elsewhere in the genome, where they participate in so-called gene silencing, a phenomenon also called RNAi. However, the boundaries between miRNAs and siRNAs are fluid.
  • small RNA may also include so-called transposable elements (TEs) and in particular retroelements, which are also understood for the purposes of the present invention by the term “small RNA”.
  • TEs transposable elements
  • retroelements which are also understood for the purposes of the present invention by the term “small RNA”.
  • RefSeq ID This name refers to entries in the NCBI
  • genomic information includes, among others, chromosomes, mRNAs, RNAs, and proteins.
  • Each RefSeq ID represents a single, naturally occurring molecule of an organism.
  • the biological sequences representing a RefSeq are derived from GenBank entries (also NCBI), but are a compilation of
  • Information elements come from primary research at the DNA, RNA and protein levels.
  • accession number represents the entry number of a polynucleotide in the NCBI gene bank known to the person skilled in the art. In this Both RefSeq IDs and less well characterized and redundant sequences are managed as entries and made available to the public (www.ncbi.nlm.nih.gov/genbank/index.html).
  • the infection is limited to the entry portal of the pathogen (e.g., wound infection)
  • the pathogen e.g., wound infection
  • Colonization The presence of microorganisms does not cause any disease symptoms in the organism.
  • Bacteremia A condition in which there are transient and short-term presence of bacteria in the blood, without this being associated with the appearance of bacterial-related clinical symptoms.
  • BLAST Basic Local Alignment Search Tool (after Altschul et al., J Mol Biol 215: 403-410; 1990). Sequence comparison algorithm, optimized for speed, is used for searching in sequence databases for optimal local adaptation to the query sequence.
  • cDNA Complementary DNA. DNA sequence, product of reverse transcription of mRNA.
  • Coding Sequence Protein-encoding portion of a gene or mRNA as distinct from introns (non-coding sequences) and 5- or 3 'untranslated portions. Coding sequences of the cDNA or mature mRNA include the region between start (AUG or ATG) and stop codon. EST: Expressed Sequence Tag. Short ssDNA sections of the cDNA (usually -300-500 bp), usually produced in large quantities. Represent the genes that are expressed in certain tissues and / or during certain stages of development. Partial coding or non-coding labels of expression for cDNA libraries. Useful for sizing complete genes and in mapping.
  • Exon Coding, the mRNA corresponding sequence region of typical eukaryotic genes. Exons may comprise the coding sequences, the 5 'untranslated region or the 3' untranslated region. Exons encode specific portions of the complete protein and are usually separated by long sections (introns), sometimes referred to as "junk DNA", whose function is not well known but likely to encode short, untranslated RNAs (snRNA) or regulatory information.
  • introns sometimes referred to as "junk DNA”
  • GenBank nucleotide sequence database with sequences from more than 100,000 organisms. Entries annotated with properties of the coding regions also include the translation products. GenBank is part of the international cooperation of the sequence databases, which also includes EMBL and DDBJ.
  • Intron Non-coding sequence region of a typical eukaryotic gene is excised from the primary transcript during RNA splicing and is thus no longer present in the mature, functional mRNA, rRNA or tRNA.
  • mRNA messenger RNA or sometimes just "message". RNA containing the sequences necessary for protein coding. The term mRNA, in contrast to the (unspliced) primary transcript, is only used for the mature transcript with polyA tail (excluding the introns removed via the splicing). Has 5'-untranslated, amino acid-encoding, 3'-untranslated regions and (almost always) a poly (A) tail. Typically represents about 2% of total cellular RNA.
  • Poly (A) tail ss adenosine extension ( ⁇ 50-200 monomers) attached to the 3 'end of the mRNA during splicing.
  • the polyA tail probably increases the stability of the mRNA (possibly protection against nucleases). Not all mRNAs have the construct, such as the histone mRNA.
  • SNPs Single Nucleotide Polymorphisms. Genetic differences between alleles of the same gene based on single nucleotide aberrations. Emerge at specific individual positions within a gene.
  • Transcript variants Alternative splicing products.
  • the exons of the primary gene transcript (pre-mRNA) were reconnected in different ways and are subsequently translated.
  • 3 'untranslated region Transcribed 3' terminal mRNA region without protein coding information (region between stop codon and polyA tail). May affect translation efficiency or stability of mRNA.
