Patent Application
20230416827
The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 2677-P33USCON_Seq_List_20230511.xml. The XML file is 979,747 bytes; was created on May 11, 2023; and is being submitted electronically via Patent Center with the filing of the specification.
The present invention relates to one or more biomarkers associated with systemic inflammatory conditions, such as Severe Inflammatory Response Syndrome (SIRS) and sepsis. More particularly, the invention relates to methods for diagnosing, monitoring and prognosing systemic inflammatory conditions, such as Severe Inflammatory Response Syndrome (SIRS), sepsis, abdominal sepsis and pulmonary sepsis, and for distinguishing between sepsis and SIRS in a patient.
Systemic inflammatory conditions such as Severe Inflammatory Response Syndrome (SIRS) and sepsis are life-threatening conditions that can result in organ failure and death.
Sepsis (or blood poisoning) is characterised by a systemic host response to infection. Sepsis affects approximately 25% of intensive care patients, and is estimated to cause over 37,000 deaths in the UK every year, with a mortality rate of between 28% and 50%. Diagnosis of sepsis is typically performed using culture-based methods, involving microbial growth followed by taxonomic identification of the pathogen. However, these culture-based techniques are time-consuming, taking over 24 hours to obtain results, and have poor sensitivity and specificity. Other more recently developed diagnostic methods involve assessment of single blood protein biomarkers such as CRP and pro-calcitonin. These methods allow quicker diagnosis, but there is growing evidence that these markers suffer from poor specificity. Furthermore, none of the available methods provide an insight into the underlying origin or aetiology of the disease, nor do they provide any way of predicting recovery from sepsis or the likelihood of progression to severe sepsis or septic shock. Treatment of sepsis typically involves administration of antimicrobials such as broad-spectrum antibiotics together with intravenous fluids. It is estimated that mortality increases by approximately 5% for every hour that treatment is delayed. Rapid diagnosis and initiation of treatment of patients having sepsis is therefore essential.
Severe Inflammatory Response Syndrome (SIRS) is also characterised by a systemic host response. However, this condition does not result from infection, but instead results from injury or trauma. Clinical symptoms of SIRS are similar to sepsis, and thus it can be difficult to distinguish between patients in the early stages of sepsis and patients who have infection-negative systemic inflammation (SIRS). Making an incorrect distinction between these two conditions has both clinical and economic implications, including inappropriate patient management, and unnecessary over-prescription of antibiotics. There is currently no clinical test available to distinguish between sepsis and non-infective SIRS. Development of a method for effectively stratifying SIRS and sepsis patients would therefore be beneficial, allowing sepsis patients to receive early, effective and aggressive treatment with antimicrobials and supportive care, and reducing the unnecessary exposure of SIRS patients to antibiotics.
There is therefore a need to provide improved ways to diagnose, monitor and/or prognose patients that have or are at risk of developing systemic inflammatory conditions (such as sepsis and SIRS) in order to facilitate early intervention and appropriate treatment. In particular, there is a need to provide for effective ways of distinguishing between sepsis and SIRS in patients in order to ensure that an appropriate treatment regimen is selected.
By conducting extensive investigations into expression patterns associated with systemic inflammatory conditions, the present inventors have identified biomarkers that may be used to evaluate various aspects of systemic inflammatory conditions, such as SIRS and sepsis. The biomarkers may be used to diagnose the presence (or absence) of a systemic inflammatory condition, and to distinguish between different types of systemic inflammatory conditions in a patient. The biomarkers may also be used to monitor a patient having a systemic inflammatory condition, and to determine whether a patient is suitable for discharge from medical care. The present invention therefore provides a solution to one or more of the above mentioned problems.
In one aspect, the present invention provides a method for distinguishing between sepsis and systemic inflammatory response syndrome (SIRS) in a patient, comprising:
In a related aspect, the present invention provides a method for distinguishing between sepsis and systemic inflammatory response syndrome (SIRS) in a patient, comprising:
In a related aspect, the present invention provides a method for distinguishing between sepsis and systemic inflammatory response syndrome (SIRS) in a patient diagnosed as having a systemic inflammatory condition, comprising:
In a related aspect, the present invention provides a method for distinguishing between sepsis and systemic inflammatory response syndrome (SIRS) in a patient diagnosed as having a systemic inflammatory condition, comprising:
In a further related aspect, the present invention provides the use of one or more of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4 and/or one or more of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124 for distinguishing between sepsis and SIRS in a patient.
In a further aspect, the present invention provides a method for diagnosing whether a patient has a systemic inflammatory condition, comprising:
In a further related aspect, the present invention provides the use of FAM20A and OLAH for diagnosing a systemic inflammatory condition in a patient.
In a further aspect, the present invention provides a method for diagnosing whether a patient has abdominal sepsis, comprising:
In a related aspect, the present invention provides a method for diagnosing whether a patient has abdominal sepsis, comprising:
In a related aspect, the present invention provides a method for diagnosing whether a patient has abdominal sepsis, comprising:
In a related aspect, the present invention provides the use of one or more of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1 as a biomarker of abdominal sepsis in a patient.
In a further aspect, the present invention provides a method for diagnosing whether a patient has pulmonary sepsis, comprising:
In a related aspect, the present invention provides a method for diagnosing whether a patient has pulmonary sepsis, comprising:
In a further related aspect, the present invention provides a method for diagnosing whether a patient has pulmonary sepsis, comprising:
In a related aspect, the present invention provides the use of one or more of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44 as a biomarker of pulmonary sepsis in a patient.
In a further aspect, the present invention provides a method for monitoring a systemic inflammatory condition in a patient, comprising:
In one embodiment, the method is for monitoring a patient having abdominal sepsis, and comprises the steps of:
In one embodiment, the method is for monitoring a patient having SIRS, and comprises the steps of:
In a related aspect, the present invention provides the use of one or more of: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, MX1, NECAB1, PKHD1 and LILRB5, as a biomarker for monitoring a patient having a systemic inflammatory condition.
In a related aspect, the invention provides a method for determining whether a patient having a systemic inflammatory condition is suitable for discharge from medical care, comprising:
In one embodiment, the method is for determining whether a patient being treated for SIRS is suitable for discharge from medical care, and comprises the steps of:
In one embodiment, the method is for determining whether a patient being treated for sepsis is suitable for discharge from medical care, and comprises the steps of:
In a related aspect, the present invention provides the use of one or more of: NECAB1, NECAB2, PKD1, PKHD1, LILRB4, and LILRB5, for determining whether a patient having a systemic inflammatory condition is suitable for discharge from medical care.
In a further aspect, the present invention provides a device for carrying out the methods and uses of the invention. In one embodiment, the device comprises one or more binding agent specific for the one or more biomarker.
As described herein, the present inventors have conducted a temporal differential gene expression study in peripheral blood leukocytes (PBLs) in patients having SIRS, abdominal sepsis and pulmonary sepsis, and in normal healthy individuals. Using this method, the inventors have identified host biomarkers associated with different systemic inflammatory conditions. In particular, the present inventors have identified biomarkers that are elevated in patients having systemic inflammatory conditions, and can thus be used for diagnosis, monitoring and/or prognosis of these conditions. The present inventors have further identified biomarkers that are differentially regulated in different types of systemic inflammatory condition (e.g., SIRS and sepsis) and biomarkers that are differentially regulated in different types of sepsis (e.g., in abdominal sepsis and pulmonary sepsis). These biomarkers can therefore be used to specifically diagnose SIRS and sepsis (e.g., abdominal sepsis and pulmonary sepsis), and can also be used to distinguish between SIRS and sepsis, and/or between abdominal sepsis and pulmonary sepsis. These biomarkers may also be used to monitor a systemic inflammatory condition in a patient (e.g., to monitor the recovery of a patient). The present inventors have also identified biomarkers that are differentially regulated in patients that recover from a systemic inflammatory condition, and those that do not recover form a systemic inflammatory condition and can thus be used to determine whether a patient is suitable for discharge from medical care. The new biomarkers for the systemic inflammatory conditions are listed in Tables 1-4 herein (together with corresponding sequence identifiers (SEQ ID NOs)). Tables 1-4 provide the HGNC gene IDs for the biomarkers of the invention. As would be understood by a person skilled in the art, the HGNC gene ID information can be used to determine the sequence of all the RNA transcripts, and thus all of the proteins which correspond to the biomarkers of the invention. Accesion numbers for each of the biomarkers are also provided in the “Sequence Information” Section of the description.
Based on these findings, the present inventors have thus developed methods and uses that allow for rapid, sensitive and accurate diagnosis, monitoring and/or prognosis of systemic inflammatory conditions (such as sepsis and/or SIRS) using one or more biological samples obtained from a patient at a single time point, or during the course of disease progression. The inventors have also developed methods and uses that allow for different systemic inflammatory conditions (such as sepsis and/or SIRS) to reliably distinguished allowing for appropriate therapeutic intervention.
Diagnosis of a Systemic Inflammatory Condition
As illustrated by Example 1 and
The present invention therefore provides a method for diagnosing a systemic inflammatory condition in a patient, comprising:
As used herein, the terms “diagnosis”, “diagnosing” and “diagnose(d)” refer to the process or act of recognising, deciding on or concluding on a disease or condition in a patient on the basis of symptoms and signs and/or from results of various diagnostic procedures (such as for example, from knowing the presence, absence or quantity of one or more biomarkers characteristic of the diagnosed disease or condition). In one embodiment, diagnosis of a systemic inflammatory condition in a patient comprises determining whether the patient has or is at risk of developing a systemic inflammatory condition.
As used herein, the term “systemic inflammatory condition” refers to a disease or condition comprising a systemic inflammatory response. In one embodiment, the term encompasses SIRS and sepsis. In one embodiment, the systemic inflammatory condition is one or more of SIRS and sepsis. In one embodiment, the systemic inflammatory condition is one or more of SIRS, abdominal sepsis and pulmonary sepsis.
As used herein, the term “systemic inflammatory response syndrome (SIRS)” refers to a systemic inflammatory response syndrome with no signs of infection. This condition may also be referred to as “non-infective SIRS” or “infection-free SIRS”. SIRS may be characterised by the presence of at least two of the four following clinical symptoms: fever or hypothermia (temperature of 38.0° C. (100.4° F.) or more, or temperature of 36.0° C. (96.8° F.) or less); tachycardia (at least 90 beats per minute); tachypnea (at least 20 breaths per minute or PaCC>2 less than 4.3 kPa (32.0 mm Hg) or the need for mechanical ventilation); and an altered white blood cell (WBC) count of 12×106 cells/mL or more, or an altered WBC count of 4×106 cells/mL or less, or the presence of more than 10% band forms (immature neutrophils).
As used herein, the term “sepsis” refers to the systemic inflammatory condition that occurs as a result of infection. Defined focus of infection is indicated by either (i) an organism grown in blood or sterile site; or (ii) an abscess or infected tissue (e.g., pneumonia, peritonitis, urinary tract, vascular line infection, soft tissue). In one embodiment, the infection may be a bacterial infection. The presence of sepsis is also characterised by the presence of at least two (of the four) systemic inflammatory response syndrome (SIRS) criteria defined above.
Sepsis may be characterised as mild sepsis, severe sepsis (sepsis with acute organ dysfunction), septic shock (sepsis with refractory arterial hypotension), organ failure, multiple organ dysfunction syndrome and death.
“Mild sepsis” can be defined as the presence of sepsis without organ dysfunction.
“Severe sepsis” can be defined as the presence of sepsis and at least one of the following manifestations of organ hypoperfusion or dysfunction: hypoxemia, metabolic acidosis, oliguria, lactic acidosis, or an acute alteration in mental status without sedation. Organ hypoperfusion or dysfunction is defined as a Sequential Organ Failure Assessment (SOFA) score ≥2 for the organ in question.
“Septic shock” can be defined as the presence of sepsis accompanied by a sustained decrease in systolic blood pressure (90 mm Hg or less, or a drop of at least 40 mm Hg from baseline systolic blood pressure) despite fluid resuscitation, and the need for vasoactive amines to maintain adequate blood pressure.
The term “sepsis” may include one or more of abdominal sepsis and pulmonary sepsis.
As used herein, the term “abdominal sepsis” refers to severe bacterial infection in the abdominal cavity (for example, but not restricted to perforated small and large bowel, pyelonephritis, spontaneous bacterial peritonitis, abscess in the peritoneal cavity, infection of the retroperitoneal space, infection in the liver, kidneys, pancreas, spleen); causing organ dysfunction. Organ hypoperfusion or dysfunction is defined as a Sequential Organ Failure Assessment (SOFA) score ≥2 for the organ in question.
As used herein, the term “pulmonary sepsis” refers to severe bacterial infection in the thoracic cavity, primarily affecting the lung and pleural space (for example, but not restricted to pneumonia, lung abscess, empyaema, mediastinitis, tracheobronchitis); causing organ dysfunction. Organ hypoperfusion or dysfunction is defined as a Sequential Organ Failure Assessment (SOFA) score ≥2 for the organ in question.
The terms “patient”, “individual”, and “subject”, are used interchangeably herein to refer to a mammalian subject for whom diagnosis, monitoring, prognosis, and/or treatment is desired. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. In one preferred embodiment, the individual, subject, or patient is a human, e.g., a male or female.
In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition (such as SIRS, sepsis, abdominal sepsis, or pulmonary sepsis). For example, the patient may be a critically ill patient, e.g., a patient admitted to an intensive care unit (ICU) or emergency department (ED), in whom the incidence of SIRS and sepsis is known to be elevated. The patient may be admitted to ICU or ED with one or more of: serious trauma, chronic obstructive pulmonary disease (COPD), patients having undergone surgery, complications from surgery, medical shock, bacterial, fungal or viral infections, Acute Respiratory Distress Syndrome (ARDS), pulmonary and systemic inflammation, pulmonary tissue injury, severe pneumonia, respiratory failure, acute respiratory failure, respiratory distress, subarachnoidal hemorrhage (SAH), (severe) stroke, asphyxia, neurological conditions, organ dysfunction, single or multi-organ failure (MOF), poisoning and intoxication, severe allergic reactions and anaphylaxis, burn injury, acute cerebral hemorrhage or infarction, and any condition for which the patient requires assisted ventilation.
In one embodiment, the patient has been previously diagnosed as having or being at risk of developing a systemic inflammatory condition (eg., SIRS, sepsis, abdominal sepsis, or pulmonary sepsis). In one embodiment, the patient may have been previously diagnosed as having or being at risk of developing a systemic inflammatory condition (eg., SIRS, sepsis, abdominal sepsis, or pulmonary sepsis) using any of the methods described herein, or any combination of methods described herein.
In one embodiment, the patient has not been previously diagnosed as having a systemic inflammatory condition (eg., SIRS, sepsis, abdominal sepsis, or pulmonary sepsis).
As used herein, the term “sample” encompasses any suitable biological material, for example blood, plasma, saliva, serum, sputum, urine, cerebral spinal fluid, cells, a cellular extract, a tissue sample, a tissue biopsy, a stool sample, and the like. Furthermore, pools or mixtures of the above-mentioned samples may be employed. Typically, the sample is blood sample. The precise biological sample that is taken from the individual may vary, but the sampling preferably is minimally invasive and is easily performed by conventional techniques. In a preferred embodiment, the sample is a whole blood sample, a purified peripheral blood leukocyte sample or a cell type sorted leukocyte sample, such as a sample of the individual's neutrophils.
The methods and uses of the present invention may utilise samples that have undergone minimal or zero processing before testing. They may also utilise samples that have been manipulated, in any way, after procurement, such as treatment with reagents, solubilisation, or enrichment for certain components.
The methods of the present invention are in vitro methods. Thus, the methods of the present invention can be carried out in vitro on an isolated sample that has been obtained from a patient.
For those embodiments described herein which involve a multi-step method, the sample used in each step of the method may be the same sample obtained from the patient. When the method comprises multiple quantification steps, all the steps may be performed at the same time using the same sample.
The sample may be obtained from the patient before, during, and/or after treatment for the systemic inflammatory condition. In one embodiment, the sample is taken before treatment for the systemic inflammatory condition has been initiated. In one embodiment, the sample is taken after treatment for the systemic inflammatory condition has been initiated (eg., so as to monitor the effectiveness of a treatment regimen).
In one embodiment, the sample may be obtained from the patient at least 1 hour (e.g., at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
In one embodiment, the sample may be obtained from the patient up to 1 hour (e.g., up to 2 hours, up to 4 hours, up to 6 hours, up to 8 hours, up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 72 hours, up to 96 hours, or up to 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition. For example, the sample may be obtained from the patient up to 24 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition. For example, the sample may be obtained from the patient up to 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition. For example, the sample may be obtained from the patient up to 72 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition. For example, the sample may be obtained from the patient up to 96 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition. For example, the sample may be obtained from the patient up to 120 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
In one embodiment, the sample may be obtained from the patient between about 1 hour and 120 hours (e.g., between about 1 hour and 96 hours, between about 1 hour and 72 hours, between about 1 hour and 48 hours, or between about 1 hour and 24 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
For example, the sample may be obtained from the patient between about 12 hours and 120 hours (e.g., between about 12 hours and 96 hours, between about 12 hours and 72 hours, between about 12 hours and 48 hours, or between about 12 hours and 24 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
For example, the sample may be obtained from the patient between about 24 hours and 120 hours (e.g., between about 24 hours and 96 hours, between about 24 hours and 72 hours, or between about 24 hours and 48 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition. For example, the sample may be obtained between about 24 hours and 48 hours. For example, the sample may be obtained between about 24 hours and 72 hours. For example, the sample may be obtained between about 24 hours and 96 hours.
For example, the sample may be obtained from the patient between about 48 hours and 120 hours (e.g., between about 48 hours and 96 hours, or between about 48 hours and 72 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
For example, the sample may be obtained from the patient between about 72 hours and 120 hours or between about 72 hours and 96 hours, after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
For example, the sample may be obtained from the patient between about 96 hours and 120 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition.
Presentation of the patient with one or more clinical symptoms of a systemic inflammatory condition means that the patient displays or presents with one or more (e.g., 2 or more, 3 or more, or all 4) clinical symptoms of a systemic inflammatory condition. The skilled person will be aware of the clinical symptoms associated with a systemic inflammatory condition. Clinical symptoms of a systemic inflammatory condition include: (i) fever (temperature of 38.0° C. (100.4° F.) or more) or hypothermia (temperature of 36.0° C. (96.8° F.) or less); (ii) tachycardia (at least 90 beats per minute); (iii) tachypnea (at least 20 breaths per minute or PaCC>2 less than 4.3 kPa (32.0 mm Hg) or the need for mechanical ventilation); and (iv) an altered white blood cell (WBC) count of 12×106 cells/mL or more, or an altered WBC count of 4×106 cells/mL or less, or the presence of more than 10% band forms.
The patient does not necessarily have to present with one or more clinical symptoms of a systemic inflammatory condition before they are tested for the presence (or absence) of a systemic inflammatory condition.
Thus, in one embodiment, the sample may be obtained from the patient at least 1 hour (e.g., at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours) after the patient is admitted to a medical care facility.
In one embodiment, the sample may be obtained from the patient up to 1 hour (e.g., up to 2 hours, up to 4 hours, up to 6 hours, up to 8 hours, up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 72 hours, up to 96 hours, or up to 120 hours) after the patient is admitted to a medical care facility. For example, the sample may be obtained from the patient up to 24 hours after the patient is admitted to a medical care facility. For example, the sample may be obtained from the patient up to 48 hours after the patient is admitted to a medical care facility. For example, the sample may be obtained from the patient up to 72 hours after the patient is admitted to a medical care facility. For example, the sample may be obtained from the patient up to 96 hours after the patient is admitted to a medical care facility. For example, the sample may be obtained from the patient up to 120 hours after the patient is admitted to a medical care facility.
In one embodiment, the sample may be obtained from the patient between about 1 hour and 120 hours (e.g., between about 1 hour and 96 hours, between about 1 hour and 72 hours, between about 1 hour and 48 hours, or between about 1 hour and 24 hours) after the patient is admitted to a medical care facility.
For example, the sample may be obtained from the patient between about 12 hours and 120 hours (e.g., between about 12 hours and 96 hours, between about 12 hours and 72 hours, between about 12 hours and 48 hours, or between about 12 hours and 24 hours) after the patient is admitted to a medical care facility.
For example, the sample may be obtained from the patient between about 24 hours and 120 hours (e.g., between about 24 hours and 96 hours, between about 24 hours and 72 hours, or between about 24 hours and 48 hours) after the patient is admitted to a medical care facility. For example, the sample may be obtained between about 24 hours and 48 hours. For example, the sample may be obtained between about 24 hours and 72 hours. For example, the sample may be obtained between about 24 hours and 96 hours.
For example, the sample may be obtained from the patient between about 48 hours and 120 hours (e.g., between about 48 hours and 96 hours, or between about 48 hours and 72 hours) after the patient is admitted to a medical care facility.
For example, the sample may be obtained from the patient between about 72 hours and 120 hours or between about 72 hours and 96 hours, after the patient is admitted to a medical care facility.
For example, the sample may be obtained from the patient between about 96 hours and 120 hours after the patient is admitted to a medical care facility.
As used herein, the phrase “after the patient is admitted to a medical care facility” refers to the admission of a patient for clinical observation and/or treatment. Admission to a medical care facility includes admittance of the patient into hospital (eg. into an intensive care unit). The term “medical care facility” is not limited hospitals but includes any environment in which a patient can be clinically monitored and/or treated (e.g., including doctors, surgeries, or expedition medical tents).
As used herein, the term “biomarker” refers to virtually any biological compound, such as a protein and a fragment thereof, a peptide, a polypeptide, a proteoglycan, a glycoprotein, a lipoprotein, a carbohydrate, a lipid, a nucleic acid, an organic or inorganic chemical, a natural polymer, and a small molecule, that is present in the biological sample and that may be isolated from, or measured in, the biological sample. Furthermore, a biomarker can be the entire intact molecule, or it can be a portion thereof that may be partially functional or recognized, for example, by an antibody or other specific binding protein.
In one embodiment, the one or more biomarker is a nucleic acid (e.g., DNA, such as cDNA or amplified DNA, or RNA, such as mRNA). The one or more biomarker may have a nucleic acid sequence as shown in the sequences in the Sequence Information section herein. The relevant sequence identifiers are also shown in Tables 1-4.
In another embodiment, the one or more biomarker is a protein. As used herein, the terms “protein”, “peptide”, and “polypeptide” are, unless otherwise indicated, interchangeable. When the presence and/or amount of two or more biomarkers are determined, the biomarkers may all be protein biomarkers or all nucleic acid biomarkers. Alternatively, the biomarkers may be both protein and nucleic acid biomarkers.
The present invention also encompasses, without limitation, polymorphisms, isoforms, metabolites, mutants, variants, derivatives, modifications, subunits, fragments, protein-ligand complexes and degradation products of the biomarkers listed in Tables 1-4.
The protein fragments can be 200, 150, 100, 50, 25, 10 amino acids or fewer in length. The nucleic acid fragments can be 1000, 500, 250 150, 100, 50, 25, 10 nucleotides or fewer in length.
Variants of the protein biomarkers of the present invention include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Variants include polypeptides that have an amino acid sequence being at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences of the polypeptides listed in Tables 1-4. Variants may be allelic variants, splice variants or any other species specific homologs, paralogs, or orthologs.
Derivatives of the protein biomarkers of the present invention are polypeptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
Variants of the nucleic acid biomarkers of the present invention may have a sequence identity of at least 80% with the corresponding nucleic acid sequence shown in the Sequence Information section. Sequence identity may be calculated as described herein. A sequence identity of at least 80% includes at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity (to each and every nucleic acid sequence presented herein and/or to each and every SEQ ID NO presented herein).
The one or more inflammation biomarker used in the method may be selected from the group consisting of: FAM20A, OLAH, CD177, ADM, IL10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1 R2, CYP19A1, MMP8, TGFA and VSTM1.
In one embodiment, the one or more inflammation biomarker may be selected from the group consisting of: IGFBP2, CYP19A1, and VSTM1. In one embodiment, the one or more biomarker may be selected from the group consisting of: CD177, IL110, IL1 R1, IL1 R2, VSTM1, ADM, and HP, wherein said biomarkers are associated with immune response and/or inflammation. In one embodiment, the one or more biomarker may be selected from the group consisting of: METTL7B, RETN, and CYP19A1, wherein said biomarkers are associated with lipid metabolism. In one embodiment, the one or more biomarker may be selected from the group consisting of: MMP9 and MMP8, wherein said biomarkers are associated with extracellular matrix maintenance or composition.
Each of the biomarkers for a systemic inflammatory condition may be used alone, or in combination with any of the biomarkers for a systemic inflammatory condition in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more 19 or more, 20 or more, up to and including all of the biomarkers may be used to diagnose a systemic inflammatory condition according to the method of the invention.
In one embodiment, the one or more biomarker is FAM20A. In one embodiment, the one or more biomarker is OLAH. In one embodiment, the one or more biomarker is CD177. In one embodiment, the one or more biomarker is ADM. In one embodiment, the one or more biomarker is IL10. In one embodiment, the one or more biomarker is METTL7B. In one embodiment, the one or more biomarker is MMP9. In one embodiment, the one or more biomarker is RETN. In one embodiment, the one or more biomarker is TDRD9. In one embodiment, the one or more biomarker is ITGA7. In one embodiment, the one or more biomarker is BMX. In one embodiment, the one or more biomarker is HP. In one embodiment, the one or more biomarker is IGFBP2. In one embodiment, the one or more biomarker is ALPL. In one embodiment, the one or more biomarker is DACH1. In one embodiment, the one or more biomarker is IL1 R1. In one embodiment, the one or more biomarker is IL1 R2. In one embodiment, the one or more biomarker is CYP19A1. In one embodiment, the one or more biomarker is MMP8. In one embodiment, the one or more biomarker is TGFA. In one embodiment, the one or more biomarker is VSTM1.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more 19 or more, 20 of more, or all 21) of the biomarkers selected from the group consisting of: FAM20A, OLAH, CD177, ADM, I-10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1R2, CYP19A1, MMP8, TGFA and VSTM1, may be used to diagnose a systemic inflammatory condition in a patient.
As demonstrated by ROC analysis performed in Example 2, the inflammation biomarkers FAM20A, OLAH and CD177 were all shown to provide highly accurate diagnosis of patients having a systemic inflammatory condition when used on their own or in combination. In one embodiment, a combination of FAM20A and OLAH may be used to diagnose a systemic inflammatory condition in a patient. In one embodiment, a combination of FAM20A, OLAH and CD177 may be used to diagnose a systemic inflammatory condition in a patient.
One or more additional biomarker for inflammation may also be used in the method of the invention to diagnose a systemic inflammatory condition. Any combination of the one or more additional biomarker may be used in combination with the one or more biomarker of the invention. For example at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 additional biomakers for inflammation may be used in combination with the one or more biomarker of the invention (as described herein). Typically, the one or more additional biomarker is selected from the group consisting of: FAM20A, OLAH, CD177, ADM, IL10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1R2, CYP19A1, MMP8, TGFA and VSTM1.
In one embodiment, the one or more biomarker is FAM20A, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, up to and including all) of the biomarkers: OLAH, CD177, ADM, I-10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1R2, CYP19A1, MMP8, TGFA and VSTM1.
In one embodiment, the one or more biomarker is OLAH, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, up to and including all) of the biomarkers: FAM20A, CD177, ADM, I-10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1 R2, CYP19A1, MMP8, TGFA and VSTM1.
In one embodiment, the one or more biomarker is CD-177, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, up to and including all) of the biomarkers: FAM20A, OLAH, ADM, IL10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1R2, CYP19A1, MMP8, TGFA and VSTM1.
A biomarker is considered to be informative if a measurable aspect or characteristic of the biomarker is associated with a given state of an individual, such as the diagnosis, monitoring or prognosis of a systemic inflammatory condition. Such a measurable aspect or characteristic may include, for example, the presence, absence, or concentration of the biomarker in the biological sample from the individual and/or its presence as part of a profile of biomarkers. Such a measurable aspect of a biomarker is defined herein as a “feature.” For example, the presence of a biomarker in a sample may be a feature. As another example, the amount of a biomarker in a sample, or the amount of a biomarker in a sample compared with a control or reference sample may be a feature. A feature may also be a ratio of two or more measurable aspects of biomarkers, which biomarkers may or may not be of known identity. A “biomarker profile” comprises at least two such features, where the features can correspond to the same or different classes of biomarkers such as, for example, two nucleic acids or a nucleic acid and a protein. A biomarker profile may also comprise at least three, four, five, 10, 20, 30 or more features. In one embodiment, a biomarker profile comprises hundreds, or even thousands, of features. In another embodiment, the biomarker profile comprises at least one measurable aspect of at least one internal standard.
A “phenotypic change” is a detectable change in a parameter associated with a given state of the individual. For instance, a phenotypic change may include an increase or decrease of a biomarker in a bodily fluid, where the change is associated with a systemic inflammatory condition (such as sepsis or SIRS) or distinguishing between sepsis and SIRS. The presence and/or amount of each of the one or more biomarkers of the invention is a feature or phenotypic change according to the present invention. For example, the presence of each of the one or more biomarkers of the invention is a feature or phenotypic change according to the present invention. For example, the amount of each of the one or more biomarkers of the invention is a feature or phenotypic change according to the present invention. In a further example, the presence and amount of each of the one or more biomarkers of the invention is a feature or phenotypic change according to the present invention.
A phenotypic change may further include a change in a detectable aspect of a given state of the individual that is not a change in a measurable aspect of a biomarker. For example, a change in phenotype may include a detectable change in body temperature, weight loss, fatigue, respiration rate or another physiological parameter. Such changes can be determined via clinical observation and measurement using conventional techniques that are well-known to the skilled artisan. As used herein, “conventional techniques” are those techniques that classify an individual based on phenotypic changes without obtaining a biomarker profile according to the present invention.
According to the present invention, systemic inflammatory conditions may be diagnosed, monitored, and/or prognosed by obtaining a profile of biomarkers from a sample obtained from a patient. As used herein, “obtain” means “to come into possession of”.
A feature as defined herein for the diagnosis, monitoring or prognosis of a systemic inflammatory condition may be detected, quantified, or determined by any appropriate means. For example, the one or more biomarker of the invention, a measurable aspect or characteristic of the one or more biomarker or a biomarker profile of the invention may be detected by any appropriate means. The presence of the one or more biomarkers of the invention may be considered together as a “biomarker profile” of the invention. The presence of the individual biomarkers within any of the biomarker combinations disclosed herein may be considered together as a “biomarker profile” of the invention.
The presence and/or amount of the one or more biomarker of the invention may be determined by quantitative and/or qualitative analysis. Measurement of the one or more biomarkers can be performed by any method that provides satisfactory analytical specificity, sensitivity, and precision. The invention encompasses the use of those methods known to a person skilled in the art to measure the presence and/or amount of one or more biomarkers.
In one embodiment, the methods described herein involve determining the “presence and amount of the one or more biomarker”. In one embodiment, the methods described herein involve determining the “presence of the one or more biomarker”. In one embodiment, the methods described herein involve determining the “amount of the one or more biomarker”.
Determining the “amount of one or more biomarker” in a sample means quantifying the biomarker by determining the relative or absolute amount of the biomarker. The amount of the one or more biomarker of the invention encompasses the mass of the one or more biomarker, the molar amount of the one or more biomarker, the concentration of the one or biomarker and the molarity of the one or more biomarker. This amount may be given in any appropriate units. For example, the concentration of the one or more biomarker may be given in pg/mL, ng/mL or μg/mL. It will be appreciated that the assay methods do not necessarily require measurement of absolute values of biomarker, unless it is desired, because relative values are sufficient for many applications of the invention. Accordingly, the “amount” can be the “absolute” total amount of the biomarker that is detected in a sample, or it can be a “relative” amount, e.g., the difference between the biomarker detected in a sample and e.g., another constituent of the sample. In some embodiments, the amount of the biomarker may be expressed by its concentration in a sample, or by the concentration of an antibody that binds to the biomarker. Thus, the actual amount of the one or more biomarker, such as the mass, molar amount, concentration, or molarity of the one or biomarker may be assessed and compared with the corresponding reference value. Alternatively, the amount of one or more biomarker may be compared with that of the reference value without quantifying the mass, molar amount, concentration or molarity of the one or more biomarker.
The presence and/or amount of the one or more biomarker can be determined at the protein or nucleic acid level using any method known in the art. The particular preferred method for determining the presence and/or amount of the one or more biomarkers will depend in part on the identity and nature of the biomarker.
The biomarkers of the invention may be detected at the nucleic acid or protein level. Thus, the biomarkers of the invention may be DNA, RNA or protein and may be detected using any appropriate technique. The presence and/or amount of the one or more biomarker of the invention may be measured directly or indirectly. Any appropriate agent may be used to determine the presence and/or amount of the one or more biomarker of the invention. For example, the presence and/or amount of the one or more biomarker of the invention may be determined using an agent selected from peptides and peptidomimetics, antibodies, small molecules and single-stranded DNA or RNA molecules, as described herein. Suitable standard techniques are known in the art.
For example, when the one or more biomarker is detected at the nucleic acid level this may be carried out using: (i) biomarker-specific oligonucleotide DNA or RNA or any other nucleic acid derivative probes bound to a solid surface; (ii) purified RNA (labelled by any method, for example using reverse transcription and amplification) hybridised to probes; (iii) whole lysed blood, from which the RNA is labelled by any method and hybridised to probes; (iv) purified RNA hybridised to probes and a second probe (labelled by any method) hybridised to the purified RNA; (v) whole lysed blood from which the RNA is hybridised to probes, and a second probe (labelled by any method) which is hybridised to the RNA; (vi) purified peripheral blood leukocytes, obtaining purified RNA (labelled by any method), and hybridising the purified labelled RNA to probes; (vii) purified peripheral blood leukocytes, obtaining purified RNA and hybridising the RNA to probes, then using a second probe (labelled by any method) which hybridises to the RNA; (viii) RT-PCR using any primer/probe combination or inter-chelating fluorescent label, for example SYBRGreen; (ix) end-point PCR; (x) digital PCR; (xi) sequencing; (xii) array cards (RT-PCR); (xiii) lateral flow devices/methodology; and/or (xiv) digital microfluidics.
In one embodiment, quantitative real-time PCR is used to determine the presence and/or amount of the one or more biomarker of the invention. Quantitative real-time PCR may be performed using forward and reverse oligonucleotide primers that amplify the target sequence (such as those described herein). Detection of the amplified product is done in real-time and may be performed using oligonucleotide probes that produce a fluorescent signal when the target DNA is amplified (e.g., Taqman® fluorgenic probes), or using SYBR Green dye that binds to double-stranded DNA and emits fluorescence when bound.
In one embodiment, oligonucleotide microarray analysis is used to detect and/or quantify the one or more biomarker of the invention using biomarker-specific oligonucleotide DNA or RNA or any other nucleic acid derivative probes bound to a solid surface.
In a preferred embodiment, RNA from a sample (either purified or unpurified) is labelled via any method (typically amplification) and used to interrogate one or more probe immobilised on a surface. Typically, the one or more probes are 50 to 100 nucleotides in length.
In another preferred embodiment, one or more probe is immobilised on a surface and the RNA from a sample is hybridised to one or more second probe (labelled by any method). The RNA hybridised with the second (labelled) probe is then used to interrogate the one or more probe immobilised on the surface. Examples of such methodology are known in the art, including the Vantix™ system.
For example, when the one or more biomarker is detected at the protein acid level this may be carried out using: (i) biomarker-specific primary antibodies or antibody fragments bound to a solid surface; (ii) whole lysed blood biomarker antigen bound to antibodies or antibody fragments; (iii) secondary biomarker-specific antibodies or antibody fragments used to detect biomarker antigen bound to primary antibody (labelled using any method); (iv) biomarker-specific primary aptamers bound to a solid surface; (v) whole lysed blood-biomarker antigen bound to aptamers; (vi) secondary biomarker-specific aptamer used to detect biomarker antigen bound to primary aptamer (labelled using any method); (vii) any antibody derivative, i.e., phage display etc. used as above; (viii) lateral flow devices/methodology; (ix) chromatography; (x) mass spectrometry; (xi) nuclear magnetic resonance (NMR); (xii) protein gels/transfers to filter; and/or (xiii) immunoprecipitation. In a preferred embodiment, a lateral flow device may be used to detect the one or more protein biomarker.
Any agent for the detection of or for the determination of the amount of the one or more biomarker of the invention may be used to determine the amount of the one or more biomarker. Similarly, any method that allows for the detecting of the one or more biomarker, the quantification, or relative quantification of the one or more biomarker may be used.
Agents for the detection of or for the determination of the amount of one or more biomarker may be used to determine the amount of the one or more biomarker in a sample obtained from the patient. Such agents typically bind to the one or more biomarker. Such agents may bind specifically to the one or more biomarker. The agent for the detection of or for the determination of the amount of the one or more biomarker may be an antibody or other binding agent specific for the one or more biomarker. By specific, it will be understood that the agent or antibody binds to the molecule of interest, in this case the one or more biomarker, with no significant cross-reactivity to any other molecule, particularly any other protein. Cross-reactivity may be assessed by any suitable method. Cross-reactivity of an agent or antibody for the one or more biomarker with a molecule other than the one or more biomarker may be considered significant if the agent or antibody binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to the one or more biomarker. Preferably, the agent or antibody binds to the other molecule at less than 20%, less than 15%, less than 10% or less than 5%, less than 2% or less than 1% the strength that it binds to the one or more biomarker.
As described herein, the presence and/or amount of the one or more biomarker, and hence the biomarker profile may be determined immunologically by reacting antibodies, or functional fragments thereof, specific to the biomarkers. A functional fragment of an antibody is a portion of an antibody that retains at least some ability to bind to the antigen to which the complete antibody binds. The fragments, which include, but are not limited to, scFv fragments, Fab fragments, F(ab) fragments and F(ab)2 fragments, can be recombinantly produced or enzymatically produced. Specific binding molecules other than antibodies, such as aptamers, may be used to bind the biomarkers.
The antibody may be monoclonal or polyclonal. The antibody may be produced by any suitable method known in the art. For example, polyclonal antibodies may be obtained by immunising a mammal, typically a rabbit or a mouse, with the one or more biomarker under suitable conditions and isolating antibody molecules from, for example, the serum of said mammal. Monoclonal antibodies may be obtained by hybridoma or recombinant methods.
Hybridoma methods may involve immunising a mammal, typically a rabbit or a mouse, with the one or more biomarker under suitable conditions, then harvesting the spleen cells of said mammal and fusing them with myeloma cells. The mixture of fused cells is then diluted, and clones are grown from single parent cells. The antibodies secreted by the different clones are then tested for their ability to bind to the one or more biomarker, and the most productive and stable clone is then grown in culture medium to a high volume. The secreted antibody is collected and purified.
Recombinant methods may involve the cloning into phage or yeast of different immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences. Those sequences which give rise to antibodies which bind to the one or more biomarker may be selected and the sequences cloned into, for example, a bacterial cell line, for production.
Typically, the antibody is a mammalian antibody, such as a primate, human, rodent (e.g., mouse or rat), rabbit, ovine, porcine, equine or camel antibody. The antibody may be a camelid antibody or shark antibody.
The antibody may be a nanobody. The antibody can be any class or isotype of antibody, for example IgM, but is preferably IgG. The antibody may be a humanised antibody. The antibody or fragment may be associated with other moieties, such as linkers which may be used to join together 2 or more fragments or antibodies. Such linkers may be chemical linkers or can be present in the form of a fusion protein with the fragment or whole antibody. The linkers may thus be used to join together whole antibodies or fragments which have the same or different binding specificities, e.g., that can bind the same or different polymorphisms. The antibody may be a bispecific antibody which is able to bind to two different antigens, typically any two of the polymorphisms mentioned herein. The antibody may be a ‘diabody’ formed by joining two variable domains back to back. In the case where the antibodies used in the method are present in any of the above forms which have different antigen binding sites of different specificities then these different specificities are typically to polymorphisms at different positions or on different proteins. In one embodiment the antibody is a chimeric antibody comprising sequence from different natural antibodies, for example a humanised antibody.
Methods to assess an amount of the one or more biomarker may involve contacting a sample with an agent or antibody capable of binding specifically to the one or more biomarker. Such methods may include dipstick assays and Enzyme-linked Immunosorbant Assay (ELISA), or similar assays, such as those using a lateral flow device. Other immunoassay types may also be used to assess the one or more biomarker amounts. Typically, dipsticks comprise one or more antibodies or proteins that specifically bind to the one or more biomarker. If more than one antibody is present, the antibodies preferably have different non-overlapping determinants such that they may bind to the one or more biomarker simultaneously.
ELISA is a heterogeneous, solid phase assay that requires the separation of reagents. ELISA is typically carried out using the sandwich technique or the competitive technique. The sandwich technique requires two antibodies. The first specifically binds the one or more biomarker and is bound to a solid support. The second antibody is bound to a marker, typically an enzyme conjugate. A substrate for the enzyme is used to quantify the one or more biomarker-antibody complex and hence the amount of the one or more biomarker in a sample. The antigen competitive inhibition assay also typically requires a one or more biomarker-specific antibody bound to a support. A biomarker-enzyme conjugate is added to the sample (containing the one or more biomarker) to be assayed. Competitive inhibition between the biomarker-enzyme conjugate and unlabelled biomarker allows quantification of the amount of the one or more biomarker in a sample. The solid supports for ELISA reactions preferably contain wells.
Antibodies capable of binding specifically to the one or more biomarker may be used in methods of immunofluorescence to detect the presence of the one or more biomarker.
The present invention may also employ methods of determining the amount of the one or more biomarker that do not comprise antibodies. High Performance Liquid Chromatography (HPLC) separation and fluorescence detection is preferably used as a method of determining the amount of the one or more biomarker. HPLC apparatus and methods as described previously may be used (Tsikas D et al. J Chromatogr. B Biomed. Sci. Appl. 1998; 705: 174-6) Separation during HPLC is typically carried out on the basis of size or charge. Prior to HPLC, endogenous amino acids and an internal standard L-homoarginine are typically added to assay samples and these are phase extracted on CBA cartridges (Varian, Harbor City, CA). Amino acids within the samples are preferably derivatized with o-phthalaldehyde (OPA). The accuracy and precision of the assay is preferably determined within quality control samples for all amino acids.
Other methods of determining the amount the one or more biomarker that do not comprise antibodies include mass spectrometry. Mass spectrometric methods may include, for example, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), surface-enhanced laser desorption/ionization mass spectrometry (SELDI MS), time of flight mass spectrometry (TOF MS) and liquid chromatography mass spectrometry (LC MS).
A separation method may be used to determine the presence and/or amount of the one or more biomarker and hence to create a profile of biomarkers, such that only a subset of biomarkers within the sample is analysed. For example, the biomarkers that are analysed in a sample may consist of mRNA species from a cellular extract, which has been fractionated to obtain only the nucleic acid biomarkers within the sample, or the biomarkers may consist of a fraction of the total complement of proteins within the sample, which have been fractionated by chromatographic techniques. One or more, two or more, three or more, four or more, or five or more separation methods may be used according to the present invention.
Determination of the presence and/or amount of the one or more biomarker, and hence the creation of a profile of biomarkers may be carried out without employing a separation method. For example, a biological sample may be interrogated with a labelled compound that forms a specific complex with a biomarker in the sample, where the intensity of the label in the specific complex is a measurable characteristic of the biomarker. A suitable compound for forming such a specific complex is a labelled antibody. A biomarker may be measured using an antibody with an amplifiable nucleic acid as a label. The nucleic acid label may become amplifiable when two antibodies, each conjugated to one strand of a nucleic acid label, interact with the biomarker, such that the two nucleic acid strands form an amplifiable nucleic acid.
The presence and/or amount of the one or more biomarker, and hence the biomarker profile may be derived from an assay, such as an array, of nucleic acids, where the biomarkers are the nucleic acids or complements thereof. For example, the biomarkers may be ribonucleic acids. The presence and/or amount of the one or more biomarker, and hence the biomarker profile may be obtained using a method selected from nuclear magnetic resonance, nucleic acid arrays, dot blotting, slot blotting, reverse transcription amplification and Northern analysis.
The determination of the presence and/or amount of the one or more biomarker, and hence a biomarker profile may be generated by the use of one or more separation methods. For example, suitable separation methods may include a mass spectrometry method, such as electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n (n is an integer greater than zero), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SLMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n. Other mass spectrometry methods may include, inter alia, quadrupole, fourier transform mass spectrometry (FTMS) and ion trap. Other suitable separation methods may include chemical extraction partitioning, column chromatography, ion exchange chromatography, hydrophobic (reverse phase) liquid chromatography, isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) or other chromatography, such as thin-layer, gas or liquid chromatography, or any combination thereof. The sample may be fractionated prior to application of the separation method.
The determination of the presence and/or amount of the one or more biomarker, and hence a biomarker profile may be generated by methods that do not require physical separation of the biomarkers themselves. For example, nuclear magnetic resonance (NMR) spectroscopy may be used to resolve a profile of biomarkers from a complex mixture of molecules. An analogous use of NMR to classify tumours is disclosed in Hagberg, NMR Biomed. 11:148-156 (1998), for example. Additional procedures include nucleic acid amplification technologies, which may be used to generate a profile of biomarkers without physical separation of individual biomarkers. (See Stordeur et al., J. Immunol. Methods 259:55-64 (2002) and Tan et al., Proc. Nat'l. Acad. Sci. USA 99: 11387-11392 (2002), for example.)
In one embodiment, laser desorption/ionization time-of-flight mass spectrometry is used to determine the presence and/or amount of the one or more biomarker, and hence create a biomarker profile where the biomarkers are proteins or protein fragments that have been ionized and vaporized off an immobilizing support by incident laser radiation. A profile is then created by the characteristic time-of-flight for each protein, which depends on its mass-to-charge (“m/z”) ratio. A variety of laser desorption/ionization techniques are known in the art. (See, e.g., Guttman et al., Anal. Chem. 73:1252-62 (2001) and Wei et al., Nature 399: 243-246 (1999).)
Laser desorption/ionization time-of-flight mass spectrometry allows the generation of large amounts of information in a relatively short period of time. A sample is applied to one of several varieties of a support that binds all of the biomarkers, or a subset thereof, in the sample. Cell lysates or samples are directly applied to these surfaces in volumes as small as 0.5 μL, with or without prior purification or fractionation. The lysates or sample can be concentrated or diluted prior to application onto the support surface. Laser desorption/ionization is then used to generate mass spectra of the sample, or samples, in as little as three hours.
In a preferred embodiment, the total mRNA from a cellular extract of the patient is assayed, and the various mRNA species that are obtained from the sample are used as biomarkers. Biomarker profiles may be obtained, for example, by hybridizing these mRNAs to an array of probes, which may comprise oligonucleotides or cDNAs, using standard methods known in the art. Alternatively, the mRNAs may be subjected to gel electrophoresis or blotting methods such as dot blots, slot blots or Northern analysis, all of which are known in the art. (See, e.g., Sambrook et al. in “Molecular Cloning, 3rd ed.,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001).) mRNA profiles also may be obtained by reverse transcription followed by amplification and detection of the resulting cDNAs, as disclosed by Stordeur et al, supra, for example. In another embodiment, the profile may be obtained by using a combination of methods, such as a nucleic acid array combined with mass spectroscopy.
Different methods have different advantages and may be preferred depending on numerous factors, such as the particular circumstances of the patients to be tested and/or the availability of reagents/equipment in the diagnostics laboratory. For example, qPCR using probe/quencher hydrolysis probes is highly specific and stringent. As another example, microarray analysis can resolve subtle differences in expression of transcript variants, which may be important in disease pathology and diagnosis.
Different one or more biomarkers may be used with different detection methods according to the present invention.
When the amount of two or more biomarkers are determined, the amount of each biomarker may be determined, or the cumulative amount of all the biomarkers may be determined. Alternatively, the amount of the two or more biomarkers can be combined with each other in a formula to form an index value.