  • 5'-untranslated region Transcribed 5'-terminal mRNA region without protein-coding information (region between initial 7-methylguanosine and the base immediately before the ATG start codon). May affect translation efficiency or stability of mRNA.
  • Polynucleotide isoforms polynucleotides having the same function, however
  • Microarray based methods are also possible.
  • PCR polymerase chain reaction
  • Deoxyribonucleotides which again arise double-stranded DNA molecules.
  • the oligodeoxyribonucleotides designated as primers define the sequence segment to be copied by hybridizing at locations of complementary sequence with the target DNA and serve as a starter for the polymerization.
  • the process of exponential product formation is limited by several factors. In the course of Therefore, the PCR ultimately returns net product formation to zero and the total amount of PCR product reaches a plateau.
  • Suitable PCR primers are, for example, primers having the sequences according to Table 3. However, it is known to the person skilled in the art that a large number of further primers can be used to carry out the present invention.
  • PCR is one of the most important methods in molecular biology and molecular medicine. Today it is used in a very broad thematic spectrum, eg. As in the detection of viruses or germs, in the
  • any sequence sections of the nucleic acid inventory of an organism can be cloned in a simple manner with the aid of the PCR.
  • the variety of developed PCR variants allows u. a. a targeted or random change in the DNA sequence and even the synthesis of larger, in this form previously nonexistent sequence sequences.
  • RNA can be detected with high sensitivity and RNA can also be qualitatively detected by reverse transcription (RT) [Wong et al., 2005;
  • Real-time PCR also called quantitative PCR (qPCR)
  • qPCR quantitative PCR
  • Amplification takes place. Based on fluorescently labeled probes, the fluorophores, amplification can be monitored in real time. Take in every reaction cycle the fluorescent PCR products and thus the intensity of the light-induced fluorescence emission. Since the increase in fluorescence and the amount of newly synthesized PCR products over a wide range are proportional to each other, the data obtained from the data obtained
  • the fluorophores used are either nucleic acid-binding fluorescent dyes such as SYBRGreen or sequence-specific fluorescent probes such as Taq-Man probes, LightCycler probes and molecular beacons [Kubista et al., 2006].
  • SYBRGreen is a nucleic acid-binding fluorescent dyes such as SYBRGreen or sequence-specific fluorescent probes such as Taq-Man probes, LightCycler probes and molecular beacons [Kubista et al., 2006].
  • the detection with fluorescence-based probes is highly specific, but also very costly.
  • the PCR approach contains, in addition to the PCR primers, a sequence-specific TaqMan hybridization probe which has a quencher and a reporter dye.
  • the probe is complementary to a sequence lying between the primers.
  • the fluorescence is suppressed by the proximity of the quencher.
  • the quencher swallows the fluorescence emission of the excited fluorophore.
  • this probe hybridizes to the target sequence, it is hydrolyzed by the Taq polymerase during PCR, the reporter dye is spatially removed from the quencher and emits detectable fluorescence upon excitation.
  • the PCR approach contains two fluorescence-labeled probes (donor and acceptor fluorescent dye) in addition to the PCR primers.
  • a fluorescence signal which can be measured outwardly only arises in the case of directly adjacent hybridization of the two probes with the specific target sequence. In a subsequent melting curve analysis, even the presence and the nature of individual
  • Point mutations are detected within the hybridization regions of the probes.
  • Another example is the Molecular Beacons. These oligonucleotides contain mutually complementary sequences at the 5 ' and 3 ' ends, which hybridize in an unbound state and form a hairpin structure.
  • Reporter fluorophore and quencher located at both ends, are in close proximity. Only when the probe binds to the template, the two
  • the quantitative determination of a template can be done by absolute or relative quantification.
  • absolute quantification finds the
  • Present invention preferably housekeeping genes are used with the sequences shown in Table 2.