In the methods and uses of the invention, the presence and/or amount of the one or more biomarker of the invention in a patient (or the profile of biomarkers in a patient) may be measured relative to a corresponding reference value. As such, the presence and/or amount of the one of more biomarker of the invention (or the profile of biomarkers) may be “compared” to a corresponding reference value.
The terms “comparison”, “comparing” and “compared” are used herein interchangeably and includes any means to discern at least one difference in the presence and/or amount of the one or more biomarker in the test sample as compared to a reference value (or as compared to a further sample obtained from the patient where monitoring of a systemic inflammatory condition takes place). In one embodiment, the methods of the invention described herein may involve comparison of “the amount of the one or more biomarker” in the test sample as compared to a reference value. In one embodiment, the methods of the invention described herein may involve comparison of “the presence and amount of the one or more biomarker” in the test sample as compared to a reference value.
A comparison may include a visual inspection of chromatographic spectra, and a comparison may include arithmetical or statistical comparisons of values assigned to the features of the profiles. Such statistical comparisons include, but are not limited to, applying a decision rule. If the biomarker profiles comprise at least one internal standard, the comparison to discern a difference in the biomarker profiles may also include features of these internal standards, such that features of the biomarker are correlated to features of the internal standards. As described herein, the comparison can be used to diagnose, monitor or prognose a systemic inflammatory condition, such as sepsis, abdominal sepsis, pulmonary sepsis, or SIRS, and can be used to distinguish between sepsis and SIRS in a patient, or it can be used to distinguish between abdominal sepsis and pulmonary sepsis in a patient.
The term “reference value” refers to a value that is representative of a control individual or population whose disease state is known. A reference value can be determined for any particular population, subpopulation, or group of individuals according to standard methods well known to those of skill in the art. The actual amount of the one or more biomarkers, such as the mass, molar amount, concentration, or molarity of the one or more biomarker of the invention may be assessed and compared with the corresponding reference population. Alternatively, the amount of one or more biomarker of the invention may be compared with that of the reference population without quantifying the mass, molar amount, concentration, or molarity of the one or more biomarker.
The reference value may be obtained from a healthy individual or a population of healthy individuals eg. by quantifying the amount of the one or more biomarker in a sample obtained from the healthy individual or the population of healthy individuals. As used herein, “healthy” refers to a subject or group of individuals who are in a healthy state, e.g., patients who have not shown any symptoms of the disease, have not been previously diagnosed with the disease and/or are not likely to develop the disease. In one embodiment, the healthy individual (or population of healthy individuals) is not on medication affecting the disease and has not been diagnosed with any other disease. In one embodiment, the healthy individual (or population of healthy individuals) has similar sex, age and body mass index (BMI) as compared with the test patient. In one embodiment, the healthy individual (or population of healthy individuals) does not have a current infection or a chronic infection. Application of standard statistical methods used in medicine permits determination of normal levels of expression, as well as significant deviations from such normal levels.
The reference value may be obtained from an individual or a population of individuals suffering from the disease eg. by quantifying the amount of the one or more biomarker in a sample obtained from the individual or the population of individuals suffering from the disease. The reference data is typically collected from individuals that present at a medical centre with clinical signs relating to the relevant disease of interest. The reference value may be obtained, for example, from an individual or population of individuals having a systemic inflammatory condition, such as those having sepsis (including those having abdominal sepsis or pulmonary sepsis) or those having SIRS. Such individual(s) may have similar sex, age and body mass index (BMI) as compared with the test patient.
In one embodiment, the reference value is obtained from an individual or population of individuals having sepsis. In one embodiment, the reference value is obtained from an individual or population of individuals having abdominal sepsis. In one embodiment, the reference value is obtained from an individual or population of individuals having pulmonary sepsis. In one embodiment, the individual (or population of individuals) presents at hospital with sepsis (such as abdominal sepsis or pulmonary sepsis) of less than 72 hours duration. The reference values may be obtained from individuals having sepsis may be obtained at any stage in the progression of sepsis, such as infection, bacteremia, severe sepsis, septic shock of multiple organ failure. For example, the reference values may be obtained from patients having severe sepsis and/or septic shock. Diagnosis of sepsis (such as severe sepsis and/or septic shock) is based on the conventional diagnosis methods defined herein.
In one embodiment, the reference value is obtained from an individual or a population of individuals having SIRS. Diagnosis of SIRS is based on the SIRS criteria defined herein. In one embodiment, the individual (or population of individuals) may have organ failure defined as SOFA score >2. In one embodiment, the individual or a population of individuals having SIRS has not been treated with antibiotics for treatment of known or suspected infection. In one embodiment, the individual or a population of individuals having SIRS have been admitted to a medical care facility following out-of hospital cardiac arrest.
The reference value may be obtained from an individual or a population of individuals who are diagnosed as having sepsis (eg. abdominal or pulmonary sepsis) or SIRS by conventional methods about 24, 48, 72, 96 or 120 hours or more after biological samples were taken for the purpose of generating a reference sample. In one embodiment, the individual or a population of individuals is diagnosed as having sepsis (eg. abdominal or pulmonary sepsis) or SIRS using conventional methods about 24-48 hours, about 48-72 hours, about 72-96 hours, or about 96-120 hours after the biological samples were taken. Conventional methods for confirming diagnosis of sepsis and SIRS are as defined herein.
The sample(s) used to generate the reference values may be obtained from the individual (or population of individuals) that present at a medical centre with clinical signs relating to the relevant disease of interest at any of the time points described herein for sample collection from the test patient. All embodiments described herein for the timing of sample collection from a test patient thus apply equally to the time point at which samples are obtained from the reference individual (or population of individuals) for the purpose of generating a reference value. For example, the sample used to generate the reference value may be obtained from an individual (or population of individuals) up to 24 hours after the individual (or population of individuals) presents with one or more clinical symptoms of a systemic inflammatory condition, or is admitted to a medical care facility. The individual (or population of individuals) from which the sample is obtained is then later on confirmed as having a systemic inflammatory condition using the conventional methods described herein.
In one embodiment, the reference values used in the comparison step of the method are generated from a sample obtained at the same time point (or time period) as the sample obtained from the test patient. For example, if a sample is obtained from a test patient up to 24 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility, then the corresponding reference value may be obtained from an individual (or population of individuals) up to 24 hours after the individual (or population of individuals) presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. Likewise, if a sample is obtained from a test patient up to 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility, then the corresponding reference value may be obtained from an individual (or population of individuals) up to 48 hours after the individual (or population of individuals) presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The individuals from which samples are obtained for generation of reference data may be subject to further follow-on consultations to confirm clinical assessments, as well as to identify further changes in biomarkers, changes in the severity of clinical signs over a period of time, and/or survival outcome. The reference data collected may include series data to indicate the progression or regression of the disease, so that the data can be used to determine if the condition of a test individual is improving, worsening or static. The reference data collected from patients that recover from the systemic inflammatory disease, can be used as a reference value that is representative of an individual having a (good) prognosis of recovery from the systemic inflammatory condition. The reference data collected from patients that do not recover from the systemic inflammatory disease, can be used as a reference value that is representative of an individual having a prognosis of non-recovery from the systemic inflammatory condition (or a poor prognosis of recovery from the systemic inflammatory condition).
Multiple separate reference values may be used in the methods of the invention. For example, reference values may include those that are representative of one or more of (eg. two or more, three or more, four or more, or all five of): (i) an individual (or a population of individuals) having sepsis, (ii) an individual (or a population of individuals) having SIRS; (iii) an individual (or a population of individuals) having abdominal sepsis; (iv) an individual (or a population of individuals) having pulmonary sepsis; and/or (v) a healthy individual (or a population of healthy individuals).
For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having sepsis; (ii) an individual (or a population of individuals) having SIRS; and (iii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having sepsis; and (ii) an individual (or a population of individuals) having SIRS. For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having sepsis; and (ii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having SIRS; and (ii) a healthy individual (or a population of healthy individuals).
For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; (ii) an individual (or a population of individuals) having pulmonary sepsis; (iii) an individual (or a population of individuals) having SIRS; and (iv) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; (ii) an individual (or a population of individuals) having pulmonary sepsis; and (iii) an individual (or a population of individuals) having SIRS. For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; (ii) an individual (or a population of individuals) having pulmonary sepsis; and (iii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; (ii) an individual (or a population of individuals) having SIRS; and (iii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having pulmonary sepsis; (ii) an individual (or a population of individuals) having SIRS; and (iii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; and (ii) an individual (or a population of individuals) having pulmonary sepsis. For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; and (ii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having pulmonary sepsis; and (ii) a healthy individual (or a population of healthy individuals). For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having abdominal sepsis; and (ii) an individual (or a population of individuals) having SIRS. For example, the methods of the invention may use reference values that are representative of: (i) an individual (or a population of individuals) having pulmonary sepsis; and (ii) an individual (or a population of individuals) having SIRS.
The reference value may be obtained from the same (test) patient, provided that the test and reference values are generated from biological samples taken at different time points and compared to one another. For example, a sample may be obtained from a patient at the start of a study period. A reference value taken from that sample may then be compared to biomarker profiles generated from subsequent samples from the same patient. Such a comparison may be used, for example, to monitor a systemic inflammatory condition (i.e., determine the progression of a systemic inflammatory condition in the patient by repeated classifications over time). Although the invention does not require a monitoring period to classify a patient, it will be understood that repeated classifications of the patient, i.e., repeated snapshots, may be taken over time until the individual is no longer at risk. Alternatively, a profile of biomarkers obtained from the patient may be compared to one or more profiles of biomarkers obtained from the same patient at different points in time.
In one embodiment, the reference value is obtained from a single individual eg. by quantifying the amount of a biomarker in a sample or samples derived from a single individual. Alternatively, the reference value may be derived by pooling data obtained from two or more (e.g., at least three, four, five, 10, 15, 20 or 25) individuals (i.e., a population of individuals) and calculating an average (for example, mean or median) amount for a biomarker. Thus, the reference value may reflect the average amount of a biomarker in a given population of individuals. Said amounts may be expressed in absolute or relative terms, in the same manner as described above in relation to the sample that is to be tested using the method of the invention. As used herein, the term “population of individuals” refers to a group of two or more individuals, such as at least three, four, five, 10, 15, 20 or 25 individuals.
When comparing between the sample and the reference value, the way in which the amounts are expressed is matched between the sample and the reference value. Thus, an absolute amount can be compared with an absolute amount, and a relative amount can be compared with a relative amount.
The reference value may be derived from the same sample as the sample that is being tested, thus allowing for an appropriate comparison between the two. Thus, by way of example, if the sample is derived from a blood sample, then the reference value will also be a blood sample.
When the amounts of two or more biomarkers are determined, the amount of each biomarker may be compared to its corresponding reference value. Alternatively, when the cumulative amount of all the biomarkers is determined, the cumulative amount the biomarkers may be compared to a cumulative corresponding reference value. Alternatively, when the amount of the two or more biomarkers are combined with each other in a formula to form an index value, the index value can be compared to a corresponding reference index value derived in the same manner.
The reference values may be obtained either within (ie. constituting a step of) or separately to (ie. not constituting a step of) the methods of the invention. In one embodiment, the methods of the invention may comprise a step of establishing a reference value for the quantity of the markers. In one embodiment, the reference values are obtained separately to the method of the invention and accessed (e.g., on a database) during the comparison step of the invention.
As illustrated in
By comparing the presence and/or amount of markers quantified in a sample obtained from a test patient to the presence and/or amount of markers quantified for a reference value (such as that obtained from a population of healthy individuals, or from a population of individuals having sepsis (e.g., abdominal sepsis or pulmonary sepsis) or SIRS), it is possible to diagnose whether the patient has a systemic inflammatory condition (such as abdominal sepsis, pulmonary sepsis or SIRS). The method permits classification of the patient as belonging to or not belonging to the reference population (i.e., by determining whether the amounts of marker quantified in the patient are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the patient's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the patient falls (or does not fall) within the reference population.
In one embodiment, a patient may be diagnosed as having or being at risk of having a systemic inflammatory condition (such as sepsis (eg. abdominal sepsis or pulmonary sepsis) or SIRS), when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having sepsis (eg. abdominal sepsis or pulmonary sepsis) and/or the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having SIRS. In one embodiment, a patient may be diagnosed as not having or not being at risk of having a systemic inflammatory condition (such as sepsis (eg. abdominal sepsis or pulmonary sepsis) or SIRS) when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals).
As used herein, the term “statistically similar” means that the amount of the one or more biomarker quantified for the test patient is similar to the amount quantified for the reference population to a statistically significant level. The term “statistically significant” means that the alteration is greater than what might be expected to happen by chance alone (p=<0.05). Statistical significance can be determined by any method known in the art.
In one embodiment, a patient may be diagnosed as having or being at risk of having a systemic inflammatory condition (such as sepsis (e.g., abdominal sepsis or pulmonary sepsis) or SIRS) when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, a patient may be diagnosed as not having or not being at risk of having a systemic inflammatory condition (such as sepsis (e.g., abdominal sepsis or pulmonary sepsis) or SIRS) when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having sepsis (e.g., abdominal sepsis or pulmonary sepsis) and/or an individual (or a population of individuals) having SIRS.
As used herein, the term “statistically deviates” means that the amount of the one or more biomarker quantified for the test patient differs from the amount quantified for the reference population to a statistically significant level. The term “statistically significant” means that the alteration is greater than what might be expected to happen by chance alone (p=<0.05). Statistical significance can be determined by any method known in the art. The deviation in biomarker abundance may be an increase or decrease. The increase or decrease may be statistically significant.
The amount of the one or more biomarker of the invention, for example in a biomarker profile, may differ by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200% or more compared with a corresponding reference value.
The amount of the one or more biomarker of the invention, for example in a biomarker profile, may differ by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, or at least 50 fold as compared to a corresponding reference value.
For example, if the amount of the one or more biomarker of the invention, typically in a biomarker profile, is reduced compared with a corresponding reference value, the expression may be reduced partially or totally compared with the corresponding reference value. Typically, the amount is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, up to total elimination of the one or more biomarker. Typically, the amount is reduced by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, or at least 50 fold as compared to a corresponding reference value. For example, the fold decrease may be at least 0.5 fold. For example, the fold decrease may be at least 1 fold. For example, the fold decrease may be at least 1.5 fold. For example, the fold decrease may be at least 2 fold. For example, the fold decrease may be at least 2.5 fold. For example, the fold decrease may be at least 3 fold. For example, the fold decrease may be at least 3.5 fold.
For example, the fold decrease may be at least 4 fold. For example, the fold decrease may be at least 4.5 fold. For example, the fold decrease may be at least 5 fold. The decrease in the amount of the marker may be statistically significant.
For example, if the amount of one or more biomarker of the invention, typically in a biomarker profile, is increased compared with a corresponding reference value, the amount may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90&, at least 100%, at least 150%, at least 200% compared with the corresponding reference value. The amount may be increased by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, or at least 50 fold as compared to a corresponding reference value. For example, the fold increase may be at least 0.5 fold. For example, the fold increase may be at least 1 fold. For example, the fold increase may be at least 1.5 fold. For example, the fold increase may be at least 2 fold. For example, the fold increase may be at least 2.5 fold. For example, the fold increase may be at least 3 fold. For example, the fold increase may be at least 3.5 fold. For example, the fold increase may be at least 4 fold. For example, the fold increase may be at least 4.5 fold. For example, the fold increase may be at least 5 fold. The increase in the amount of the marker may be statistically significant.
The amount of the one or more biomarker may be altered compared with a corresponding reference value for at least 12 hours, at least 24 hours, at least 30 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks or more.
As described herein, the present inventors observed that all of biomarkers shown in Table 1 increased in abundance in samples obtained from patients having a systemic inflammatory condition, as compared to healthy individuals. Detecting elevated levels of one or more of these biomarkers in a patient can thus be used to diagnose the presence of a systemic inflammatory condition in a patient.
Thus, in one embodiment, when the reference value is representative of a healthy individual (or population of healthy individuals), an increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has or is at risk of having a systemic inflammatory condition. Likewise, no increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have a systemic inflammatory condition.
The present inventors observed that the overall increase in biomarker abundance observed in patients having a systemic inflammatory condition varied between different biomarkers, with some biomarkers showing very significant increases in abundance, and others showing more subtle changes.
In one embodiment, the patient may be diagnosed as having a systemic inflammatory condition, or being at risk of developing a systemic inflammatory condition, when the one or more biomarker (or the one or more additional biomarker) increases by at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual.
The ‘comparison’ step of the methods of the invention may comprise applying a decision rule or using a decision tree. A “decision rule” or a “decision tree” is a method used to classify individuals. This rule can take on one or more forms that are known in the art, as exemplified in Hastie et al., in “The Elements of Statistical Learning” Springer-Nerlag (Springer, New York (2001)). Analysis of biomarkers in the complex mixture of molecules within the sample generates features in a data set. A decision rule or a decision tree may be used to act on a data set of features to diagnose, monitor, or prognose a systemic inflammatory condition (such as sepsis or SIRS), to distinguish between sepsis and SIRS in a patient, or to distinguish between abdominal sepsis and pulmonary sepsis.
The decision rule or decision tree can comprise a data analysis algorithm, such as a computer pattern recognition algorithm. Other suitable algorithms include, but are not limited to, logistic regression or a nonparametric algorithm that detects differences in the distribution of feature values (e.g., a Wilcoxon Signed Rank Test). The decision rule may be based upon one, two, three, four, five, 10, 20 or more features. In one embodiment, the decision rule or decision tree is based on hundreds or more of features. Applying the decision rule or decision tree may also comprise using a classification tree algorithm. For example, the reference value (or reference biomarker profile) may comprise at least three features or biomarkers, where the features are predictors in a classification tree algorithm. The data analysis algorithm predicts membership within a population (or class) with an accuracy of at least about 60%, at least about 70%, at least about 80% and at least about 90%.
Suitable algorithms are known in the art, some of which are reviewed in Hastie et al., supra. Such algorithms classify complex spectra from biological materials, such as a blood sample, to distinguish individuals as normal or as possessing biomarker expression levels characteristic of a particular disease state. While such algorithms may be used to increase the speed and efficiency of the application of the decision rule and to avoid investigator bias, one of ordinary skill in the art will realise that computer-based algorithms are not required to carry out the methods of the present invention.
Algorithms may be applied to the comparison of the one or more biomarker or the biomarker profiles, regardless of the method that was used to generate the data for the one or more biomarker or the biomarker profile. For example, suitable algorithms can be applied to biomarker profiles generated using gas chromatography, as discussed in Harper, “Pyrolysis and GC in Polymer Analysis” Dekker, New York (1985). Further, Wagner et al., Anal. Chem. 74:1824-1835 (2002) disclose an algorithm that improves the ability to classify individuals based on spectra obtained by static time-of-flight secondary ion mass spectrometry (TOF-SIMS). Additionally, Bright et al., J. Microbiol. Methods 48: 127-38 (2002) disclose a method of distinguishing between bacterial strains with high certainty (79-89% correct classification rates) by analysis of MALDI-TOF-MS spectra. Dalluge, Fresenius J. Anal. Chem. 366: 701-711 (2000) discusses the use of MALDI-TOF-MS and liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) to classify profiles of biomarkers in complex biological samples.
The methods and uses of the invention may thus comprise applying a decision rule as described herein. Applying the decision rule may comprise using a data analysis algorithm, also as described herein. The data analysis algorithm may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 25, at least 50 or more input parameters. The data analysis algorithm may use any of the biomarkers of the invention, or combination of biomarkers of the invention as input parameters. Typically, the data analysis algorithm uses at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 25, at least 50 of the biomarkers of the invention (e.g., as listed in any one of Tables 1 to 4) as input parameters.
The application of the decision rule or the decision tree does not require perfect classification. A classification may be made with at least about 90% certainty, or even more, in one embodiment. In other embodiments, the certainty is at least about 80%, at least about 70%, or at least about 60%. The useful degree of certainty may vary, depending on the particular method of the present invention. “Certainty” is defined as the total number of accurately classified individuals divided by the total number of individuals subjected to classification. As used herein, “certainty” means “accuracy”.
Classification may also be characterized by its “sensitivity”. The “sensitivity” of classification relates to the percentage of individuals who were correctly identified as having a particular disease or condition e.g., the percentage of individuals who were correctly identified as having a systemic inflammatory condition (such as sepsis or SIRS). “Sensitivity” is defined in the art as the number of true positives divided by the sum of true positives and false negatives.
The “specificity” of a method is defined as the percentage of patients who were correctly identified as not having particular disease or condition, e.g., the percentage of individuals who were correctly identified as not having a systemic inflammatory condition (such as sepsis or SIRS). That is, “specificity” relates to the number of true negatives divided by the sum of true negatives and false positives.
Typically, the accuracy, sensitivity and/or specificity of the methods and uses of the invention is at least about 90%, at least about 80%, at least about 70% or at least about 60%.
The method for diagnosing a systemic inflammatory condition in a patient as described herein can be used in a decision tree process to investigate the health of a patient having or suspected of having a systemic inflammatory condition. For example, the method for diagnosing a systemic inflammatory condition in a patient can be performed before, after, or in addition to any of the other methods described herein.
In one embodiment, the method for diagnosing a systemic inflammatory condition in a patient is performed as described herein. If the patient tests positive for a systemic inflammatory condition, they may be tested using the method for distinguishing between sepsis and SIRS described herein to determine whether the patient has sepsis and/or SIRS. In one embodiment, the above combination of methods are performed as described, and if the patient tests positive for sepsis, the patient may be further tested for sepsis, abdominal sepsis and/or pulmonary sepsis using the diagnostic methods described herein, so as to confirm whether the patient has or is at risk of developing sepsis, and/or determine whether the patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis. If the patient tests positive for SIRS, the patient may be further tested for SIRS using the diagnostic method described herein, so as to confirm whether the patient has or is at risk of developing SIRS. If the patient tests positive for sepsis using the method for diagnosis of sepsis (as described herein), the patient may be further tested for abdominal sepsis and/or pulmonary sepsis using the methods described herein.
In one embodiment, the method of the invention for diagnosing a systemic inflammatory condition in a patient is performed as described herein. If the patient tests positive for a systemic inflammatory condition, they may be tested for sepsis, abdominal sepsis, pulmonary sepsis and/or SIRS using the diagnostic methods described herein. The methods for diagnosis of sepsis, abdominal sepsis, pulmonary sepsis and/or SIRS may be performed simultaneously or sequentially in any order.
The above described combination of methods may also be performed in parallel to determine the disease status of a patient by simultaneously (or substantially simultaneously) investigating the expression of all the biomarkers in a sample obtained from the patient, and determining whether the patient has or is at risk of having a systemic inflammatory condition, sepsis (such as abdominal or pulmonary sepsis) and/or SIRS.
When performing these different methods in a decision tree process, the sample used in each step of the method may be the same sample obtained from the patient. When the method comprises multiple quantification steps, these multiple steps may be performed at the same time (e.g., in parallel) and/or using the same sample. When the method comprises multiple comparison steps, these multiple steps may be performed at the same time (e.g., in parallel).
In all methods described herein, any appropriate technique may be used to confirm the diagnosis. Standard techniques are known in the art. For example, confirmation of a diagnosis of a systemic inflammatory condition in a patient may include: testing for the presence of other known biomarkers of a systemic inflammatory condition including: C-reactive protein (CRP), Procalcitonin (PCT), lactate, Cystatin C (CYTC), Neutrophil gelatinase-associated lipocalin (NGAL) and interleukin 6 (IL6).
Additional clinical parameters that may be used to confirm the diagnosis also include: white blood cell count, kidney function tests (such as serum creatinine, or urine output), respiratory system function tests (such as PaO2/FiO2), nervous system function tests (expressed as Glasgow coma scale), cardiovascular function tests (expressed as mean arterial pressure), liver function tests (such as bilirubin concentration), and coagulation function tests (such as platelet concentration). The methods and uses of the invention may further comprise determining such clinical parameters in the patient.