  • the generated experiment data are evaluated using the device's own software. For the plot, the measured fluorescence intensity is plotted against the number of cycles. The resulting curve is divided into three areas. In the first phase, ie at the beginning of the reaction, the background noise predominates, a signal of the PCR product is not yet detectable. The second phase corresponds to the exponential growth phase. In this
  • This Threshold Cycle (CT) value or Crossing Point provides the basis for calculating the starting amount of existing target DNA. For example, the software determines the crossing points of the different reference dilutions for an absolute quantification and quantifies the values based on the calculated standard curve
  • Quantitative PCR is an important tool for gene expression studies in clinical research. With the ability to accurately quantify mRNA, the search for new drugs can be used to analyze the effects of certain factors on cells, to observe the differentiation of precursor cells into different cell types, or to respond to gene expression in host cells
  • a preferred method for selecting multigene biomarker sequences comprises the following steps: a. Patient selection is based on the extreme group procedure; b. Generation of at least one multigene biomarker; c. Determination of final multigene biomarkers.
  • a preferred method of the assay similar to the "in vitro diagnostic multivariate index assay” comprises the following steps: a) isolation of sample nucleic acids from a patient-derived sample b detection of gene activities using sequences from at least one condition and / or C) detecting gene activities for at least one internal reference gene to normalize the gene activities detected in b);
  • a preferred embodiment of the present invention is also in a use in which the gene activities are determined by means of a hybridization method, in particular on at least one microarray.
  • the advantage of a microarray lies in the higher information density of the biochip compared to the amplification method. So it is e.g. It is easily possible to provide several 100 probes on a microarray in order to examine several questions simultaneously in a single examination.
  • the gene activity data obtained by means of the invention can also be used advantageously for electronic further processing, e.g. B. be used for recording in the electronic medical record.
  • a further embodiment of the invention consists of the use of recombinantly or synthetically produced, specific nucleic acid sequences, partial sequences, individually or in part, as multigene biomarkers in sepsis assays and / or for evaluating the effect and toxicity in drug screening and / or for the preparation of therapeutics and of substances and mixtures intended as therapeutic agents for the prevention and treatment of SIRS and sepsis.
  • the sample is selected from: tissue,
  • Body fluids in particular blood, serum, plasma, urine, saliva or cells or cell components; or a mixture of them.
  • samples especially cell samples, have a lytic
  • Classification procedures are mainly used in 2 different tasks, in delineating previously unknown classes (unlearned learning, class discovery) and in assigning certain data / samples / patients to a ready-defined class
  • Class prediction uses data / samples / patients assigned to existing or defined classes / groups (so-called training data set) to develop an analytical method (classification algorithm) that reflects the differences between the groups. Independent samples (so-called test data set) are used to determine the separation quality of the To evaluate the classification rule.
  • the procedure can be divided into the following steps:
  • Profiles for the training record ideally contain data that has a
  • Sensitivity places the data in the right class.
  • Typical representatives of such algorithms in the field of supervised learning are the
  • DA Discriminant Analysis
  • Random Forests Random Forests
  • GPLS Generalized Partial Least Squares
  • SVM Support Vector Machines
  • kNN k-Nearest Neighbors
  • DA Discriminant Analysis
  • QDA quadratic discriminant analysis
  • Random Forests The Random Forests classification is based on the combination of decision trees, [Breiman, 2001]. The process of the algorithm is something like this: 1 . Drag and drop to select a training data set (out-of-bag data).
  • GPLS Generalized Partial Least Squares
  • Support Vector Machine The Support Vector Machine classifier is a generalized linear classifier. The input data is mapped into a higher dimensional space and in this space an optimal separating (hyper) plane is constructed. These barriers, linear in higher-dimensional space, transform into nonlinear barriers in the space of input data, [Vapnik, 1999].
  • k-nearest neighbors k-nearest neighbors, kNN: In the k-nearest neighbor method, the class affiliation of an observation (a
  • the neighborhood is usually determined with the help of the Euclidean distance and the class membership then on the basis of a
  • biomarker Based on gene expression data, a method for the determination of a multigene biomarker should be developed, which reflects an infectious complication such as sepsis.
  • the biomarker and associated index value also known as “score” form the basis of a so-called “in vitro diagnostic multivariate index assay” [IVDMIA, FDA Guidelines, 2003] for improving the diagnosis of systemic infections.
  • IVDMIA in vitro diagnostic multivariate index assay
  • the classification rule should allow differentiation of SIRS and sepsis patients with improved sensitivity and specificity compared to the established biomarker procalcitonin, but is not limited to this question.
  • Classification genes will be suitable filter options such as the threshold of
  • 3rd step Classification procedure. Different classification methods are regarding their ability to separate with respect to the differentiated
  • Classification error is selected, with the smallest necessary number of genes being co-determined. As a reasonable rule has been found that the number of genes should always be smaller than the number of samples in the
  • Patient Selection The patient selection is used when setting up the
  • extreme groups can be useful. Thereafter, in a study, only those patient groups are taken into account that reflect the examined effect as clearly as possible. The selected samples represent an idealized case in which many effects occurring in practice (eg the frequency of the disease) not considered.
  • Liu Liu [Liu et al., 2005] suggested forming extreme groups for the training data set of a microarray-based classifier. Using cancer survivor survival as an example, it has been shown that the use of extreme groups (patients who died in a short time vs.
  • Example of U.S. Patent Application No. 20060246495 also used clinical sepsis diagnosis for the selection of the sepsis group.
  • Applicant's patient database has included 400 ITS patients who were suspected to be at risk for sepsis over a two-and-a-half-year timeframe and detailed clinical data throughout their stay
  • RNA samples were collected for approximately 7-14 sepsis-relevant days.
  • Approaching the PIRO concept [Levy et al., 2003], patients were retrospectively stratified according to the following criteria: (i) which indication led to the transfer to the intensive care unit (postoperative complication, trauma or polytrauma, acute suspected sepsis), ( ii) was diagnosed with an infectious complication, what was the infection focus, (iii) how was the response of the organism (number of SIRS criteria present, shock treatment, PCT, CRP levels), (iv) how severe was the disease (SOFA, M ODS score).
  • the database research revealed that infectious complications (sepsis) in particular included patients after pneumonia (40%) and after peritonitis (23%) in the study.
  • the values correspond to the number or, marked with an asterisk, the median (interquartile distance) of the values.
  • 3rd step Rankinq: In order to arrange the gene markers according to their separation quality, the linear discriminant analysis (LDA) [Hastie et al., 2001] was used together with the method of forward selection, the separability being evaluated by the F value [Hocking, RR, 1976). This analysis step was for 1000
  • Step 4 Classification: For the markers that gave the best results in the ranking analysis, a discriminant function was determined based on the LDA. The associated weights are set forth in Table 9.
  • Step 5 Internal validation: To assess the quality of the classification for growing number of markers, the simple cross-validation was used.
  • Step 6 Establishment of the SIQ score: Based on the discriminant function, a sepsis-related diagnostic parameter, a so-called SIQ score (SIQ), was introduced as follows. For a new independent sample, one generally obtains a dimension-free value of the discriminant function as a classification result. A positive value classifies the sample as infectious and a negative value as non-infectious. absolutely higher values are obtained for typical representatives of the respective group, hard-to-classify samples reach values close to zero. The spreading area The discriminant values generally correspond to the variability of the data matrix. Discriminant values of about -5 to 5 were achieved in the classification. To emphasize the differences more clearly, the SIQ score (SIQ) was introduced as the 10-fold value of the discriminant function with the weights in Table 9.
  • SIQ SIQ score
  • the SIQ values of the test data varied from about -50 to 50.
  • This independent test dataset consisted of 13 samples from 65 individuals (see Tables 4 and 5). Samples from 38 sepsis patients representing a broad range of clinical phenotypes at risk of generalized infection were examined. In addition, samples were analyzed on the SIRS course of 22 postoperative patients and 5 healthy volunteers.
  • FIG. 2 (patient 81-12) shows the course of the SIQ score for a patient who has developed sepsis postoperatively. From Fig. 2 it can be seen that SIQ score exceeds the diagnostic-relevant threshold already 2 days before the clinical manifestation of sepsis. The course of further sepsis-relevant clinical parameters (PCT, CRP, SOFA, body temperature,
  • Shock treatment is shown as a comparison with. In this comparison will be It can be seen that SIQ score is the only parameter that prematurely reflects the infectious complication. This demonstrates that the described
  • FIG. 3 (Patient 7084) shows the course of the SIQ score for a patient who developed postoperative sepsis, fell into septic shock, but has recovered from an acute phase through relevant treatment. From Figure 3, it can be seen that SIQ score increases above the diagnostic threshold one day before the clinical manifestation of sepsis and remains above the threshold in the acute phase. After the acute phase, the SIQ score falls below this threshold. This demonstrates that the invention described for the
  • Fig. 2 is an illustration of an exemplary course of an inventive
  • Fig. 3 is an illustration of an exemplary course of an inventive
  • Fig. 1 shows an ROC curve for the classification of the test data by means of the SIQ score.