In a related aspect, the present invention also provides the use of one or more of: FAM20A, OLAH, CD177, ADM, IL10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1R2, CYP19A1, MMP8, TGFA and VSTM1 as a biomarker for a systemic inflammatory condition. For example, the one or more biomarker may be selected from: FAM20A, OLAH and/or CD177.
In one embodiment, the use is of the one or more biomarker in the diagnosis of a systemic inflammatory condition in a patient. For example, the use may comprise (i) determining the presence and/or amount of one or more biomarker in a sample obtained from a patient; and (ii) comparing the presence and/or amount of the one or more biomarker determined in said sample in (i) to a corresponding reference value to determine whether the patient has a systemic inflammatory condition.
All embodiments described above for the method of diagnosing a systemic inflammatory condition in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “systemic inflammatory condition”, “patient”, “sample”, and “the one or more biomarker”.
Diagnosis of SIRS
When investigating gene expression patterns in patients having systemic inflammatory conditions, the inventors observed that certain biomarkers (see Table 2) were particularly elevated in abundance in patients having SIRS, as compared to patients having other systemic inflammatory conditions, and healthy individuals. As a result of these findings, the inventors thus observed that PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3 can be used as biomarkers for diagnosis of SIRS.
The present invention therefore provides a method for diagnosing SIRS in a patient, comprising:
All embodiments described above for the “method for diagnosing a systemic inflammatory condition in a patient” apply equally to the “method for diagnosing SIRS in a patient”. This includes all embodiments relating to the “sample”, “patient”, “biomarker”, and “reference value”, and all embodiments relating to the quantification step for “determining the presence and/or amount of one or more biomarker in a sample” and the “comparison” step used to make a conclusion about the disease state of the patient.
As used herein, the phrase “diagnosis of SIRS in a patient” means determining whether the patient has or is risk of developing SIRS. The systemic inflammatory condition “SIRS” diagnosed using the method of the invention is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
The “patient” for which diagnosis is performed is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having a systemic inflammatory condition using the method described herein. In one embodiment, the patient has been diagnosed as having or being at risk of developing SIRS and/or sepsis using the method of the invention for distinguishing between sepsis and SIRS in a patient as described herein. In one embodiment, the patient is suspected of having or being at risk of developing SIRS.
The “sample” obtained from the patient is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”, including all embodiments relating to the time point at which the sample is obtained.
The “one or more biomarker” of the invention is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
As illustrated in Example 1, the present inventors observed that PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3 are elevated in patients having SIRS, and can thus be used as biomarkers for diagnosis of SIRS.
The reference to the biomarker MYCL throughout the entire description, includes the transcript variant 1 of MYCL (as encoded by SEQ ID NO: 37) and the transcript variant 3 of MYCL (as encoded by SEQ ID NO: 38). In one embodiment, the reference to the biomarker MYCL is a reference to the transcript variant 1 of MYCL (as encoded by SEQ ID NO: 37). In one embodiment, the reference to the biomarker MYCL is a reference to the transcript variant 3 of MYCL (as encoded by SEQ ID NO: 38).
In one embodiment, the one or more biomarker may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3. For example, the one or more biomarker may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, RBP4, and MPP3. For example, the one or more biomarker may be selected from the group consisting of: MYCL, TGFBI, GPR124, NLRP3, and MPP3. For example, the one or more biomarker may be selected from the group consisting of: ARHGEF10L, TGFBI, GPR124, IL1RN, NLRP3, RBP4, and MPP3. For example, the one or more biomarker may be selected from the group consisting of: TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3. For example, the one or more biomarker may be selected from the group consisting of: GPR124, IL1 RN, NLRP3, RBP4, and MPP3. For example, the one or more biomarker may be selected from the group consisting of: GPR124, NLRP3, and MPP3.
The present inventors observed that a sub-set of the biomarkers for SIRS (GPR124, TGFBI, PLA2G7, MYCL, and ARHGEF10L) increase in abundance in patients having SIRS compared to healthy individuals, but do not increase in abundance in patients having sepsis as compared to healthy individuals. These markers therefore provide highly specific biomarkers for diagnosing SIRS. Thus, in one embodiment, the one or more biomarker may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124. For example, the one or more biomarker may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
Each of the biomarkers of SIRS may be used alone, or in combination with any of the SIRS biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, up to and including all of the SIRS biomarkers may be used to diagnose SIRS in a patient according to the method of the invention.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) of the biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3, may be used to diagnose SIRS in a patient. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, or all 5) of the biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124, may be used to diagnose SIRS in a patient. For example, any combination of 1 or more (e.g., 2 or more, 3 or more, or all 4) of the biomarkers selected from the group consisting of: ARHGEF10L, MYCL, TGFBI, and GPR124 may be used to diagnose SIRS in a patient. For example, any combination of 1 or more (e.g., 2 or more, 3 or more, or all 4) of the biomarkers selected from the group consisting of: ARHGEF10L, MYCL, TGFBI, and GPR124, may be used to diagnose SIRS in a patient. For example, any combination of 1 or more (e.g., 2 or more, or all 3) of the biomarkers selected from the group consisting of: MYCL, TGFBI and GPR124, may be used to diagnose SIRS in a patient. For example, any combination of 1 or more (e.g., 2 or more, or all 3) of the biomarkers selected from the group consisting of: PLA2G7, TGFBI and GPR124, may be used to diagnose SIRS in a patient. For example, any combination of 1 or more (or both) of the biomarkers selected from the group consisting of: MYCL and GPR124, may be used to diagnose SIRS in a patient. For example, any combination of 1 or more (or both) of the biomarkers selected from the group consisting of: PLA2G7 and GPR124, may be used to diagnose SIRS in a patient.
In a further example, the following combinations of SIRS biomarkers may be used in the method of the invention to diagnose SIRS: (i) TGFBI and PLA2G7; (ii) TGFBI and GPR124; (iii) TGFBI and MYCL; (iv) TGFBI and ARHGEF10L; (v) PLA2G7 and GPR124; (vi) PLA2G7 and MYCL; (vii) PLA2G7 and ARHGEF10L; (viii) GPR124 and MYCL; (ix) GPR124 and ARHGEF10L; (x) MYCL and ARHGEF10L.
As described in Example 2, a subset of the SIRS biomarkers (PLA2G7, ARHGEF10L, MYCL, and TGFBI) were shown to be particularly effective in diagnosing SIRS when tested by ROC analysis. Specifically, AUC values of 0.89, 0.8, 0.8, 0.79 were achieved for PLA2G7, ARHGEF10L, MYCL, and TGFBI. Thus, in one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, or all 4) of the biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI, may be used to diagnose SIRS in a patient. For example, 2 or more of the biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI, may be used to diagnose SIRS in a patient. For example, 3 or more of the biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI, may be used to diagnose SIRS in a patient. In one embodiment, the method of the invention may be preferably performed using the combination of: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
In one embodiment, the one or more biomarker is TGFBI. In one embodiment, the one or more biomarker is PLA2G7. In one embodiment, the one or more biomarker is MYCL. In one embodiment, the one or more biomarker is ARHGEF10L. In one embodiment, the one or more biomarker is GPR124. In one embodiment, the one or more biomarker is IL1 RN. In one embodiment, the one or more biomarker is NLRP3. In one embodiment, the one or more biomarker is RBP4. In one embodiment, the one or more biomarker is MPP3.
One or more additional biomarker for SIRS may also be used in the diagnosis of SIRS according to the method of the invention. Any combination of the one or more additional biomarker may be used in combination with the one or more biomarker of the invention. For example at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 8 additional biomakers for SIRS may be used in combination with the one or more biomarker of the invention (as described herein). Typically, the one or more additional biomarker is selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3. For example, the one or more additional biomarker is selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI and GPR124.
In one embodiment, the one or more biomarker is TGFBI, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, GPR124, IL1RN, NLRP3, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, and GPR124. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L and MYCL.
In one embodiment, the one or more biomarker is PLA2G7, and the one or more additional biomarker is selected from at least 1 (eg. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3.
In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: ARHGEF10L, MYCL, TGFBI, and GPR124. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, up to and including all) of the biomarkers: ARHGEF10L, MYCL and TGFBI.
In one embodiment, the one or more biomarker is MYCL, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, TGFBI, GPR124, IL1RN, NLRP3, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, TGFBI and GPR124. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L and TGFBI.
In one embodiment, the one or more biomarker is ARHGEF10L, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, MYCL, TGFBI, GPR124, IL1RN, NLRP3, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: PLA2G7, MYCL, TGFBI, and GPR124. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, up to and including all) of the biomarkers: PLA2G7, MYCL and TGFBI.
In one embodiment, the one or more biomarker is GPR124, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, IL1 RN, NLRP3, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
In one embodiment, the one or more biomarker is IL1 RN, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, NLRP3, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124.
In one embodiment, the one or more biomarker is NLRP3, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124.
In one embodiment, the one or more biomarker is RBP4, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1RN, NLRP3, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124.
In one embodiment, the one or more biomarker is MPP3, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1RN, NLRP3, and RBP4. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124.
As illustrated in
For example, by comparing the presence and/or amount of markers quantified in a sample obtained from a patient to the presence and/or amount of markers quantified for a reference value (such as a reference value that is representative of a healthy individual (or a population of healthy individuals), a reference value that is representative of an individual (or a population of individuals) having sepsis (e.g., abdominal and/or pulmonary sepsis), or a reference value that is representative of an individual (or a population of individuals) having SIRS, it is possible to diagnose the presence (or absence) of SIRS in a patient. The method permits classification of the patient as belonging to or not belonging to the reference population (i.e., by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the patient's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the patient falls (or does not fall) within the reference population.
In one embodiment, a patient may be diagnosed as having or being at risk of having SIRS, when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having SIRS. In one embodiment, a patient may be diagnosed as not having or not being at risk of having SIRS when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, a patient may be diagnosed as not having or not being at risk of having SIRS when the amount of the one or more biomarker quantified is statistically similar to the amount determined for the corresponding reference value representative of an individual having sepsis (or a population of individuals having sepsis).
In one embodiment, a patient may be diagnosed as having or being at risk of having SIRS when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, a patient may be diagnosed as having or being at risk of having SIRS when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual having sepsis (or a population of individuals having sepsis). In one embodiment, a patient may be diagnosed as not having or not being at risk of having SIRS when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having SIRS.
All embodiments described above for the classification of a patient as having or being at risk of having a systemic inflammatory condition (or as not having or not being at risk of having a systemic inflammatory condition) apply equally to the method for diagnosing whether a patient has or is at risk of having SIRS. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may as defined above for the method of diagnosing a systemic inflammatory condition in a patient. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value is representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, the reference value is representative of an individual having sepsis (or a population of individuals having sepsis).
As described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the method of the invention may involve the use of multiple separate reference values. For example, the reference value may include one of more (e.g., two or more, or all 3) of the reference values selected from: a reference value that is representative of a healthy individual (or a population of healthy individuals); a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS); and a reference value that is representative of an individual having sepsis (or a population of individuals having sepsis).
The present inventors observed that the SIRS biomarkers described herein each increase in abundance in samples obtained from patients having SIRS, as compared to healthy individuals. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of SIRS. In one embodiment, when the reference value is representative of a healthy individual (or population of healthy individuals), an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value indicates that the patient has or is at risk of developing SIRS. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value indicates that the patient does not have SIRS.
For some of the SIRS biomarkers identified by the present inventors, increased levels of these markers were also observed in patients having sepsis as compared to healthy individuals, although much bigger increases were observed for these biomarkers in the patients having SIRS. The accuracy of SIRS diagnosis can thus be improved by looking for a “minimum” fold change or % change in the levels of the one or more biomarkers as compared to the corresponding reference value that is representative of a healthy individual. The fold change or % change may be as defined above for the method for diagnosis of a systemic inflammatory condition.
In one embodiment, the patient may be diagnosed as having SIRS, or being at risk of developing SIRS, when the one or more biomarker (or the one or more additional biomarker) increases by at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual.
For example, an increase of at least 1.1 (e.g., at least 1.2, at least 1.3, at least 1.4, at least 1.5) fold in GPR124 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 1.6) fold in GPR124 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
For example, an increase of at least 1.5 (e.g., at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2) fold in TGFBI in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase or an increase of less than 1.5 (e.g., less than 1.6, less than 1.7, or less than 1.8) fold in TGFBI in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
For example, an increase of at least 1.1 (e.g., at least 1.2, at least 1.3, at least 1.4, at least 1.5, or at least 1.6) fold in PLA2G7 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, less than 1.5, or less than 1.6) fold in PLA2G7 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.1 (e.g., at least 1.2, at least 1.3, or at least 1.4) fold in MYCL in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase or an increase of less than 1.1 (e.g., less than 1.2, or less than 1.3, or less than 1.4) fold in MYCL in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS. In one embodiment, when detecting this biomarker, the method is performed using a sample obtained from a patient at least 24 (e.g., at least 36, at least 48, at least 72, at least 96, or at least 120) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.1 (e.g., at least 1.2, at least 1.3, at least 1.4, at least 1.5) fold in ARHGEF10L in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase, or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, or less than 1.5) fold in ARHGEF10L in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
For example, an increase of at least 4 (e.g., at least 4.1, at least 4.2, at least 4.3, at least 4.4, or at least 4.5) fold in IL1 RN in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase, or an increase of less than 4 (e.g., less than 4.1, less than 4.2, less than 4.3, less than 4.4, or less than 4.5) fold in IL1 RN in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
For example, an increase of at least 2 (e.g., at least 2.1, at least 2.1, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3) fold in NLRP3 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase, or an increase of less than 2 (e.g., less than 2.1, less than 2.2, less than 2.3, less than 2.4, or less than 2.5) fold in NLRP3 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
For example, an increase of at least 3.5 (e.g., at least 3.6, at least 3.7, at least 3.8, at least 3.9, or at least 4) fold in RBP4 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase, or an increase of less than 3.5 (eg. less than 3.6, less than 3.7, less than 3.8, less than 3.9, or less than 4,) fold in RBP4 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS. In one embodiment, when detecting this biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 2 (e.g., at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5) fold in MPP3 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase, or an increase of less than 2 (e.g., less than 2.1, less than 2.2, less than 2.3, less than 2.4, less than 2.5) fold in MPP3 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
As described herein, the present inventors observed that the levels of the one or more SIRS biomarkers were elevated in patients having SIRS as compared to patients having sepsis. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients having sepsis can thus be used to diagnose the presence of SIRS.
Thus, in one embodiment, the reference value used in the method of the invention is representative of an individual (or population of individuals) having sepsis (such as abdominal sepsis and/or pulmonary sepsis). The reference value that is representative of an individual having sepsis is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
In one embodiment, an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having sepsis, indicates that the patient has or is at risk of developing SIRS. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having sepsis, indicates that the patient does not have SIRS.
In one embodiment, the patient may be diagnosed as having SIRS, or being at risk of developing SIRS, when the one or more biomarker (or the one or more additional biomarker) increases by at least 1 (e.g. at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value of an individual having sepsis.
The method for diagnosis of SIRS as described herein can be used in a decision tree process to investigate the health of a patient having or suspected of having a systemic inflammatory condition. For example, the method for diagnosis of SIRS in a patient can be performed before, after, or in addition to any of the other methods described herein.
In one embodiment, the method for diagnosing SIRS in a patient (as described herein) can be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). If the patient tests positive for a systemic inflammatory condition (using the method of the invention for diagnosing whether a patient has a systemic inflammatory condition), they may be tested for SIRS using the diagnostic method described herein. Furthermore, the method for diagnosis of SIRS may be performed before, after, or in addition to the method for diagnosis of sepsis in a patient, as described herein.
In one embodiment, the method of the invention for diagnosing SIRS in a patient (as described herein) can be performed subsequent to (or in addition to) the method for distinguishing between sepsis and SIRS in a patient (as described herein). If the patient tests positive for SIRS using the distinguishing method of the invention, they may then be tested for SIRS using the diagnostic method described herein, so as to further confirm the diagnosis of SIRS in the patient.
In one embodiment, the method for diagnosis of SIRS may be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein), and the method for distinguishing between sepsis and SIRS in a patient (as described herein). For example, the patient may be tested first using the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). If the patient tests positive for a systemic inflammatory condition, they may be tested using the distinguishing method of the invention (as described herein) to determine whether they have sepsis and/or SIRS. If the patient tests positive for SIRS using the distinguishing method of the invention, they may be tested for SIRS using the diagnostic method described herein, so as to further confirm the diagnosis of SIRS in the patient. Furthermore, the method for diagnosis of SIRS may be performed before, after, or in addition to the method for diagnosis of sepsis in a patient, as described herein.
The methods for diagnosis of a systemic inflammatory condition, sepsis, abdominal sepsis, pulmonary sepsis and/or SIRS may be performed simultaneously or sequentially in any order. The above described combination of methods may be performed in parallel to determine the disease status of a patient by simultaneously (or substantially simultaneously) investigating the expression of all the biomarkers in a sample obtained from the patient, and determining whether the patient has or is at risk of having a systemic inflammatory condition, sepsis (such as abdominal or pulmonary sepsis) and/or SIRS.
When performing these different methods in a decision tree process, the sample used in each step of the method may be the same sample obtained from the patient (as described herein). When the method comprises multiple quantification steps, all the steps may be performed at the same time (e.g., in parallel) and/or using the same sample.
In a related aspect, the present invention also provides the use of one or more of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3, as a biomarker for SIRS. In one embodiment, the use is of the one or more biomarker in the diagnosis of SIRS in a patient. In one embodiment, the use is of one or more of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124. In one embodiment, the use is of one or more of: PLA2G7, ARHGEF10L, MYCL, and TGFBI. For example, the use may be of the combination of: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
For example, the use may comprise (i) determining the presence and/or amount of one or more biomarker in a sample obtained from a patient; and (ii) comparing the presence and/or amount of the one or more biomarker determined in said sample in (i) to a corresponding reference value to determine whether the patient has or is at risk of developing SIRS.
All embodiments described above for the method of diagnosing SIRS in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “SIRS”, “patient”, “sample”, and “the one or more biomarker”.
Diagnosis of Sepsis
When investigating gene expression patterns in patients having systemic inflammatory conditions, the inventors identified a subset of biomarkers (see Table 3) that were expressed at different levels in patients having sepsis, as compared to patients having other systemic inflammatory conditions, and healthy individuals. As a result of these findings, the inventors thus observed that ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, CLEC1B, MRAS, PCOLCE2, TMEM37, SLC39A8, KIF2C, CIQC, CIQB, CIQA, TNF, IFI44, IFIT1, RPGRIP1, EPSTI1, DISC1, CXCR1, and HCAR2, can be used as biomarkers for diagnosis of sepsis.
In particular, the present inventors have identified that ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B, are elevated in all types of sepsis tested, and thus can be used as biomarkers for sepsis including abdominal sepsis and pulmonary sepsis.
The inventors also observed that the levels of SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF are elevated in patients having abdominal sepsis, compared to patients having pulmonary sepsis or SIRS, and healthy individuals. The inventors also observed that the levels of IFI44, IFIT1, and RPGRIP1 were decreased in patients having abdominal sepsis, compared to patients having pulmonary sepsis or SIRS, and healthy individuals. SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1 can thus be used as biomarkers for abdominal sepsis. Likewise, the inventors observed that the levels of HCAR2, CXCR1, DISC1, EPSTI1, and IFI44 are elevated in patients having pulmonary sepsis, compared to patients having abdominal sepsis and/or SIRS, and healthy individuals. HCAR2, CXCR1, DISC1, EPSTI1, and IFI44 can thus be used as biomarkers for pulmonary sepsis.
The present invention therefore provides a method for diagnosing sepsis in a patient, comprising:
All embodiments described above for the “method for diagnosing a systemic inflammatory condition in a patient” apply equally to the “method for diagnosing sepsis in a patient”. This includes all embodiments relating to the “sample”, “patient”, “biomarker”, and “reference value”, and all embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step for making a conclusion about the disease status of the patient.
As used herein, the phrase “diagnosis of sepsis in a patient” means determining whether the patient has or is risk of developing sepsis. The systemic inflammatory condition “sepsis” diagnosed using the method of the invention is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”. In one embodiment, the method is for diagnosing one or more of: abdominal sepsis and pulmonary sepsis. In one embodiment, the method is for diagnosing abdominal sepsis. In one embodiment, the method is for diagnosing pulmonary sepsis.
The “patient” for which diagnosis is performed is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having a systemic inflammatory condition using the method described herein. In one embodiment, the patient is suspected of having or being at risk of developing sepsis. In one embodiment, the patient has been diagnosed as having or being at risk of developing sepsis using the method described herein for distinguishing between sepsis and SIRS in a patient.
The “sample” obtained from the patient is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”, including all embodiments relating to the time point at which the sample is obtained.
The optimum time point at which a sample is obtained from a patient may depend on the biomarker being tested. For example, when testing for any one or more of the biomarkers MAP1A, SELP, NEXN, ITGA2B, MYL9, CMTM5, PPBP, TREML1, PF4, CLEC1B or ITGB3, the sample may be obtained up to 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition, or is admitted to a medical care facility. Preferably, the sample is obtained up to 24 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. Preferably, the sample is obtained up to 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The “one or more biomarker” of the invention is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
As illustrated in Example 1, the present inventors observed that ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, CLEC1B, SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, RPGRIP1, HCAR2, CXCR1, DISC1, and EPSTI1, are biomarkers of sepsis, and thus can be used in the diagnosis of sepsis.
The reference to the biomarker SLC39A8 throughout the entire description, includes the transcript variant 1 of SLC39A8 (as encoded by SEQ ID NO: 70) and the transcript variant 3 of SLC39A8 (as encoded by SEQ ID NO: 71). In one embodiment, the reference to the biomarker SLC39A8 is a reference to the transcript variant 1 of SLC39A8 (as encoded by SEQ ID NO: 70). In one embodiment, the reference to the biomarker SLC39A8 is a reference to the transcript variant 3 of SLC39A8 (as encoded by SEQ ID NO: 71).
In one embodiment, the one or more biomarker may be selected from the group consisting of ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, CLEC1B, SLC39A8, CIQC CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, RPGRIP1, HCAR2, CXCR1, DISC1, and EPST11.
In one embodiment, the one or more biomarker may be selected from the group consisting of ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, MAP1A, SELP, NLRC4, IFI44, HCAR2, CXCR1, DISC1, and EPSTI1. When detecting one or more biomarker selected from this sub-group, the systemic inflammatory condition diagnosed using the method may be pulmonary sepsis.
The present inventors observed that a sub-set of the biomarkers for sepsis specifically increased in abundance in all types of sepsis tested (including abdominal and pulmonary sepsis) as compared to healthy individuals and patients having SIRS. These markers are therefore useful for diagnosis of sepsis in a patient. In one embodiment, the one or more biomarker may be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B.
The present inventors also observed that a sub-set of the biomarkers for sepsis (ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4) increase in abundance in patients having sepsis as compared to healthy individuals, and show no increase (or a decrease) in patients having SIRS as compared to healthy individuals (e.g., in patients tested at days 1 and 2 post-hospitalisation). These markers therefore provide highly specific biomarkers for diagnosing sepsis. Thus, in one embodiment, the one or more biomarker may therefore be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4.
Furthermore, of this sub-set of sepsis biomarkers, the inventors also observed that the specific biomarkers ITGB3, ITGA2B, MYL9, LCN2, and TREML1 were particularly effective at diagnosing sepsis when tested using ROC analysis, as described in Example 2. Specifically, AUC values of 0.86, 0.83, 0.82, 0.82 and 0.8 were observed for ITGB3, ITGA2B, MYL9, LCN2, and TREML1. Thus, in one embodiment, the one or more biomarker is preferably selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1.
Each of the biomarkers of sepsis may be used alone, or in combination with any of the sepsis biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, up to and including all of the sepsis biomarkers may be used to diagnose sepsis in a patient according to the method of the invention.
In one embodiment, the one or more biomarker is LCN2. In one embodiment, the one or more biomarker is ITGA2B. In one embodiment, the one or more biomarker is MYL9. In one embodiment, the one or more biomarker is ITGB3. In one embodiment, the one or more biomarker is TREML1. In one embodiment, the one or more biomarker is LCN15. In one embodiment, the one or more biomarker is CMTM5. In one embodiment, the one or more biomarker is PPBP. In one embodiment, the one or more biomarker is PF4. In one embodiment, the one or more biomarker is KIF2C. In one embodiment, the one or more biomarker is MAP1A. In one embodiment, the one or more biomarker is SELP. In one embodiment, the one or more biomarker is NEXN. In one embodiment, the one or more biomarker is NLRC4. In one embodiment, the one or more biomarker is CLEC1B. In one embodiment, the one or more biomarker is MRAS. In one embodiment, the one or more biomarker is CIQC. In one embodiment, the one or more biomarker is CIQB. In one embodiment, the one or more biomarker is PCOLCE2. In one embodiment, the one or more biomarker is CIQA. In one embodiment, the one or more biomarker is TMEM37. In one embodiment, the one or more biomarker is SLC39A8. In one embodiment, the one or more biomarker is TNF. In one embodiment, the one or more biomarker is IFI44. In one embodiment, the one or more biomarker is IFIT1. In one embodiment, the one or more biomarker is RPGRIP1. In one embodiment, the one or more biomarker is EPSTI1. In one embodiment, the one or more biomarker is DISC1. In one embodiment, the one or more biomarker is CXCR1. In one embodiment, the one or more biomarker is HCAR2.
In one embodiment, any combination of 1 or more (eg. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, or all 29) of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, CLEC1B, SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, RPGRIP1, HCAR2, CXCR1, DISC1, and EPSTI1, may be used to diagnose sepsis in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or all 15) of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B, may be used to diagnose sepsis in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4, may be used to diagnose sepsis in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, or all 5) of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1, may be used to diagnose sepsis in a patient. For example, the method may be performed using 2 or more of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using 3 or more of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using 4 or more of the biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using all 5 biomarkers: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. This combination of sepsis biomarkers was shown to be particularly effective in diagnosing sepsis when tested by ROC analysis described in Example 2.
For example, the following combinations of sepsis biomarkers may be used in the method of the invention to diagnose sepsis: (i) LCN15 and ITGA2B; (ii) LCN15 and MYL9; (iii) LCN15 and CMTM5; (iv) LCN15 and PPBP; (v) LCN15 and TREML1; (vi) LCN15 and PF4; (vii) LCN15 and LCN2; (viii) LCN15 and ITGB3; (ix) ITGA2B and MYL9; (x) ITGA2B and CMTM5; (xi) ITGA2B and PPBP; (xii) ITGA2B and TREML1; (xiii) ITGA2B and PF4; (xiv) ITGA2B and LCN2; (xv) ITGA2B and ITGB3; (xvi) MYL9 and CMTM5; (xvii) MYL9 and PPBP; (xviii) MYL9 and TREML1; (xix) MYL9 and PF4; (xx) MYL9 and LCN2; (xxi) MYL9 and ITGB3; (xxii) CMTM5 and PPBP; (xxiii) CMTM5 and TREML1; (xxiv) CMTM5 and PF4; (xxv) CMTM5 and LCN2; (xxvi) CMTM5 and ITGB3; (xxvii) PPBP and TREML1; (xxviii) PPBP and PF4; (xxix) PPBP and LCN2; (xxx) PPBP and ITGB3; (xxxi) TREML1 and PF4; (xxxii) TREML1 and LCN2; (xxxiii) TREML1 and ITGB3; (xxxiv) PF4 and LCN2; (xxxv) PF4 and ITGB3; and (xxxvi) LCN2 and ITGB3.
One or more additional biomarker for sepsis may also be used in the diagnosis of sepsis according to the method of the invention. Any combination of the one or more additional biomarker may be used in combination with the one or more biomarker of the invention. For example at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 additional biomakers for sepsis may be used in combination with the one or more biomarker of the invention (as described herein). Typically, the one or more additional biomarker is selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. For example, the one or more additional biomarker may be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4. For example, the one or more additional biomarker may be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1.
In one embodiment, the one or more biomarker is LCN2, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, TREML1, LCN15, CMTM5, PPBP, and PF4. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, and TREML1.
In one embodiment, the one or more biomarker is ITGA2B, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (eg. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, MYL9, LCN2, TREML1, LCN15, MYL9, ITGB3, CMTM5, PPBP, and PF4. In one embodiment, the one or more additional biomarker is selected from at least 1 (eg. at least 2, at least 3, up to and including all) of the biomarkers: ITGB3, MYL9, LCN2, and TREML1.
In one embodiment, the one or more biomarker is MYL9, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, LCN2, TREML1, LCN15, ITGA2B, ITGB3, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, LCN2, TREML1, LCN15, ITGA2B, ITGB3, CMTM5, PPBP, and PF4. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: ITGB3, ITGA2B, LCN2, and TREML1.
In one embodiment, the one or more biomarker is ITGB3, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, CMTM5, PPBP, and PF4. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: ITGA2B, MYL9, LCN2, and TREML1.
In one embodiment, the one or more biomarker is TREML1, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, LCN15, CMTM5, PPBP, and PF4. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, and LCN2.
In one embodiment, the one or more biomarker is LCN15, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2 TREML1, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, CMTM5, PPBP, and PF4.
In one embodiment, the one or more biomarker is CMTM5, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, PPBP, and PF4.
In one embodiment, the one or more biomarker is PPBP, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, and PF4.
In one embodiment, the one or more biomarker is PF4, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, and PPBP.
In one embodiment, the one or more biomarker is KIF2C, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, LCN15, TREML1, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, PF4, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, and PF4.
In one embodiment, the one or more biomarker is MAP1A, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, PF4, KIF2C, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, and PF4.
In one embodiment, the one or more biomarker is SELP, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, PF4, KIF2C, MAP1A, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, LCN15, TREML1, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, and PF4.
In one embodiment, the one or more biomarker is NEXN, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, and PF4.
In one embodiment, the one or more biomarker is NLRC4, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, and PF4.
In one embodiment, the one or more biomarker is CLEC1B, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, and NLRC4. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, and PF4.
As illustrated in
For example, by comparing the presence and/or amounts of markers quantified in a sample obtained from a patient to the presence and/or amounts of markers quantified for a reference value (such as a reference value that is representative of a healthy individual (or a population of healthy individuals), or a reference value that is representative of an individual (or a population of individuals) having sepsis e.g., abdominal and/or pulmonary sepsis, or a reference value that is representative of an individual (or a population of individuals) having SIRS, it is possible to diagnose the presence (or absence) of sepsis in a patient. The method permits classification of the individual as belonging to or not belonging to the reference population (i.e., by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the individual's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the individual falls (or does not fall) within the reference population.
In one embodiment, an individual may be diagnosed as having or being at risk of having sepsis, when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having sepsis. In one embodiment, an individual may be diagnosed as not having or not being at risk of having sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, an individual may be diagnosed as not having or not being at risk of having sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual having SIRS (or a population of individuals having SIRS).
In one embodiment, an individual may be diagnosed as not having or not being at risk of having sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference values representative of an individual (or a population of individuals) having sepsis. In one embodiment, an individual may be diagnosed as having or being at risk of having sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, an individual may be diagnosed as having or being at risk of having sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference values representative of an individual having SIRS (or a population of individuals having SIRS).
All embodiments described above for the classification of a patient as having or being at risk of having a systemic inflammatory condition (or as not having or not being at risk of having a systemic inflammatory condition) apply equally to the method for diagnosing whether a patient has or is at risk of having sepsis. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may be as defined above for the method of diagnosing a systemic inflammatory condition in a patient. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value is representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, the reference value is representative of an individual having sepsis (or a population of individuals having sepsis). As described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the method of the invention may involve the use of multiple separate reference values. For example, the reference value may include one of more (e.g., two or more, or all 3) of the reference values selected from: a reference value that is representative of a healthy individual (or a population of healthy individuals); a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS); and a reference value that is representative of an individual having sepsis (or a population of individuals having sepsis). The reference value that is representative of an individual having sepsis (or a population of individuals having sepsis) may be representative of an individual (or a population of individuals) having abdominal sepsis and/or pulmonary sepsis.
The present inventors observed that the sepsis biomarkers described herein each increase in abundance in samples obtained from patients having sepsis, as compared to healthy individuals. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of sepsis. In one embodiment, when the reference value is representative of a healthy individual (or population of healthy individuals), an increase in the one or more biomarker for sepsis in the sample obtained from the patient relative to the corresponding reference value indicates that the patient has sepsis or is at risk of developing sepsis. Likewise, no increase in the one or more biomarker for sepsis in the sample obtained from the patient relative to the corresponding reference value indicates that the patient does not have sepsis.
For some of the sepsis biomarkers identified by the present inventors, increased levels of these markers were also observed in patients having SIRS as compared to healthy individuals, although much bigger increases were observed for these biomarkers in the patients having sepsis. The accuracy of sepsis diagnosis can thus be improved by looking for a “minimum” fold change or % change in the levels of the one or more biomarkers as compared to the corresponding reference value that is representative of a healthy individual. The fold change or % change may be as defined above for the method for diagnosis of a systemic inflammatory condition.
In one embodiment, the patient is diagnosed as having sepsis, or being at risk of developing sepsis, when the one or more biomarker for sepsis (or the one or more additional biomarker) increases by at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, or at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual.
For example, an increase of at least 1 (e.g., at least 1.1, at least 1.2, at least 1.3, at least 1.4, or at least 1.5) fold in LCN15 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1 (e.g., less than 1.1, less than 1.2, less than 1.3, less than 1.4, less than 1.5) fold in LCN15 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis.
For example, an increase of at least 2 (e.g., at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5) fold in LCN2 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 2.1 (e.g., less than 2.2, less than 2.3) fold in LCN2 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis.
For example, an increase of at least 1.5 (e.g., at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.5, at least 3, or at least 3.5) fold in ITGA2B in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 1.6, less than 1.7, less than 1.8, less than 1.9, less than 2, less than 2.5, or less than 3) fold in ITGA2B in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1 (e.g., at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or at least 2.5) fold in MYL9 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase or an increase of less than 1 (e.g., less than 1.1, less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 2) fold in MYL9 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 2 (e.g., at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5) fold in ITGB3 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase or an increase of less than 2.1 (e.g., less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2) fold in ITGB3 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.1 (e.g., at least 1.2 at least 1.3, at least 1.4, at least 1.5, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or at least 2.5) fold in CMTM5 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, less than 1.5) fold in CMTM5 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition, or is admitted to a medical care facility.
For example, an increase of at least 1 (e.g., at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2 or at least 2.5) fold in PPBP in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase or an increase of less than 1 (e.g., less than 1.1, less than 1.2, less than 1.3) fold in PPBP in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1 (e.g., at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or at least 2.5) fold in TREML1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1 (e.g., less than 1.1, less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 2) fold in TREML1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1 (e.g., at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2) fold in PF4 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1 (e.g., less than 1.1, less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 1.6, less than 1.7, or less than 1.8) fold in PF4 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.5 (e.g., at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5) fold in KIF2C in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis (such as abdominal sepsis). In one embodiment, no increase, or an increase of less than 1.5 (e.g., less than 1.6, less than 1.7, less than 1.8, less than 1.9, less than 2) fold in KIF2C in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis (such as abdominal sepsis). In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient at least 36 (e.g., at least 48) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient between about 24 and 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, when detecting this level of fold change in the biomarker, the method is for diagnosing abdominal sepsis in a patient.
For example, an increase of at least 1.2 (e.g., at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or at least 2.5) fold in MAP1A in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1.2 (e.g., less than 1.3, less than 1.4, less than 1.5, less than 1.6, less than 1.7, less than 1.8) fold in MAP1A in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.5 (e.g., at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, or at least 3) fold in SELP in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1.2 (e.g., less than 1.3, less than 1.4, less than 1.5, less than 2) fold in SELP in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.8 (e.g., at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or at least 2.5) fold in the amount of NEXN in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 1.6, less than 1.7, less than 1.8, less than 1.9, less than 2) fold in NEXN in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 3.2 (e.g., at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4) fold in NLRC4 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 2.5 (e.g., less than 2.6, less than 2.7, less than 2.8, less than 2.9, less than 3, less than 3.1, less than 3.2) fold in NLRC4 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 2.7 (e.g., at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 4) fold in CLEC1B in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1.8 (e.g., less than 1.9, less than 2, less than 2.1, less than 2.2, less than 2.3, less than 2.4, less than 2.5, less than 2.6, less than 2.7, less than 2.8, less than 2.9, less than 3) fold in CLEC1B in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
As described herein, the present inventors observed that the levels of the one or more sepsis biomarkers were elevated in patients having sepsis as compared to patients having SIRS. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients having SIRS can thus be used to diagnose the presence of sepsis. Thus, in one embodiment, when the reference value is representative of an individual having SIRS, an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient has or is at risk of developing sepsis. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient does not have or is not at risk of having sepsis. In one embodiment, the increase may be a minimum fold increase or a minimum % increase as defined above for the method for diagnosis of a systemic inflammatory condition.
In a related aspect, the present invention provides the use of one or more of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, CLEC1B, SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, RPGRIP1, HCAR2, CXCR1, DISC1, and EPSTI1, as a biomarker for sepsis. For example, the use is of one or more of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. For example, the use is of one or more of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4. For example, the use is of one or more of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the use is of the combination of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. In one embodiment, the use is of the one or more biomarker in the diagnosis of sepsis in a patient. In one embodiment, the sepsis is abdominal sepsis and/or pulmonary sepsis. For example, the use may comprise (i) determining the presence and/or amount of one or more biomarker in a sample obtained from a patient; and (ii) comparing the presence and/or amount of the one or more biomarker determined in said sample in (i) to a corresponding reference value to determine whether the patient has sepsis.
All embodiments described above for the method of diagnosing sepsis in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “sepsis”, “patient”, “sample”, and “the one or more biomarker” described above.
As discussed herein, the present inventors observed that MRAS, PCOLCE2, TMEM37, SLC39A8, KIF2C, CIQC, CIQB, CIQA, TNF, IFI44, IFIT1, and RPGRIP1, are biomarkers specific for abdominal sepsis (see Table 3).
The present invention thus also provides a method for diagnosing abdominal sepsis in a patient, comprising:
All embodiments described above for the “method for diagnosing a systemic inflammatory condition in a patient” apply equally to the “method for diagnosing abdominal sepsis in a patient”. This includes all embodiments relating to the “sample”, “patient”, “biomarker”, and “reference value”, and all embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step for making a conclusion about the disease status of the patient.
As used herein, the phrase “diagnosis of abdominal sepsis in a patient” means determining whether the patient has or is risk of developing abdominal sepsis. The systemic inflammatory condition “abdominal sepsis” diagnosed using the method of the invention is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
The “patient” for which diagnosis is performed is as described above for the “method for diagnosing a systemic inflammatory condition in a patient” and “the method for diagnosing sepsis in a patient”. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having a systemic inflammatory condition using the method described herein. In one embodiment, the patient is suspected of having or being at risk of developing sepsis. In one embodiment, the patient has been diagnosed as having or being at risk of developing sepsis (eg. using the method of diagnosing sepsis in a patient as described herein and/or using the method of distinguishing between sepsis and SIRS in a patient as described herein). In one embodiment, the patient is suspected of having or being at risk of developing abdominal sepsis.
The “sample” obtained from the patient is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”, including all embodiments relating to the time point at which the sample is obtained. All embodiments of the “sample” defined above for the method for diagnosing sepsis also apply to the method for diagnosing pulmonary sepsis.
The optimum time point at which a sample is obtained from a patient may depend on the biomarker being tested. For example, when testing for the biomarkers CIQC, CIQB, CIQA, and MRAS, the sample may be obtained up to 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the sample is obtained up to 24 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the sample is obtained up to 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The “one or more biomarker” of the invention is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”.
As illustrated in Example 1, the present inventors observed that SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2 KIF2C, TNF, IFI44, IFIT1, and RPGRIP1 are biomarkers of abdominal sepsis, and thus can be used in the diagnosis of abdominal sepsis.
In one embodiment, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1.
A sub-set of the biomarkers identified (IFI44, IFIT1, and RPGRIP1) were found at decreased levels in abdominal sepsis patients as compared to healthy individuals, individuals having SIRS, and/or individuals having pulmonary sepsis. In one embodiment, the one or more biomarker may be selected from the group consisting of: IFI44, IFIT1, and RPGRIP1.
A sub-set of the biomarkers identified were found at elevated levels in abdominal sepsis patients as compared to healthy individuals, individuals having SIRS, and/or individuals having pulmonary sepsis. In one embodiment, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF.
As described Example 2, a sub-set of the markers tested (SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB) was observed to provide particularly accurate diagnosis of abdominal sepsis (see the ROC curve data presented in Example 2). In one embodiment, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB. In one embodiment, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, and CIQA.
The present inventors observed that the biomarker TNF is elevated in patients having abdominal sepsis as compared to healthy individuals and patients having pulmonary sepsis. However, the inventors observed that TNF is also elevated in patients having SIRS, and thus this marker is most useful in diagnosing abdominal sepsis when a patient has already been diagnosed as having sepsis (eg. using the methods described herein for diagnosis of sepsis, or using the method described herein for distinguishing between abdominal sepsis and pulmonary sepsis). Thus, in one embodiment, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, CIQA MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and optionally TNF. For example, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, CIQA MRAS, TMEM37, CIQB, PCOLCE2, and KIF2C. In one embodiment, the one or more biomarker may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, and optionally TNF.
In one embodiment, the one or more biomarker may be selected from the group consisting of: MRAS, CIQC, CIQB, and CIQA. In one embodiment, the one or more biomarker may be selected from the group consisting of: PCOLCE2, TMEM37, SLC39A8, KIF2C and TNF. In one embodiment, the one or more biomarker may be selected from the group consisting of: PCOLCE2, TMEM37, SLC39A8 and KIF2C.
Each of the biomarkers of abdominal sepsis may be used alone, or in combination with any of the abdominal sepsis biomarkers in the methods and uses of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or up to and including all of the abdominal sepsis biomarkers may be used to diagnose abdominal sepsis in a patient according to the methods and uses of the invention.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1 may be used to diagnose abdominal sepsis in a patient. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more or all 9) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF may be used to diagnose abdominal sepsis in a patient. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more or all 8) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, and KIF2C, may be used to diagnose abdominal sepsis in a patient. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more or all 5) of the biomarkers selected from the group consisting of: MRAS, CIQC, CIQB, CIQA, and TNF may be used to diagnose abdominal sepsis in a patient. In one embodiment, when detecting one or more biomarker selected from this group, the sample is obtained from the patient up to 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, the following combinations of abdominal sepsis biomarkers may be used to diagnose abdominal sepsis: (i) MRAS and CIQC; (ii) MRAS and CIQB; (iii) MRAS and PCOLCE2; (iv) MRAS and CIQA; (v) MRAS and TMEM37; (vi) MRAS and TNF; (vii) MRAS and SLC39A8; (viii) CIQC and CIQB; (ix) CIQC and PCOLCE2; (x) CIQC and CIQA; (xi) CIQC and TMEM37; (xii) CIQC and TNF; (xiii) CIQC and SLC39A8; (xiv) CIQB and PCOLCE2; (xv) CIQB and CIQA; (xvi) CIQB and TMEM37; (xvii) CIQB and TNF; (xviii) CIQB and SLC39A8; (xix) PCOLCE2 and CIQA; (xx) CIQA and TMEM37; (xxi) PCOLCE2 and TMEM37; (xxii) PCOLCE2 and TNF; (xxiii) PCOLCE2 and SLC39A8; (xxiv) CIQA and TNF; (xxv) CIQA and SLC39A8; (xxvi) TMEM37 and TNF; (xxvii) TMEM37 and SLC39A8; and (xxviii) TNF and SLC39A8; (xxxxix) MRAS and KIF2C; (xl) CIQC and KIF2C; (xli) CIQB and KIF2C; (xlii) CIQA and KIF2C; (xliii) TNF and KIF2C; (xliv) PCOLCE2 and KIF2C; (xlv) TMEM37 and KIF2C; (xlvi) SLC39A8 and KIF2C.
As described in Example 2, a sub-set of the biomarkers tested (SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB) was observed to provide particularly accurate diagnosis of abdominal sepsis (see the ROC curve data in Example 2). In one embodiment, any combination of 1 or more (eg. 2 or more, 3 or more, 4 or more, 5 or more, or all 6) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used to diagnose abdominal sepsis in a patient. For example, the combination of SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB may be used to diagnose abdominal sepsis in a patient. For example, 2 or more biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used to diagnose abdominal sepsis in a patient. For example, 3 or more biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used to diagnose abdominal sepsis in a patient. For example, 4 or more biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used to diagnose abdominal sepsis in a patient. For example, the combination of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used to diagnose abdominal sepsis in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, or all 3) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, and CIQA may be used to diagnose abdominal sepsis in a patient. SLC39A8, CIQC, and CIQA may be used to diagnose abdominal sepsis in a patient. For example, 2 or more biomarkers selected from the group consisting of: SLC39A8, CIQC, and CIQA, may be used to diagnose abdominal sepsis in a patient. For example, the combination of the biomarkers: SLC39A8, CIQC, and CIQA may be used to diagnose abdominal sepsis in a patient.
In one embodiment, the one or more biomarker is SLC39A8. In one embodiment, the one or more biomarker is CIQC. In one embodiment, the one or more biomarker is CIQA. In one embodiment, the one or more biomarker is CIQB. In one embodiment, the one or more biomarker is MRAS. In one embodiment, the one or more biomarker is TMEM37. In one embodiment, the one or more biomarker is PCOLCE2. In one embodiment, the one or more biomarker is KIF2C. In one embodiment, the one or more biomarker is TNF. In one embodiment, the one or more biomarker is IFI44. In one embodiment, the one or more biomarker is IFIT1. In one embodiment, the one or more biomarker is RPGRIP1.
One or more additional biomarker for abdominal sepsis may also be used in the diagnosis of abdominal sepsis according to the method of the invention. Any combination of the one or more additional biomarker may be used in combination with the one or more biomarker of the invention. For example at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or all 11 additional biomakers for abdominal sepsis may be used in combination with the one or more biomarker of the invention (as described herein). Typically, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB.
In one embodiment, the one or more biomarker is MRAS, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, TMEM37, and CIQB.
In one embodiment, the one or more biomarker is PCOLCE2, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1.
In one embodiment, the one or more biomarker is TMEM37, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, and CIQB.
In one embodiment, the one or more biomarker is SLC39A8, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: CIQC, CIQA, MRAS, TMEM37, and CIQB.
In one embodiment, the one or more biomarker is KIF2C, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, TNF, IFI44, IFIT1, and RPGRIP1.
In one embodiment, the one or more biomarker is CIQA, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, MRAS, TMEM37, and CIQB.
In one embodiment, the one or more biomarker is CIQC, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQA, MRAS, TMEM37, and CIQB.
In one embodiment, the one or more biomarker is CIQB, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. In one embodiment, the one or more additional biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, and TMEM37.
In one embodiment, the one or more biomarker is TNF, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, IFI44, IFIT1, and RPGRIP1.
In one embodiment, the one or more biomarker is IFI44, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, MRAS, CIQA, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFIT1, and RPGRIP1.
In one embodiment, the one or more biomarker is IFIT1, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, and RPGRIP1.
In one embodiment, the one or more biomarker is RPGRIP1, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, up to and including all) of the biomarkers: SLC39A8, CIQC, CIQA, MRAS, CIQB, TMEM37, PCOLCE2, KIF2C, TNF, IFI44, and IFIT1.
As illustrated in
For example, by comparing the presence and/or amount of markers quantified in a sample obtained from a patient to the presence and/or amount of markers quantified for a reference value (such as a reference value that is representative of a healthy individual (or a population of healthy individuals), a reference value that is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis), a reference value that is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis), and/or a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS)), it is possible to diagnose the presence (or absence) of abdominal sepsis in a patient. The method permits classification of the individual as belonging to or not belonging to the reference population (i.e., by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the individual's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the individual falls (or does not fall) within the reference population.