  • the relationship between true positives (sensitivity) and false positives (1 specificity) is marked, dashed gray for the threshold of zero and dashed black for the best achieved classification of 81, 4%.
  • FIG. 2 shows a profile of the SIQ score of an exemplary patient and the sepsis-relevant clinical parameters PCT, CRP, SOFA, body temperature and the dose of catecholamines (norepinephrine), which reflects the shock treatment.
  • the scale of each parameter was adjusted so that the black horizontal center line marks the diagnostically relevant threshold. Sepsis was diagnosed on the 6th day, the SIQ score rises above the -4.9 threshold on the 4th day.
  • FIG. 3 shows a course of the SIQ score of another patient and the sepsis-relevant clinical parameters PCT, CRP, SOFA, body temperature and the dosage of catecholamines (norepinephrine), which reflects the shock treatment.
  • the scale of each parameter was adjusted so that the black horizontal center line marks the diagnostically relevant threshold.
  • the sepsis was diagnosed on the 4th ITS day, the SIQ score rises above the threshold of -4.9 already on the 3rd day. After the acute phase, which ends with catecholamine discontinuation (shock treatment) on the 8th day, the SIQ score falls below the -4.9 threshold.
  • FIG. 4 shows ROC curves for the classification of the test data by means of the parameter PCT or CRP.
  • the ROC curve is shown in PCT in black and the ROC curve in CRG is shown in CRP.
  • the area under the curve that indicates the quality the classification is 56.8% for PCT and 66.9% for CRP.
  • Table 3 gives, for each of the marker polynucleotides of the invention, the primers (forward and reverse) for quantitative PCR and the resulting Amplicon and their one-to-one assignment to the respective SEQ ID of the sequence listing again.
  • the described biomarkers are functionally associated with high significance immunological and inflammatory signaling pathways.
  • a knowledge-based biomarker population analysis was performed using the Ingenuity Pathways Analysis software (Ingenuity Systems, USA, www ngenuity.com) to clarify the functional context of the identified markers.
  • the markers are categorized into functional networks and categories.
  • the major categories of the present marker population are the complement system, Toll-like receptor signal transduction, communication between cells of the innate and adaptive immune defense, TREM-1 signal transduction, ceramide signal transduction. Accordingly, the markers are of high significance for immunological and inflammatory processes, which underlines the relevance for the clinical picture of sepsis. Thus, an important prerequisite for biomarkers, the presence of biological plausibility, could be demonstrated.
  • M6 and M9 are functional roles for M6 and M9 in the context of coagulation and fibrinolysis. Both processes are among the most deregulated physiological functions in septic patients.
  • a treatment option for patients with severe sepsis and organ failure is treatment with activated protein C or thrombomodulin.
  • M6 is overexpressed in septic patients and at the same time negative Subjected to transcriptional control by activated protein C.
  • M9 is suppressed in septic patients and may not fulfill the attributed activity of prothrombin cleavage to provide thrombin.
  • Thrombin is an important factor for the activation of protein C after association with thrombomodulin.

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Abstract

L'invention concerne l'utilisation de polynucléotides définis pour former au moins un biomarqueur multigénique afin de produire un dosage multiplexe servant de moyen auxiliaire pour détecter et/ou identifier précocement et/ou différencier et/ou évaluer des états physiopathologiques d'un patient et/ou en observer l'évolution, ledit état physiopathologique étant sélectionné dans le groupe comprenant: le syndrome de réponse inflammatoire systémique (SIRS), la septicémie et ses degrés de gravités, les états analogues à la septicémie, le choc septique, la bactériémie, la défaillance multiorganique infectieuse ou non-infectieuse; la détection précoce de ces états, le contrôle du foyer d'infection, le contrôle des mesures sanitaires chirurgicales s'appliquant au foyer d'infection, la réponse/non réponse à une thérapie donnée, le contrôle thérapeutique, la différenciation entre une genèse infectieuse et non infectieuse lors de réactions systémiques de l'organisme, comme par exemple, le SIRS, la septicémie, des complications post-opératoires, un dysfonctionnement organique chronique ou aigu, une réaction de choc, une réaction inflammatoire et/ou un traumatisme.
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