In one embodiment, an individual may be diagnosed as having or being at risk of having abdominal sepsis, when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having abdominal sepsis. In one embodiment, an individual may be diagnosed as not having or not being at risk of having abdominal sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, an individual may be diagnosed as not having or not being at risk of having abdominal sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, an individual may be diagnosed as not having or not being at risk of having abdominal sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
In one embodiment, an individual may be diagnosed as not having or not being at risk of having abdominal sepsis when the amount of the one or more biomarker quantified statistically deviates from the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having abdominal sepsis. In one embodiment, an individual may be diagnosed as having or being at risk of having abdominal sepsis when the amount of the one or more biomarker quantified statistically deviates from the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, an individual may be diagnosed as having or being at risk of having abdominal sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, an individual may be diagnosed as having or being at risk of having abdominal sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
All embodiments described above for the classification of a patient as having or being at risk of having a systemic inflammatory condition (or as not having or not being at risk of having a systemic inflammatory condition) apply equally to the method for diagnosing whether a patient has or is at risk of having abdominal sepsis. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may be as defined above for the method of diagnosing a systemic inflammatory condition in a patient. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value is representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, the reference value is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis). In one embodiment, the reference value is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
As described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the method of the invention may involve the use of multiple separate reference values. For example, the reference value may include one of more (e.g., two or more, three or more, or all 4) of the reference values selected from: a reference value that is representative of a healthy individual (or a population of healthy individuals); a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS); and a reference value that is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis); and a reference value that is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
The present inventors observed that the biomarkers for abdominal sepsis described herein (MRAS, CIQC, CIQB, PCOLCE2, CIQA, TMEM37, SLC39A8, KIF2C, TNF) each increase in abundance in samples obtained from patients having abdominal sepsis, as compared to healthy individuals. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of abdominal sepsis. Thus, in one embodiment, when the reference value is representative of a healthy individual (or population of healthy individuals), an increase in the one or more biomarker for abdominal sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has abdominal sepsis, or may be at risk of developing abdominal sepsis. Likewise, no increase in the one or more biomarker for abdominal sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have abdominal sepsis.
For some of the abdominal sepsis biomarkers identified by the present inventors, increased levels of these markers were also observed in patients having other systemic inflammatory conditions (such as pulmonary sepsis and SIRS) as compared to healthy individuals, although typically much bigger increases were observed for these biomarkers in the patients having abdominal sepsis. The accuracy of abdominal sepsis diagnosis can thus be improved by looking for a “minimum” fold change or % change in the levels of the one or more biomarkers as compared to the corresponding reference value that is representative of a healthy individual. The fold increase or % increase may be as defined above for the method for diagnosis of a systemic inflammatory condition.
For example, an increase of at least 50 (e.g., at least 55, at least 60, at least 70, at least 80, at least 90, at least 95 at least 100, at least 125, at least 150) fold in PCOLCE2 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 50 (e.g., less than 55, less than 60, less than 70, less than 80, less than 90, less than 95) fold in PCOLCE2 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 7 (e.g., at least 7.5, at least 8, at least 8.5) fold in TMEM37 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 3.5 (e.g., less than 4, less than 5, less than 6, less than 7) fold in TMEM37 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis.
For example, an increase of at least 3 (e.g., at least 3.5, at least 4, at least 4.5, at least 5) fold in SLC39A8 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 2 (e.g., less than 2.5, less than 3, less than 3.5, less than 4) fold in SLC39A8 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis.
For example, an increase of at least 2.6 (e.g., at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1) fold in KIF2C in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 2.6 (e.g., less than 2.7, less than 2.8, less than 2.9, less than 3) fold in KIF2C in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis.
For example, an increase of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20) fold in CIQC in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 7 (e.g., less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20) fold in CIQC in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25) fold in CIQB in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or may be at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 6 (e.g., less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20) fold in CIQB in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 10 (e.g., at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16) fold in CIQA in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase or an increase of less than 4 (e.g., less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, or less than 16) fold in CIQA in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.3 (e.g., at least 1.4, or at least 1.5) fold in TNF in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 1.3 (e.g., less than 1.4) fold in TNF in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, the patient from which a sample is obtained has been diagnosed as having or being at risk of developing sepsis (e.g., using the method of diagnosing sepsis in a patient as described herein and/or using the method of distinguishing between sepsis and SIRS in a patient as described herein).
For example, an increase of at least 1.1 (e.g., at least 1.2, at least 1.3, at least 1.4, at least 1.5, or at least 1.6) fold in MRAS in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no increase, or an increase of less than 1.1 (less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5) fold in MRAS in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The present inventors observed that the biomarkers IFI44, IFIT1, and RPGRIP1 each decrease in abundance in samples obtained from patients having abdominal sepsis, as compared to healthy individuals. Detection of decreased levels of these biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of abdominal sepsis.
In one embodiment, when the reference value is representative of a healthy individual, a decrease in the one or more biomarker for abdominal sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has abdominal sepsis, or may be at risk of developing abdominal sepsis. Likewise, no decrease in the one or more biomarker for abdominal sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have abdominal sepsis.
For the biomarkers IFIT1 and RPGRIP1, decreased levels of these markers were also observed in patients having other systemic inflammatory conditions (such as pulmonary sepsis and SIRS) as compared to healthy individuals, although typically much bigger decreases were observed for these biomarkers in the patients having abdominal sepsis. The accuracy of abdominal sepsis diagnosis can thus be improved by looking for a “minimum” fold decrease or % decrease in the levels of the one or more biomarkers as compared to the corresponding reference value that is representative of a healthy individual. The fold decrease or % decrease may be as defined above for the method for diagnosis of a systemic inflammatory condition.
For example, a decrease of at least 0.5 (e.g., at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1) fold in IFI44 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no decrease in IFI44 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, a decrease of at least 2.5 (e.g., at least 2.6, at least 2.7, at least 2.8, at least 2.9, or at least 3) fold in IFIT1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no decrease, or a decrease of less than 1.9 (e.g., less than 2, less than 2.1, less than 2.2, less than 2.3, less than 2.4, less than 2.5) fold in IFIT1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, a decrease of at least 1.75 (e.g., at least 1.8, at least 1.9, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, or at least 5) fold in RPGRIP1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing abdominal sepsis. In one embodiment, no decrease, or a decrease of less than 1.4 (e.g., less than 1.5, less than 1.6, less than 1.7, less than 1.8, less than 1.9, less than 2) fold in RPGRIP1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing abdominal sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
As described herein, the present inventors observed that the levels of the one or more sepsis biomarkers (MRAS, PCOLCE2, TMEM37, SLC39A8, KIF2C, CIQC, CIQB, CIQA, and TNF) were elevated in patients having abdominal sepsis as compared to patients having other systemic inflammatory conditions such as pulmonary sepsis or SIRS (with the exception of TNF which is increased in abundance as compared to patients having pulmonary sepsis only). Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients having one or more of these other systemic inflammatory conditions can thus be used to diagnose the presence of abdominal sepsis.
Thus, in one embodiment, an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis, indicates that the patient has or is at risk of developing abdominal sepsis. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis, indicates that the patient does not have abdominal sepsis.
In one embodiment, an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient has or is at risk of developing abdominal sepsis. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient does not have abdominal sepsis.
In one embodiment, the patient may be diagnosed as having abdominal sepsis, or being at risk of developing abdominal sepsis, when the one or more biomarker (or the one or more additional biomarker) increases by at least 1 (e.g., at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis and/or an individual having SIRS.
As described herein, the present inventors observed that the levels of the biomarkers IFI44, IFIT1, and RPGRIP1 were decreased in patients having abdominal sepsis as compared to patients having other systemic inflammatory conditions such as pulmonary sepsis or SIRS. Detection of decreased levels of these biomarkers in a patient as compared to the levels detected for patients having one or more of these other systemic inflammatory conditions can thus be used to diagnose the presence of abdominal sepsis.
Thus, in one embodiment, a decrease in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis, indicates that the patient has or is at risk of developing abdominal sepsis. Likewise, no decrease in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis, indicates that the patient does not have abdominal sepsis.
In one embodiment, a decrease in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient has or is at risk of developing abdominal sepsis. Likewise, no decrease in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient does not have abdominal sepsis.
In one embodiment, the patient may be diagnosed as having abdominal sepsis, or being at risk of developing abdominal sepsis, when the one or more biomarker (or the one or more additional biomarker) decreases by at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis and/or an individual having SIRS.
In a related aspect, the present invention also provides the use of one or more of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1 as a biomarker for abdominal sepsis. In one embodiment, the one or more biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF. In one embodiment, the one or more biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, and KIF2C. In one embodiment, the one or more biomarker is selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB. In one embodiment, the one or more biomarker is selected from the group consisting of: SLC39A8, CIQC, and CIQA. In one embodiment, the use is of the one or more biomarker in the diagnosis of abdominal sepsis in a patient.
All embodiments described above for the method of diagnosing abdominal sepsis in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “abdominal sepsis”, “patient”, “sample”, and “the one or more biomarker”.
As discussed herein, the present inventors have also observed that the levels of HCAR2, CXCR1, DISC1, EPSTI1, and IFI44, are elevated in patients having pulmonary sepsis, and are thus suitable for use as biomarkers for pulmonary sepsis (see Table 3).
The present invention therefore also provides a method for diagnosing pulmonary sepsis in a patient, comprising:
All embodiments described above for the “method for diagnosing a systemic inflammatory condition in a patient” apply equally to the “method for diagnosing pulmonary sepsis in a patient”. This includes all embodiments relating to the “sample”, “patient”, “biomarker”, and “reference value”, and all embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step for determining whether the patient has or is at risk of developing pulmonary sepsis.
As used herein, the phrase “diagnosis of pulmonary sepsis in a patient” means determining whether the patient has or is risk of developing pulmonary sepsis. The term “pulmonary sepsis” is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
The “patient” for which diagnosis is performed is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”, and the “method for diagnosing sepsis in a patient”. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having a systemic inflammatory condition using the method described herein. In one embodiment, the patient is suspected of having or being at risk of developing sepsis. In one embodiment, the patient has been diagnosed as having or being at risk of developing sepsis (eg. using the method of diagnosing sepsis in a patient as described herein and/or using the method of distinguishing between sepsis and SIRS in a patient as described herein). In one embodiment, the patient is suspected of having or being at risk of developing pulmonary sepsis.
The “sample” obtained from the patient is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”, including all embodiments relating to the time point at which the sample is obtained. All embodiments of the “sample” described above for the method for diagnosing sepsis also apply to the method for diagnosing pulmonary sepsis.
The “one or more biomarker” of the invention is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”.
In one embodiment, the one or more biomarker may be selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44. For example, the one or more biomarker may be selected from the group consisting of: EPSTI1 and DISC1. For example, the one or more biomarker may be selected from the group consisting of: CXCR1, HCAR2, and IF144.
The present inventors observed that the biomarkers CXCR1, HCAR2, and IFI44 are elevated in patients having pulmonary sepsis as compared to patients having abdominal sepsis. However, these biomarkers were also observed as being elevated in patients having SIRS, and thus these biomarkers are particularly useful for diagnosing pulmonary sepsis in patients already diagnosed as having sepsis (eg. using the methods described herein for diagnosis of sepsis, or using the method described herein for distinguishing between abdominal sepsis and pulmonary sepsis). Thus, when the patient has already been diagnosed as having sepsis, the one or more biomarker may be selected from the group consisting of: CXCR1, HCAR2, and IF144.
Each of the biomarkers of pulmonary sepsis may be used alone, or in combination with any of the pulmonary sepsis biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, or up to and including all of the pulmonary sepsis biomarkers may be used to diagnose pulmonary sepsis in a patient according to the method of the invention.
In one embodiment, the one or more biomarker is HCAR2. In one embodiment, the one or more biomarker is CXCR1. In one embodiment, the one or more biomarker is DISC1. In one embodiment, the one or more biomarker is EPSTI1. In one embodiment, the one or more biomarker is IF144.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, or all 5) of the biomarkers selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44, may be used to diagnose pulmonary sepsis in a patient. In one embodiment, any combination of 1 or more (e.g., 2 or more, or all 3) of the biomarkers selected from the group consisting of: CXCR1, HCAR2, and IFI44 may be used to diagnose pulmonary sepsis in a patient. In one embodiment, any combination of 1 or more (e.g., or both) of the biomarkers selected from the group consisting of: EPSTI1 and DISC1, may be used to diagnose pulmonary sepsis in a patient.
For example, the following combinations of pulmonary sepsis biomarkers may be used to diagnose pulmonary sepsis: (i) EPSTI1 and HCAR2; (ii) EPSTI1 and DISC1; (iii) EPSTI1 and CXCR1; (iv) EPSTI1 and IFI44; (v) DISC1 and CXCR1; (vi) DISC1 and HCAR2; (vii) DISC1 and IFI44; (viii) CXCR1 and HCAR2; (ix) CXCR1 and IFI44; (x) HCAR2 and IF144.
As described in Example 2, a sub-set of the biomarkers tested (HCAR2, CXCR1, DISC1) was observed to provide particularly accurate diagnosis of pulmonary sepsis (see the ROC curve data in Example 2). In one embodiment, any combination of 1 or more (e.g., 2 or more, or all 3) of the biomarkers selected from the group consisting of: HCAR2, CXCR1, DISC1, may be used to diagnose pulmonary sepsis in a patient. For example, the combination of HCAR2, CXCR1, and DISC1 may be used to diagnose abdominal sepsis in a patient. For example, the combination of HCAR2 and CXCR1 may be used to diagnose abdominal sepsis in a patient.
One or more additional biomarker for pulmonary sepsis may also be used in the diagnosis of pulmonary sepsis according to the method of the invention. Any combination of the one or more additional biomarker may be used in combination with the one or more biomarker of the invention. For example at least 1, at least 2, at least 3, or all 4 additional biomakers for pulmonary sepsis may be used in combination with the one or more biomarker of the invention (as described herein). Typically, the one or more additional biomarker is selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44. In one embodiment, one or more additional biomarker is selected from the group consisting of: HCAR2, CXCR1, and DISC1.
In one embodiment, the one or more biomarker is HCAR2, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: EPSTI1, DISC1, CXCR1, and IFI44. In one embodiment, the one or more additional biomarker is selected from CXCR1 and/or DISC1.
In one embodiment, the one or more biomarker is CXCR1, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: EPSTI1, DISC1, HCAR2, and IFI44. In one embodiment, the one or more additional biomarker is selected from HCAR2 and/or DISC1.
In one embodiment, the one or more biomarker is DISC1, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: EPSTI1, HCAR2, CXCR1, and IFI44. In one embodiment, the one or more additional biomarker is selected from HCAR2 and/or CXCR1.
In one embodiment, the one or more biomarker is EPSTI1, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: DISC1, CXCR1, HCAR2, and IF144.
In one embodiment, the one or more biomarker is IFI44, and the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: EPSTI1, DISC1, CXCR1, and HCAR2.
As illustrated in
For example, by comparing the amount of markers quantified in a sample obtained from a patient to the amount of markers quantified for a reference value (such as a reference value that is representative of a healthy individual (or a population of healthy individuals), a reference value that is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis), a reference value that is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis), and/or a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS)), it is possible to diagnose the presence (or absence) of pulmonary sepsis in a patient. The method permits classification of the individual as belonging to or not belonging to the reference population (ie. by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the individual's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the individual falls (or does not fall) within the reference population.
In one embodiment, an individual may be diagnosed as having or being at risk of having pulmonary sepsis, when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having pulmonary sepsis. In one embodiment, an individual may be diagnosed as not having or not being at risk of having pulmonary sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, an individual may be diagnosed as not having or not being at risk of having pulmonary sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis). In one embodiment, an individual may be diagnosed as not having or not being at risk of having pulmonary sepsis when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference values representative of an individual having SIRS (or a population of individuals having SIRS).
In one embodiment, an individual may be diagnosed as not having or not being at risk of having pulmonary sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having pulmonary sepsis. In one embodiment, an individual may be diagnosed as having or being at risk of having pulmonary sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference values representative of a healthy individual (or a population of healthy individuals). In one embodiment, an individual may be diagnosed as having or being at risk of having pulmonary sepsis when the amount of the one or more biomarkers statistically deviates from the amount determined for the corresponding reference value representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis). In one embodiment, an individual may be diagnosed as having or being at risk of having pulmonary sepsis when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference values representative of an individual having SIRS (or a population of individuals having SIRS).
All embodiments described above for the classification of a patient as having or being at risk of having a systemic inflammatory condition (or as not having or not being at risk of having a systemic inflammatory condition) apply equally to the method for diagnosing whether a patient has or is at risk of having pulmonary sepsis. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may be as defined above for the method of diagnosing a systemic inflammatory condition in a patient. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value is representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, the reference value is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis). In one embodiment, the reference value is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
As described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the method of the invention may involve the use of multiple separate reference values. For example, the reference value may include one of more (e.g., two or more, three of more, or all 4) of the reference values selected from: a reference value that is representative of a healthy individual (or a population of healthy individuals); a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS); a reference value that is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis), and a reference value that is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
The present inventors observed that the pulmonary sepsis biomarkers EPSTI1, DISC1, CXCR1, HCAR2 and IFI44 each increase in abundance in samples obtained from patients having pulmonary sepsis, as compared to healthy individuals. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of pulmonary sepsis.
In one embodiment, an increase in the one or more biomarker for pulmonary sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has pulmonary sepsis, or is at risk of developing pulmonary sepsis. Likewise, no increase in the one or more biomarker for pulmonary sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have pulmonary sepsis.
For some of the pulmonary sepsis biomarkers identified by the present inventors (DISC1, CXCR1, HCAR2, and IFI44), increased levels of these markers were also observed in patients having other systemic inflammatory conditions (abdominal sepsis or SIRS) as compared to healthy individuals, although much bigger increases were observed for these biomarkers in the patients having pulmonary sepsis. The accuracy of pulmonary sepsis diagnosis can thus be improved by looking for a “minimum” fold increase or % increase in the levels of the one or more biomarkers as compared to the corresponding reference value that is representative of a healthy individual. The fold increase or % increase may be as defined above for the method for diagnosis of a systemic inflammatory condition.
For example, an increase of at least 1 (e.g., at least 1.05, at least 1.1, at least 1.15. at least 1.2, at least 1.25, at least 1.3) fold in EPSTI1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing pulmonary sepsis. In one embodiment, no increase, or an increase of less than 1 (e.g., less than 1.05, less than 1.1) fold in EPSTI1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing pulmonary sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.8 (e.g., at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3) fold in DISC1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing pulmonary sepsis. In one embodiment, no increase or an increase of less than 1.3 (e.g., less than 1.4, less than 1.5, less than 1.6, less than 1.7, less than 1.8, less than 1.9, less than 2) fold in DISC1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing pulmonary sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 3.5 (e.g., at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4) fold in CXCR1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing pulmonary sepsis. In one embodiment, no increase, or an increase of less than 3.5 (e.g., less than 3, less than 2.5, less than 2) fold in CXCR1 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing pulmonary sepsis. In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the patient from which a sample is obtained has been diagnosed as having or being at risk of developing sepsis (e.g., using the method of diagnosing sepsis in a patient as described herein and/or using the method of distinguishing between sepsis and SIRS in a patient as described herein).
For example, an increase of at least 1.4 (e.g., at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2) fold in HCAR2 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing pulmonary sepsis. In one embodiment, no increase, or an increase of less than 1.5 (e.g., less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1) fold in HCAR2 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing pulmonary sepsis. In one embodiment, the patient from which a sample is obtained has been diagnosed as having or being at risk of developing sepsis (e.g., using the method of diagnosing sepsis in a patient as described herein and/or using the method of distinguishing between sepsis and SIRS in a patient as described herein).
For example, an increase of at least 1.4 (e.g., at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least fold 1.9, or at least 2) fold in IFI44 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing pulmonary sepsis. In one embodiment, no increase or an increase of less than 1.7 (e.g., less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1) fold in IFI44 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing pulmonary sepsis. In one embodiment, the patient from which a sample is obtained has been diagnosed as having or being at risk of developing sepsis (e.g., using the method of diagnosing sepsis in a patient as described herein and/or using the method of distinguishing between sepsis and SIRS in a patient as described herein). In one embodiment, when detecting this level of fold change in the biomarker, the method is performed using a sample obtained from a patient up to 24 (e.g., up to 36, up to 48, up to 72, or up to 96) hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
As described herein, the present inventors observed that the levels of the one or more “pulmonary sepsis” biomarkers were elevated in patients having pulmonary sepsis as compared to patients having other systemic inflammatory conditions such as abdominal sepsis or SIRS. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients having one or more of these other systemic inflammatory conditions can thus be used to diagnose the presence of pulmonary sepsis.
In one embodiment, when the reference value is representative of an individual having abdominal sepsis, an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value indicates that the patient has or is at risk of developing pulmonary sepsis. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have pulmonary sepsis.
In one embodiment, when the reference value is representative of an individual having SIRS, an increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has or is at risk of developing pulmonary sepsis. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have pulmonary sepsis.
In one embodiment, the patient may be diagnosed as having pulmonary sepsis, or being at risk of developing pulmonary sepsis, when the one or more biomarker (or the one or more additional biomarker) increases by at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, or at least 50) fold in the sample obtained from the patient relative to the corresponding reference value representative of an individual having abdominal sepsis and/or an individual having SIRS.
In a related aspect, the present invention also provides the use of one or more of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44 as a biomarker for pulmonary sepsis. In one embodiment, the one or more biomarker is selected from the group consisting of: HCAR2, CXCR1, and DISC1. In one embodiment, the one or more biomarker is selected from the group consisting of: HCAR2, and CXCR1. In one embodiment, the present invention provides the use of a combination of HCAR2, CXCR1, and optionally DISC1 as biomarkers for pulmonary sepsis. In one embodiment, the use is of the one or more biomarker in the diagnosis of pulmonary sepsis in a patient.
All embodiments described above for the method of diagnosing pulmonary sepsis in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “pulmonary sepsis”, “patient”, “sample”, and “the one or more biomarker”.
The method for diagnosis of sepsis, the method for diagnosis of abdominal sepsis and/or the method for diagnosis of pulmonary sepsis as described herein can be used in a decision tree process to investigate the health of a patient having or suspected of having a systemic inflammatory condition. For example, the method for diagnosis of sepsis, the method for diagnosis of abdominal sepsis and/or the method for diagnosis pulmonary sepsis as described herein in a patient can be performed before, after, or in addition to any of the methods of the invention described herein.
In one embodiment, the method of the invention for diagnosing sepsis in a patient (as described herein) can be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). If the patient tests positive for a systemic inflammatory condition (using the method for diagnosing whether a patient has a systemic inflammatory condition), they may be tested for sepsis using the diagnostic method described herein. In one embodiment, the above combination of methods are performed as described, and if the patient tests positive for sepsis, the patient may be further tested for abdominal sepsis and/or pulmonary sepsis using the diagnostic methods of the invention described herein, so as to determine whether the patient has or is at risk of developing abdominal and/or pulmonary sepsis.
In one embodiment, the method of the invention for diagnosing sepsis in a patient (as described herein) can be performed subsequent to (or in addition to) the method for distinguishing between sepsis and SIRS in a patient (as described herein). If the patient tests positive for sepsis using the distinguishing method of the invention, they may be tested for sepsis using the diagnostic method described herein, so as to further confirm whether the patient has or is at risk of developing sepsis. In one embodiment, the above combination of methods are performed as described, and if the patient tests positive for sepsis, the patient may be further tested for abdominal sepsis and/or pulmonary sepsis using the diagnostic methods of the invention described herein, so as to determine whether the patient has or is at risk of developing abdominal and/or pulmonary sepsis.
In one embodiment, the method for diagnosis of sepsis may be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein), and the method for distinguishing between sepsis and SIRS in a patient (as described herein). For example, the patient may be tested using the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). If the patient tests positive for a systemic inflammatory condition, they may be tested using the distinguishing method of the invention (as described herein) to determine whether them patient has or is at risk of developing sepsis and/or SIRS. If the patient tests positive for sepsis using the distinguishing method of the invention, they may be tested for sepsis using the diagnostic method described herein, so as to further confirm the diagnosis. In one embodiment, the above combination of methods are performed as described, and if the patient tests positive for sepsis, the patient may be further tested for abdominal sepsis and/or pulmonary sepsis using the diagnostic methods of the invention described herein, so as to determine whether the patient has or is at risk of developing abdominal and/or pulmonary sepsis.
In one embodiment, the method for diagnosing abdominal sepsis and/or pulmonary sepsis in a patient (as described herein) may be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). If the patient tests positive for a systemic inflammatory condition (using the method of the invention for diagnosing whether a patient has a systemic inflammatory condition), they may be tested for abdominal sepsis and/or pulmonary sepsis using the diagnostic methods described herein to determine whether the patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis.
In one embodiment, the method of the invention for diagnosing abdominal sepsis and/or pulmonary sepsis in a patient (as described herein) can be performed subsequent to (or in addition to) the method for distinguishing between sepsis and SIRS in a patient (as described herein). If the patient tests positive for sepsis using the distinguishing method of the invention, they may be tested for abdominal sepsis and/or pulmonary sepsis using the diagnostic methods described herein, so as to further determine whether the patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis.
In one embodiment, the method for diagnosing abdominal sepsis and/or pulmonary sepsis in a patient (as described herein) may be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein), and the method for distinguishing between sepsis and SIRS in a patient (as described herein). For example, the patient may be tested using the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein).
If the patient tests positive for a systemic inflammatory condition, they may be tested using the distinguishing method of the invention (as described herein) to determine whether the patient has or is at risk of developing sepsis and/or SIRS. If the patient tests positive for sepsis using the distinguishing method of the invention, they may be tested for abdominal sepsis and/or pulmonary sepsis using the diagnostic methods described herein, so as to determine whether the patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis.
Furthermore, in one embodiment, each of above combination of methods may be performed as described, and if the patient tests postive for a systemic inflammatory condition, they may be tested for SIRS using the diagnostic method described herein, in addition to being tested for sepsis, abdominal sepsis and/or pulmonary sepsis using the methods described herein.
The above described combination of methods may be performed in parallel to determine the disease status of a patient by simultaneously (or substantially simultaneously) investigating the expression of all the biomarkers in a sample obtained from the patient, and determining whether the patient has or is at risk of having sepsis (such as abdominal or pulmonary sepsis).
When performing these different methods in a decision tree process, the sample used in each step of the method may be the same sample obtained from the patient (as described herein). When the method comprises multiple quantification steps, all the steps may be performed at the same time (e.g., in parallel) and/or using the same sample.
Methods for Distinguishing Between Different Types of Systemic Inflammatory Conditions Sepsis and SIRS are both systemic inflammatory conditions associated with overlapping clinical symptoms. Distinguishing between these conditions is important, because different treatments are required for the two conditions. As described herein, the present inventors have identified a set of biomarkers that is predictive of sepsis and a separate set of biomarkers that is predictive of SIRS in patients. Using these distinct sets of biomarkers, the present inventors have developed a rapid and sensitive way to distinguish between SIRS and sepsis in a patient by quantifying one or more biomarker for sepsis and/or one or more biomarker for SIRS in a sample obtained from a patient, so as to determine whether the patient has a biomarker profile that is predictive of sepsis or SIRS.
The present invention therefore provides a method for distinguishing between sepsis and SIRS in a patient, comprising:
As illustrated in Example 2, the distinguishing method of the invention can be performed using only one or more of the sepsis biomarker described herein, or only one or more of the SIRS biomarkers described herein. These biomarkers can be used on their own to distinguish sepsis and SIRS because their expression correlates with the patient's disease condition (i.e., the presence and/or amount of these biomarkers depends on whether a patient has sepsis or SIRS or is healthy). Determining the presence and/or amount of either of these biomarkers and comparing this to a corresponding reference value (such as a reference value that is representative of a healthy individual, a sepsis patient and/or a SIRS patient) therefore allows the disease status of the patient to be determined.
In one embodiment, the present invention provides a method for distinguishing between sepsis and SIRS in a patient, comprising:
In one embodiment, the present invention provides a method for distinguishing between sepsis and SIRS in a patient, comprising:
Alternatively, the one or more biomarker for sepsis may used in combination with the one or more biomarker for SIRS to distinguish between sepsis and SIRS in a patient.
In one embodiment, the present invention provides a method for distinguishing between sepsis and SIRS in a patient, comprising:
All embodiments described above for the “method for diagnosing a systemic inflammatory condition”, the “method for diagnosing SIRS”, and the “method for diagnosing sepsis” (including the methods for diagnosing abdominal sepsis and pulmonary sepsis) apply equally to the “method for distinguishing between sepsis and SIRS in a patient”. This includes all embodiments relating to the “sample”, “patient”, “biomarker”, and “reference value”, and all embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step for making a conclusion about the disease status of the patient.
As used herein, “distinguishing between sepsis and SIRS” means to determine whether a patient has or is at risk of developing sepsis and/or SIRS. For example, it may involve determining whether a patient has or is at risk of developing sepsis or SIRS. For example, it may involve determining whether a patient has or is at risk of developing sepsis and SIRS. This may involve distinguishing between a group (ie. one or more) of patients having sepsis and a group (ie. one or more) of patients having SIRS. In one embodiment, this may involve diagnosing or determining whether a patient has or is at risk of developing one or more systemic inflammatory condition selected from: sepsis and SIRS.
The systemic inflammatory conditions “sepsis” and “SIRS” are as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
The “patient” for which diagnosis is performed is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition using the method described herein for diagnosis of a systemic inflammatory condition in a patient. In one embodiment, the patient is suspected of having or being at risk of developing sepsis and/or SIRS. In one embodiment, the patient is suspected of having or being at risk of developing sepsis. In one embodiment, the patient is suspected of having or being at risk of developing SIRS.
The “sample” obtained from the patient is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”, the “method for diagnosing SIRS in a patient” and the “method for diagnosing sepsis in a patient”, including all embodiments relating to the time point at which the sample is obtained.
The “one or more biomarker” of the invention is as defined above for the “method for diagnosing a systemic inflammatory condition in a patient”.
The “one or more biomarker for sepsis” may be as defined above for the method for diagnosing sepsis in a patient and includes any of the one or more sepsis biomarkers described herein (with or without the one or more additional biomarker) and further includes any of the combinations of sepsis biomarkers described herein.
For example, the one or more biomarker for sepsis may be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In a further example, the one or more biomarker for sepsis may be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4. In a further example, the one or more biomarker for sepsis may be selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1.
Each of the biomarkers of sepsis may be used alone, or in combination with any of the sepsis biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, up to and including all of the sepsis biomarkers may be used to distinguish between sepsis and SIRS in a patient.
For example, the method may be performed using 1 or more biomarker for sepsis selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using 2 or more biomarkers for sepsis selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using 3 or more biomarkers for sepsis selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using 4 or more biomarkers for sepsis selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. For example, the method may be performed using the combination of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. This combination of sepsis biomarkers was shown to be particularly effective in distinguishing sepsis from SIRS when tested by ROC analysis (see Example 2).
In one embodiment, the one or more biomarker for sepsis is LCN2. In one embodiment, the one or more biomarker for sepsis is ITGA2B. In one embodiment, the one or more biomarker for sepsis is MYL9. In one embodiment, the one or more biomarker for sepsis is ITGB3. In one embodiment, the one or more biomarker for sepsis is TREML1. In one embodiment, the one or more biomarker for sepsis is LCN15. In one embodiment, the one or more biomarker for sepsis is CMTM5. In one embodiment, the one or more biomarker for sepsis is PPBP. In one embodiment, the one or more biomarker for sepsis is PF4. In one embodiment, the one or more biomarker for sepsis is MAP1A. In one embodiment, the one or more biomarker for sepsis is SELP. In one embodiment, the one or more biomarker for sepsis is NEXN. In one embodiment, the one or more biomarker for sepsis is NLRC4. In one embodiment, the one or more biomarker for sepsis is CLEC1B.
As described above for the method for diagnosis of sepsis in a patient, one or more additional biomarker for sepsis may also be used in the distinguishing method. All embodiments described above for the one or more additional biomarker used in the method for diagnosis of sepsis in a patient apply equally to the method for distinguishing between sepsis and SIRS in a patient. For example, if the one or more biomarker is LCN15, the one or more additional biomarker may be selected from at least 1 (eg. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: ITGB3, ITGA2B, MYL9, LCN2, TREML1, CMTM5, PPBP, and PF4.
In a preferred embodiment, the method for distinguishing between sepsis and SIRS in a patient may comprise:
Preferably, the one or more biomarker for sepsis comprises the combination of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1. The patient used in these methods has preferably been diagnosed as having a systemic inflammatory condition (e.g., preferably using the method described for diagnosis of a systemic inflammatory condition).
The “one or more biomarker for SIRS” is as defined above for the method for diagnosing SIRS in a patient and includes any of the one or more SIRS biomarkers described herein (with or without the one or more additional biomarker), and further includes any of the combinations of SIRS biomarkers described herein.
For example, the one or more biomarker for SIRS may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1 RN, NLRP3, RBP4, and MPP3. For example, the one or more biomarker for SIRS may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124. For example, the one or more biomarker for SIRS may be selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
Each of the biomarkers of SIRS may be used alone, or in combination with any of the SIRS biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, up to and including all of the SIRS biomarkers may be used to distinguish between sepsis and SIRS in a patient.
For example, the distinguishing method may be performed using 1 or more biomarker for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI. For example, the distinguishing method may be performed using 2 or more biomarkers for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI. For example, the distinguishing method may be performed using 3 or more biomarkers for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI. For example, the distinguishing method may be performed using all 4 biomarkers for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI. This combination of SIRS biomarkers was shown to be particularly effective in distinguishing sepsis from SIRS when tested by ROC analysis (see Example 2).
In one embodiment, the one or more biomarker for SIRS is TGFBI. In one embodiment, the one or more biomarker for SIRS is PLA2G7. In one embodiment, the one or more biomarker for SIRS is MYCL. In one embodiment, the one or more biomarker for SIRS is ARHGEF10L. In one embodiment, the one or more biomarker for SIRS is GPR124. In one embodiment, the one or more biomarker for SIRS is IL1 RN. In one embodiment, the one or more biomarker for SIRS is NLRP3. In one embodiment, the one or more biomarker for SIRS is RBP4. In one embodiment, the one or more biomarker for SIRS is MPP3.
As described above for the method for diagnosis of SIRS in a patient, one or more additional biomarker for SIRS may also be used in the distinguishing method. All embodiments described above for the one or more additional biomarker used in the method for diagnosis of SIRS in a patient apply equally to the method for distinguishing between sepsis and SIRS in a patient. For example, if the one or more biomarker is GPR124, the one or more additional biomarker may be selected from at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, TGFBI, IL1 RN, NLRP3, RBP4, and MPP3. In one embodiment, the one or more additional biomarker is selected from at least 1 (e.g., at least 2, at least 3, up to and including all) of the biomarkers: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
In a preferred embodiment, the method for distinguishing between sepsis and SIRS in a patient may comprise:
Preferably, the one or more biomarker for SIRS comprises the combination of: PLA2G7, ARHGEF10L, MYCL, and TGFBI. The patient used in this method has preferably been diagnosed as having a systemic inflammatory condition (e.g., preferably using the method described for diagnosis of a systemic inflammatory condition).
Any combination of the one or more biomarker for sepsis described herein (including the one or more additional biomarker for sepsis) may be used in conjunction with any combination of the one or more biomarker for SIRS described herein (including the one or more additional biomarker for SIRS) in the method of the invention for distinguishing between sepsis and SIRS in a patient.
For example, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, up to and including all) of the sepsis biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, up to and including all) of the SIRS biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1RN, NLRP3, RBP4, and MPP3, to distinguish between sepsis and SIRS in a patient according to the method described herein.
For example, any combination of 1 or more (e.g., 2 or more, or all 3) of the sepsis biomarkers selected from the group consisting of: LCN15, LCN2, and NLRC4, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, up to and including all) of the SIRS biomarkers selected from the group consisting of: GPR124, TGFBI, PLA2G7, MYCL, and ARHGEF10L, to distinguish between sepsis and SIRS in a patient according to the method described herein.
For example, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, up to and including all) of the sepsis biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, up to and including all) of the SIRS biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, up to and including all) of the sepsis biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, 3 or more, up to and including all) of the SIRS biomarkers selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, up to and including all) of the sepsis biomarkers selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, up to and including all) of the SIRS biomarkers selected from the group consisting of: ARHGEF10L, MYCL, and TGFBI, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, the combination of preferred sepsis biomarkers: ITGB3, ITGA2B, MYL9, LCN2, and TREML1, may be used in conjunction with the combination of preferred SIRS biomarkers: PLA2G7, ARHGEF10L, MYCL, and TGFBI, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, the combination of preferred sepsis biomarkers: ITGB3, ITGA2B, MYL9, LCN2, and TREML1, may be used in conjunction with the combination of preferred SIRS biomarkers: ARHGEF10L, MYCL, and TGFBI, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In a further example, the following combinations of sepsis and SIRS biomarkers may be used to distinguish sepsis and SIRS according to the method described herein: (i) LCN15 and TGFBI; (ii) LCN15 and PLA2G7; (iii) LCN15 and GPR124; (iv) LCN15 and MYCL; (v) LCN15 and ARHGEF10L; (vi) ITGA2B and TGFBI; (vii) ITGA2B and PLA2G7; (viii) ITGA2B and GPR124; (ix) ITGA2B and MYCL; (x) ITGA2B and ARHGEF10L; (xi) MYL9 and TGFBI; (xii) MYL9 and PLA2G7; (xiii) MYL9 and GPR124; (xiv) MYL9 and MYCL; (xv) MYL9 and ARHGEF10L; (xvi) CMTM5 and TGFBI; (xvii) CMTM5 and PLA2G7; (xviii) CMTM5 and GPR124; (xix) CMTM5 and MYCL; (xx) CMTM5 and ARHGEF10L; (xxi) PPBP and TGFBI; (xxii) PPBP and PLA2G7; (xxiii) PPBP and GPR124; (xxiv) PPBP and MYCL; (xxv) PPBP and ARHGEF10L; (xxvi) TREML1 and TGFBI; (xxvii) TREML1 and PLA2G7; (xxviii) TREML1 and GPR124; (xxxix) TREML1 and MYCL; (xl) TREML1 and ARHGEF10L; (xli) PF4 and TGFBI; (xlii) PF4 and PLA2G7; (xliii) PF4 and GPR124; (xliv) PF4 and MYCL; and (xlv) PF4 and ARHGEF10L; (xlvi) LCN2 and GPR124; (xlvii) LCN2 and TGFBI; (xlviii) LCN2 and PLA2G7; (xlix) LCN2 and MYCL; (1) LCN2 and ARHGEF10L; (li) ITGB3 and GPR124; (lii) ITGB3 and TGFBI; (liii) ITGB3 and PLA2G7; (liv) ITGB3 and MYCL; and (lv) ITGB3 and ARHGEF10L.
The one or more additional biomarker for sepsis (described herein) and/or the one or more additional biomarker SIRS (described herein) may also be used together with these combinations of biomarkers in the distinguishing method described herein.
In a preferred embodiment, the method for distinguishing between sepsis and SIRS in a patient may comprise:
Preferably, the one or more biomarker for sepsis comprises the combination of ITGB3, ITGA2B, MYL9, LCN2, and TREML1 and the one or more biomarker for SIRS comprises the combination of: PLA2G7, ARHGEF10L, MYCL, and TGFB. The patient used in this method has preferably been diagnosed as having a systemic inflammatory condition (e.g preferably using the method described for diagnosis of a systemic inflammatory condition).
All embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step for making a conclusion about the disease status of the patient as defined above for the “method for diagnosing SIRS” and the “method for diagnosing sepsis” apply equally to the “method for distinguishing between sepsis and SIRS in a patient”. This includes all embodiments relating to the reference value used in these methods.
As described herein, the present inventors observed that the sepsis biomarkers increased in abundance in patients having sepsis as compared to patients having SIRS, as well as healthy individuals. Likewise, the SIRS biomarkers were observed to increase in abundance in patients having SIRS as compared to patients having sepsis, as well as healthy individuals. These differences in marker abundance can be used to determine whether an individual has or is at risk of developing sepsis and/or SIRS.
For example, by comparing the presence and/or amount of markers quantified in a sample obtained from a patient to the presence and/or amount of markers quantified for a reference value (such as a reference value that is representative of a healthy individual (or a population of healthy individuals), a reference value that is representative of an individual having sepsis (or a population of individuals having sepsis), and/or a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS)), it is possible to diagnose the presence (or absence) of sepsis and/or SIRS in a patient. The method permits classification of the individual as belonging to or not belonging to the reference population (ie. by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the individual's marker profile (ie. the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the individual falls (or does not fall) within the reference population.
All embodiments described above (in the context of the methods for diagnosis of sepsis) for the classification of a patient as having or being at risk of having sepsis (or not having or not being at risk of having sepsis) in the method for diagnosis of sepsis in a patient apply equally to the method for distinguishing between sepsis and SIRS in a patient. Likewise, all embodiments described above (in the context of the methods for diagnosis of SIRS) for the classification of a patient as having or being at risk of having SIRS (or not having or not being at risk of having SIRS) in the method for diagnosis of SIRS in a patient apply equally to the method for distinguishing between sepsis and SIRS in a patient. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may be as defined above for the “method of diagnosing a systemic inflammatory condition in a patient”, the “method for diagnosing sepsis in a patient” and the “method for diagnosing SIRS in a patient”. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value is representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, the reference value is representative of an individual having sepsis (or a population of individuals having sepsis). For example, the reference value may be representative of an individual having abdominal sepsis and/or an individual having pulmonary sepsis (or a population of individuals having abdominal sepsis and/or a population of individuals having pulmonary sepsis).
As described herein, the present inventors observed that the “SIRS” biomarkers described herein each increase in abundance in samples obtained from patients having SIRS, as compared to healthy individuals. Likewise, the “sepsis” biomarkers were also observed to increase in abundance in samples obtained from patients having sepsis, as compared to healthy individuals. Detection of increased levels of the “SIRS” biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of SIRS. Whilst detection of increased levels of the “sepsis” biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to diagnose the presence of sepsis. By combining the results from these analyses, a patient can be diagnosed as having sepsis or SIRS.
Thus, in one embodiment, when the reference value is representative of a healthy individual (or a population of healthy individuals), an increase in the one or more biomarker (and/or one or more additional biomarker) for SIRS in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has or is at risk of developing SIRS. Likewise, no increase or a decrease in the one or more SIRS biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have SIRS. Further confirmation of the diagnosis may be obtained when no increase is observed in the one or more biomarker for sepsis, in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual. This indicates that the patient has or is at risk of developing SIRS but does not have sepsis. Furthermore, no increase or a decrease in the one or more SIRS biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, may indicate that the patient has or is at risk of having sepsis (e.g. where the patient has already been diagnosed as having a systemic inflammatory condition).
An increase in the one or more biomarker (and/or one or more additional biomarker) for sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has or is at risk of developing sepsis. Likewise, no increase or a decrease in the one or more sepsis biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have sepsis. Further confirmation of the diagnosis may be obtained when no increase is observed in the one or more biomarker for SIRS, in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual. This indicates that the patient has or is at risk of developing sepsis but does not have SIRS. Furthermore, no increase or a decrease in the one or more sepsis biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, may indicate that the patient has or is at risk of having SIRS (e.g., where the patient has already been diagnosed as having a systemic inflammatory condition).
Furthermore, the patient may be diagnosed as having sepsis and SIRS. The patient may be diagnosed as having sepsis and SIRS when an increase is observed in the one or more biomarker for sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual; and an increase is observed in the one or more biomarker for SIRS in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual.
For some of the SIRS biomarkers identified by the present inventors, increased levels of these markers were also observed in patients having sepsis as compared to healthy individuals, although much bigger increases were observed for these biomarkers in the patients having SIRS. Similarly, for some of the sepsis biomarkers identified by the present inventors, increased levels of these markers were also observed in patients having SIRS as compared to healthy individuals, although much bigger increases were observed for these biomarkers in the patients having sepsis. The accuracy of the method for distinguishing between sepsis and SIRS in a patient can thus be improved by looking for a “minimum” fold increase or % increase in the levels of the one or more sepsis biomarker and the one or more SIRS biomarker as compared to the corresponding reference value that is representative of a healthy individual. The fold increase or % increase may be as defined above for the method for diagnosis of a systemic inflammatory condition, the method for diagnosis of sepsis and the method for diagnosis of SIRS.
In one embodiment, the minimum fold increase for the one or more sepsis biomarker (e.g., LCNI5, LCN2, ITGA2B, MYL9, ITGB3, CMTM5, PPBP, TREML1, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B) is as defined above for the method for diagnosing sepsis in a patient. For example, for the biomarker LCN15, an increase of at least 1 (e.g., at least 1.1, at least 1.2, at least 1.3, at least 1.4, or at least 1.5) fold in LCN15 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing sepsis. In one embodiment, no increase, or an increase of less than 1 (e.g., less than 1.1, less than 1.2, less than 1.3, less than 1.4, less than 1.5) fold in LCN15 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing sepsis.
In one embodiment, the minimum fold increase for the one or more SIRS biomarker (e.g., GPR124, TGFBI, PLA2G7, MYCL, ARHGEF10L, IL1 RN, NLRP3, RBP4, and MPP3) is as defined above for the method for diagnosing SIRS in a patient. For example, for the GRP124 biomarker, an increase of at least 1.1 (e.g., at least 1.2, at least 1.3, at least 1.4, at least 1.5) fold in GPR124 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has, or is at risk of developing SIRS. In one embodiment, no increase, or an increase of less than 1.1 (e.g., less than 1.2, less than 1.3, less than 1.4, less than 1.5, less than 1.6) fold in GPR124 in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient does not have, or is not at risk of developing SIRS.
As described herein, the present inventors observed that the levels of the one or more SIRS biomarkers were elevated in patients having SIRS as compared to patients having sepsis. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients having sepsis can thus be used to diagnose the presence of SIRS. Thus, in one embodiment, when the reference value is representative of an individual (or population of individuals) having sepsis (such as abdominal sepsis and/or pulmonary sepsis), an increase in the one or more biomarker for SIRS (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having sepsis, indicates that the patient has or is at risk of developing SIRS. The increase in the one or more SIRS biomarker may be as defined above for the method for diagnosing SIRS described above. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having sepsis, indicates that the patient does not have SIRS.
As described herein, the present inventors observed that the levels of the one or more sepsis biomarkers were elevated in patients having sepsis as compared to patients having SIRS. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients having SIRS can thus be used to diagnose the presence of sepsis. Thus, in one embodiment, when the reference value is representative of an individual (or population of individuals) having SIRS, an increase in the one or more sepsis biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient has or is at risk of developing sepsis (such as abdominal sepsis and/or pulmonary sepsis). The increase in the one or more sepsis biomarker may be as defined above for the method for diagnosing sepsis described above. Likewise, no increase in the one or more biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS, indicates that the patient does not have sepsis.
As described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the method of the invention may involve the use of multiple separate reference values. All combinations of reference values defined above for the “method for diagnosing a systemic inflammatory condition in a patient” apply equally to the method for distinguishing between sepsis and SIRS.
For example, the reference value used in the method may comprise: (i) a reference value that is representative of an individual (or population of individuals) having sepsis and a separate reference value that is representative of an individual (or population of individuals) having SIRS. In one embodiment, the patient may be diagnosed as having sepsis and SIRS, when an increase is observed in the one or more biomarker for sepsis in the sample obtained from the patient relative to the corresponding reference value representative of an individual having SIRS; and an increase is observed in the one or more biomarker for SIRS in the sample obtained from the patient relative to the corresponding reference value representative of an individual having sepsis.
The method for distinguishing between sepsis and SIRS in a patient as described herein can be used in a decision tree process to investigate the health of a patient having or suspected of having a systemic inflammatory condition. For example, the method for distinguishing sepsis and SIRS in a patient can be performed before, after, or in addition to any of the other methods of the invention described herein.
In one embodiment, the method for distinguishing sepsis and SIRS in a patient is performed as described herein. If the patient tests positive for sepsis, the patient may be further tested for sepsis, abdominal sepsis and/or pulmonary sepsis using the diagnostic methods described herein, so as to confirm whether the patient has or is at risk of developing sepsis, and/or determine whether the patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis. If the patient tests positive for SIRS, the patient may be further tested for SIRS using the diagnostic method described herein, so as to confirm whether the patient has or is at risk of developing SIRS.
In one embodiment, the method for distinguishing sepsis and SIRS in a patient (as described herein) can be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). For example, if the patient tests positive for a systemic inflammatory condition (using the method for diagnosing whether a patient has a systemic inflammatory condition), they may be tested using the distinguishing method described herein to determine whether they have sepsis and/or SIRS. In one embodiment, the above combination of methods are performed as described, and if the patient tests positive for sepsis, the patient may be further tested for sepsis, abdominal sepsis and/or pulmonary sepsis using the diagnostic methods described herein, so as to confirm whether the patient has or is at risk of developing sepsis, and/or to determine whether the patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis. In one embodiment, the above combination of methods are performed as described, and if the patient tests positive for SIRS, the patient may be further tested for SIRS using the diagnostic method described herein, so as to confirm whether the patient has or is at risk of developing SIRS.
The above described combination of methods may also be performed in parallel to determine the disease status of a patient by simultaneously (or substantially simultaneously) investigating the expression of all the biomarkers in a sample obtained from the patient, and determining whether the patient has or is at risk of having a systemic inflammatory condition, sepsis (such as abdominal or pulmonary sepsis) and/or SIRS.
When performing these different methods in a decision tree process, the sample used in each step of the method may be the same sample obtained from the patient (as described herein). When the method comprises multiple quantification steps, these multiple steps may be performed at the same time (e.g., in parallel) and/or using the same sample. When the method comprises multiple comparison steps, these multiple steps may be performed at the same time (e.g., in parallel).
For example, the method for distinguishing between sepsis and SIRS in a patient may comprise:
In a further example, a method may be performed to distinguish between sepsis and SIRS in a patient, comprising;
In a related aspect, the present invention also provides the use of one or more biomarker for sepsis (as described herein), and/or one or more biomarker for SIRS (as described herein) for distinguishing between sepsis and SIRS in a patient.
In one embodiment, the invention provides the use of one or more biomarker for sepsis selected from the group consisting of: ITGA2B, ITGB3, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, and CLEC1B, and/or one or more biomarker for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, GPR124, IL1RN, NLRP3, RBP4, and MPP3, for distinguishing between sepsis and SIRS in a patient. In one embodiment, the invention provides the use of one or more biomarker for sepsis selected from the group consisting of: ITGA2B, ITGB3, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4, and/or one or more biomarker for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124, for distinguishing between sepsis and SIRS in a patient. In one embodiment, the invention provides the use of one or more biomarker for sepsis selected from the group consisting of: ITGA2B, ITGB3, MYL9, LCN2, and TREML1, and/or one or more biomarker for SIRS selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL and TGFBI, for distinguishing between sepsis and SIRS in a patient. In one embodiment, the invention provides the use of the sepsis biomarkers: ITGA2B, ITGB3, MYL9, LCN2, and TREML1, and/or the SIRS biomarkers PLA2G7, ARHGEF10L, MYCL, and TGFBI, for distinguishing between sepsis and SIRS in a patient. In one embodiment, the invention provides the use of the sepsis biomarkers: ITGA2B, ITGB3, MYL9, LCN2, and TREML1, and/or the SIRS biomarkers ARHGEF10L, MYCL, and TGFBI, for distinguishing between sepsis and SIRS in a patient.
All embodiments described above for the method of distinguishing between sepsis and SIRS in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “sepsis”, “SIRS”, “patient”, “sample”, “the one or more biomarker for sepsis”, and “the one or more biomarker for SIRS” (including all combinations of sepsis and SIRS biomarkers described above).
At present, there is no clinical test available for distinguishing between abdominal sepsis and pulmonary sepsis. Rapid diagnosis of the physiological origin of sepsis in a patient would however be useful for selecting the most appropriate treatment for patients having sepsis. The present inventors have identified a set of biomarkers that is predictive of abdominal sepsis and a separate set of biomarkers that is predictive of pulmonary sepsis in patients. Using these distinct sets of biomarkers, the present inventors have developed a rapid and sensitive way to distinguish between abdominal sepsis and pulmonary sepsis in a patient by simultaneously quantifying one or more biomarker for abdominal sepsis and/or one or more biomarker for pulmonary sepsis in a sample obtained from a patient, so as to determine whether the patient has a biomarker profile that is predictive of abdominal or pulmonary sepsis.
The present invention therefore provides a method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, comprising:
As illustrated in Example 2, the distinguishing method of the invention can be performed using only one or more of the abdominal sepsis biomarker described herein, or only one or more of the pulmonary sepsis biomarkers described herein. These biomarkers can be used on their own to distinguish abdominal and pulmonary sepsis because their expression correlates with the patient's disease condition (i.e., the presence and/or amount of these biomarkers depends on whether a patient has abdominal sepsis or pulmonary sepsis or is healthy). Determining the presence and/or amount of either of these biomarkers and comparing this to a corresponding reference value (such as a reference value that is representative of a healthy individual, an abdominal sepsis patient and/or a pulmonary sepsis patient) therefore allows the disease status of the patient to be determined.
In one embodiment, the present invention therefore provides a method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, comprising:
In one embodiment, the present invention therefore provides a method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, comprising:
Alternatively, the abdominal and pulmonary sepsis biomarkers can be used in combination to distinguish between abdominal sepsis and pulmonary sepsis.
In one embodiment, the present invention provides a method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, comprising:
All embodiments described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the “method for diagnosing abdominal sepsis” and the “method for diagnosing pulmonary sepsis” apply equally to the “method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient”. This includes all embodiments relating to the “sample”, “patient”, “biomarker”, and “reference value”, and all embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step for making a conclusion as to the disease status of the patient.
As used herein, “distinguishing between abdominal sepsis and pulmonary sepsis” means to determine whether a patient has or is at risk of developing abdominal sepsis and/or pulmonary sepsis. For example, it may involve determining whether a patient has or is at risk of developing abdominal sepsis or pulmonary sepsis. For example, it may involve determining whether a patient has or is at risk of developing abdominal sepsis and pulmonary sepsis. This may involve distinguishing between a group (ie. one or more) of patients having abdominal sepsis and a group (i.e., one or more) of patients having pulmonary sepsis. In one embodiment, this may involve diagnosing or determining whether a patient has or is at risk of developing one or more systemic inflammatory condition selected from: abdominal sepsis and pulmonary sepsis.
The systemic inflammatory conditions “abdominal sepsis” and “pulmonary sepsis” are as described above for the “method for diagnosing a systemic inflammatory condition in a patient”.
The “patient” for which diagnosis is performed is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having a systemic inflammatory condition using the method described herein. In one embodiment, the patient is suspected of having or being at risk of developing sepsis. In one embodiment, the patient has been diagnosed as having sepsis (eg. using the methods described herein for diagnosis of sepsis, or for distinguishing between sepsis and SIRS). In one embodiment, the patient is suspected of having or being at risk of developing abdominal sepsis and/or pulmonary sepsis. In one embodiment, the patient is suspected of having or being at risk of developing abdominal sepsis. In one embodiment, the patient is suspected of having or being at risk of developing pulmonary sepsis.
The “sample” obtained from the patient is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the “method for diagnosing abdominal sepsis” and the “method for diagnosing pulmonary sepsis”, including all embodiments relating to the time point at which the sample is obtained.
The “one or more biomarker” of the invention is as described above for the “method for diagnosing a systemic inflammatory condition in a patient”. In one embodiment, the “one or more biomarker” is a nucleic acid, as defined herein. In one embodiment, the “one or more biomarker” is a protein, as defined herein.
The “one or more biomarker for abdominal sepsis” is as described above for the method for diagnosis of abdominal sepsis in a patient and includes any of the one or more abdominal sepsis biomarkers described herein (with or without the one or more additional biomarker) and further includes any of the combinations of abdominal sepsis biomarkers described herein.
For example, the one or more biomarker for abdominal sepsis may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. For example, the one or more biomarker for abdominal sepsis may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF. For example, the one or more biomarker for abdominal sepsis may be selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB. For example, the one or more biomarker for abdominal sepsis may be selected from the group consisting of: SLC39A8, CIQC, and CIQA.
Each of the biomarkers of abdominal sepsis may be used alone, or in combination with any of the abdominal sepsis biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or up to and including all of the abdominal sepsis biomarkers may be used to diagnose abdominal sepsis in a patient according to the method of the invention.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or all 6) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used as a biomarker for abdominal sepsis in the distinguishing method. For example, the combination of SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB may be used. In one embodiment, any combination of 1 or more (e.g., 2 or more, or all 3) of the biomarkers selected from the group consisting of: SLC39A8, CIQC, and CIQA may be used as a biomarker for abdominal sepsis in the distinguishing method. For example, the combination of SLC39A8, CIQC, and CIQA may be used.
As described above for the method for diagnosis of abdominal sepsis in a patient, one or more additional biomarker for abdominal sepsis may also be used in the distinguishing method for determining the presence (or absence) of abdominal sepsis in a patient. All embodiments described above for the one or more additional biomarker used in the method for diagnosis of abdominal sepsis in a patient apply equally to the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient.
In a preferred embodiment, the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, may comprise:
The “one or more biomarker for pulmonary sepsis” is as described above for the method for diagnosis of pulmonary sepsis in a patient and includes any of the one or more pulmonary sepsis biomarkers described herein (with or without the one or more additional biomarker), and further includes any of the combinations of pulmonary sepsis biomarkers described herein.
For example, the one or more biomarker for pulmonary sepsis may be selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44. For example, the one or more biomarker for pulmonary sepsis may be selected from the group consisting of: HCAR2, CXCR1, and DISC1. For example, the one or more biomarker for pulmonary sepsis may be selected from the group consisting of: HCAR2 and CXCR1.
Each of the biomarkers of pulmonary sepsis may be used alone, or in combination with any of the pulmonary sepsis biomarkers in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, or up to and including all of the pulmonary sepsis biomarkers may be used to diagnose pulmonary sepsis in a patient according to the method of the invention.
In one embodiment, any combination of 1 or more (eg. 2 or more, or all 3) of the biomarkers selected from the group consisting of: HCAR2, CXCR1, and DISC1, may be used as a biomarker for pulmonary sepsis in the distinguishing method. For example, the combination of HCAR2, CXCR1, and DISC1 may be used. In one embodiment, HCAR2 and/or CXCR1 may be used as a biomarker for pulmonary sepsis in the distinguishing method.
As described above for the method for diagnosis of pulmonary sepsis in a patient, one or more additional biomarker for pulmonary sepsis may also be used in the distinguishing method for determining the presence (or absence) of pulmonary sepsis in a patient. All embodiments described above for the one or more additional biomarker used in the method for diagnosis of pulmonary sepsis in a patient apply equally to the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient.
In a preferred embodiment, the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, may comprise:
As illustrated in Example 2, effective results for distinguishing between abdominal and pulmonary sepsis can be achieved by using the abdominal sepsis in conjunction with the pulmonary sepsis biomarkers. Any combination of the one or more biomarker for abdominal sepsis described herein (including the one or more additional biomarker for abdominal sepsis) may be used in conjunction with any combination of the one or more biomarker for pulmonary sepsis described herein (including the one or more additional biomarker for pulmonary sepsis) in the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more up to and including all) of the abdominal sepsis biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1, may be used in conjunction with any combination of 1 or more (eg. 2 or more, 3 or more, 4 or more, up to and including all) of the pulmonary sepsis biomarkers selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more up to and including all) of the abdominal sepsis biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, and KIF2C, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, up to and including all) of the pulmonary sepsis biomarkers selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, up to and including all) of the abdominal sepsis biomarkers selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used in conjunction with any combination of 1 or more (e.g., 2 or more, up to and including all) of the pulmonary sepsis biomarkers selected from the group consisting of: HCAR2, CXCR1, and DISC1, to distinguish between sepsis and SIRS in a patient according to the method described herein. For example, the combination of abdominal sepsis biomarkers SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, may be used in conjunction with the combination of pulmonary sepsis biomarkers HCAR2, CXCR1, and DISC1, to distinguish between sepsis and SIRS in a patient according to the method described herein.
In one embodiment, any combination of 1 or more (e.g., 2 or more, up to and including all) of the abdominal sepsis biomarkers selected from the group consisting of: SLC39A8, CIQC, and CIQA, may be used in conjunction with the pulmonary sepsis biomarkers HCAR2 and/or CXCR1, to distinguish between sepsis and SIRS in a patient according to the method described herein. For example, the combination of the abdominal sepsis biomarkers SLC39A8, CIQC, and CIQA may be used in conjunction with the combination of the pulmonary sepsis biomarkers HCAR2, CXCR1, and DISC1, to distinguish between sepsis and SIRS in a patient according to the method described herein. For example, the combination of abdominal sepsis biomarkers SLC39A8, CIQC, and CIQA may be used in conjunction with the combination of pulmonary sepsis biomarkers HCAR2 and CXCR1 to distinguish between sepsis and SIRS in a patient according to the method described herein.
For example, the following combinations of abdominal sepsis and pulmonary sepsis biomarkers may be used to distinguish between abdominal sepsis and pulmonary sepsis according to the method described herein: (i) MRAS and EPSTI1; (ii) MRAS and DISC1; (iii) MRAS and CXCR1; (iv) MRAS and HCAR2; (v) MRAS and IFI44; (vi) PCOLCE2 and EPSTI1; (vii) PCOLCE2 and DISC1; (viii) PCOLCE2 and CXCR1; (ix) PCOLCE2 and HCAR2; (x) PCOLCE2 and IFI44; (xi) (xii) TMEM37 and EPSTI1; (xiv) TMEM37 and DISC1; (xv) TMEM37 and CXCR1; (xvi) TMEM37 and HCAR2; (xvii) TMEM37 and IFI44; (xviii) SLC39A8 and EPSTI1; (xix) SLC39A8 and DISC1; (xx) SLC39A8 and CXCR1; (xxi) SLC39A8 and HCAR2; (xxii) SLC39A8 and IFI44; (xxiii) KIF2C and EPSTI1; (xxiv) KIF2C and DISC1; (xxv) KIF2C and CXCR1; (xxvi) KIF2C and HCAR2; (xxvii) KIF2C and IFI44; (xxviii) CIQC and EPSTI1; (xxxix) CIQC and DISC1; (xl) CIQC and CXCR1; (xli) CIQC and HCAR2; (xlii) CIQC and IFI44; (xliii) CIQB and EPSTI1; (xliv) CIQB and DISC1; (xlivi) CIQB and CXCR1; (xlivii) CIQB and HCAR2; (xliviii) CIQB and IFI44; (xlix) CIQA and EPSTI1; (1) CIQA and DISC1; (li) CIQA and CXCR1; (lii) CIQA and HCAR2; (liii) CIQA and IFI44; (liv) TNF and EPSTI1; (lv) TNF and DISC1; (lvi) TNF and CXCR1; (lvii) TNF and HCAR2; (lviii) TNF and IF144.
In one embodiment, the biomarker IFI44 may be used to distinguish between abdominal sepsis and pulmonary sepsis in a patient (such as patient that has been diagnosed as having sepsis).
The one or more additional biomarker for abdominal sepsis (described herein) and/or the one or more additional biomarker pulmonary sepsis (described herein) may also be used together with these combinations of biomarkers in the distinguishing method described herein.
In a preferred embodiment, the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient, may comprise:
All embodiments relating to the step for “determining the presence and/or amount of one or more biomarker in a sample” and for the “comparison” step as described above for the “method for diagnosing abdominal sepsis” and the “method for diagnosing pulmonary sepsis” apply equally to the “method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient”. This includes all embodiments relating to the reference value used in these methods.
As described herein, the present inventors observed that some of the “abdominal sepsis” biomarkers (MRAS, PCOLCE2, TMEM37, SLC39A8, KIF2C, CIQC, CIQB, CIQA, TNF) described herein each increase in abundance in samples obtained from patients having abdominal sepsis, as compared to patients having pulmonary sepsis and/or healthy individuals, whilst others (IFI44, IFIT1, and RPGRIP1) decreased in abundance in samples obtained from patients having abdominal sepsis, as compared to patients having pulmonary sepsis and/or healthy individuals. Likewise, the “pulmonary sepsis” biomarkers described herein were also observed to increase in abundance in samples obtained from patients having pulmonary sepsis, as compared to patients having abdominal sepsis and/or healthy individuals. These differences in marker abundance can be used to determine whether an individual has or is at risk of developing abdominal sepsis and/or pulmonary sepsis.
For example, by comparing the amount of markers quantified in a sample obtained from a patient to the amount of markers quantified for a reference value (such as a reference value that is representative of a healthy individual (or a population of healthy individuals), a reference value that is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis), a reference value that is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis), and/or a reference value that is representative of an individual having SIRS (or a population of individuals having SIRS)), it is possible to diagnose the presence (or absence) of abdominal sepsis and/or pulmonary sepsis in a patient. The method permits classification of the individual as belonging to or not belonging to the reference population (ie. by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the individual's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the individual falls (or does not fall) within the reference population.
All embodiments described above (in the context of the method for diagnosis of abdominal sepsis) for the classification of a patient as having or being at risk of having (or not having or not being at risk of having) abdominal sepsis in the context of the method for diagnosis of abdominal sepsis apply equally to the method for distinguishing between abdominal sepsis and pulmonary sepsis. All embodiments described above (in the context of the method for diagnosis of pulmonary sepsis) for the classification as a patient as having or being at risk of having (or not having or not being at risk of having) pulmonary sepsis in the context of the method for diagnosis of pulmonary sepsis apply equally to the method for distinguishing between abdominal sepsis and pulmonary sepsis. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may be as defined above for the “method of diagnosing a systemic inflammatory condition in a patient”, the “method for diagnosing abdominal sepsis”, and the “method for diagnosing pulmonary sepsis”. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value is representative of an individual having SIRS (or a population of individuals having SIRS). In one embodiment, the reference value is representative of an individual having abdominal sepsis (or a population of individuals having abdominal sepsis). In one embodiment, the reference value is representative of an individual having pulmonary sepsis (or a population of individuals having pulmonary sepsis).
As described herein, the present inventors observed that the “abdominal sepsis” biomarkers SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF each increase in abundance in samples obtained from patients having abdominal sepsis, as compared to healthy individuals. Thus, in one embodiment, when the reference value is representative of a healthy individual (or a population of healthy individuals), an increase in the one or more biomarker (and/or one or more additional biomarker) for abdominal sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has or is at risk of developing abdominal sepsis. Likewise, no increase in the one or more abdominal sepsis biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have abdominal sepsis.
The inventors also observed a decrease in the abdominal sepsis biomarkers IFI44, IFIT1, and RPGRIP1 in samples obtained from patients having abdominal sepsis, as compared to healthy individuals. Thus, in one embodiment, when the reference value is representative of a healthy individual (or a population of healthy individuals), a decrease in the one or more biomarker (and/or one or more additional biomarker) for abdominal sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has or is at risk of developing abdominal sepsis. Likewise, no decrease in the one or more abdominal sepsis biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have abdominal sepsis.
Further confirmation of the diagnosis may be obtained when no increase is observed in the one or more biomarker for pulmonary sepsis, in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual. This indicates that the patient has or is at risk of developing abdominal sepsis but does not have pulmonary sepsis.
As described above, the inventors observed an increase in the “pulmonary sepsis” biomarkers (HCAR2, CXCR1, DISC1, EPSTI1, and IFI44) in samples obtained from patients having pulmonary sepsis, as compared to healthy individuals. Thus, in one embodiment, when the reference value is representative of a healthy individual (or a population of healthy individuals), an increase in the one or more biomarker (and/or one or more additional biomarker) for pulmonary sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient has or is at risk of developing pulmonary sepsis. Likewise, no increase in the one or more pulmonary sepsis biomarker (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient does not have pulmonary sepsis.
Further confirmation of the diagnosis may be obtained when no increase (e.g., in any one or more of MRAS, PCOLCE2, TMEM37, SLC39A8, KIF2C, CIQC, CIQB, CIQA, TNF) and/or no decrease (e.g., in any one or more of IFI44, IFIT1, and RPGRIP1) is observed in the one or more biomarker for abdominal sepsis, in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual. This indicates that the patient has or is at risk of developing pulmonary sepsis but does not have abdominal sepsis.
Furthermore, the patient may be diagnosed as having abdominal sepsis and pulmonary sepsis when an increase is observed in any one or more of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, and TNF, and/or a decrease is observed in any one or more of: IFI44, IFIT1, and RPGRIP1, in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual; and an increase is observed in the one or more biomarker for pulmonary sepsis in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual.
The accuracy of the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient can be improved by looking for a “minimum” fold change in the levels of the one or more abdominal sepsis biomarker and the one or more pulmonary sepsis biomarker as compared to the corresponding reference value that is representative of a healthy individual. In one embodiment, the minimum fold change or % change for the abdominal sepsis biomarkers is as defined above for the method for diagnosing abdominal sepsis in a patient. In one embodiment, the minimum fold change increase or % increase for the pulmonary sepsis biomarkers is as defined above for the method for diagnosing pulmonary sepsis in a patient.
In one embodiment, the reference value used in the distinguishing method may include a reference value that is representative of an individual having pulmonary sepsis. All embodiments described above for the method of diagnosing abdominal sepsis when using a reference value that is representative of an individual having pulmonary sepsis apply equally to the distinguishing method described herein.
For example, an increase or decrease in the one or more biomarker for abdominal sepsis (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis, indicates that the patient has or is at risk of developing abdominal sepsis. The increase or decrease may be as defined above for the method for diagnosing abdominal sepsis in a patient. No increase in the one or more biomarker for abdominal sepsis (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis, indicates that the patient may have or is at risk of developing pulmonary sepsis (e.g where the patient has already been diagnosed as having sepsis).
In one embodiment, the reference value used in the distinguishing method may include a reference value that is representative of an individual having abdominal sepsis. All embodiments described above for the method of diagnosing pulmonary sepsis when using a reference value that is representative of an individual having abdominal sepsis apply equally to the distinguishing method described herein.
For example, an increase in the one or more biomarker for pulmonary sepsis (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having abdominal sepsis, indicates that the patient has or is at risk of developing pulmonary sepsis. The increase may be as defined above for the method for diagnosing pulmonary sepsis in a patient. No increase in the one or more biomarker for pulmonary sepsis (and/or one or more additional biomarker) in the sample obtained from the patient relative to the corresponding reference value representative of an individual having abdominal sepsis, indicates that the patient may have or is at risk of developing abdominal sepsis (e.g., where the patient has already been diagnosed as having sepsis).
As described above for the “method for diagnosing a systemic inflammatory condition in a patient”, the method of the invention may involve the use of multiple separate reference values. All combinations of reference values defined above for the “method for diagnosing a systemic inflammatory condition in a patient” apply equally to the method for distinguishing between abdominal sepsis and pulmonary sepsis. For example, the reference value used in the method may comprise: (i) a reference value that is representative of an individual (or population of individuals) having abdominal sepsis and a separate reference value that is representative of an individual (or population of individuals) having pulmonary sepsis.
In one embodiment, the patient may be diagnosed as having abdominal sepsis and pulmonary sepsis when an increase is observed in any one or more of: MRAS, PCOLCE2, TMEM37, SLC39A8, KIF2C, CIQC, CIQB, CIQA, and TNF, and/or a decrease is observed in any one or more of: IFI44, IFIT1, and RPGRIP1, in the sample obtained from the patient relative to the corresponding reference value representative of an individual having pulmonary sepsis; and an increase is observed in the one or more biomarker for pulmonary sepsis in the sample obtained from the patient relative to the corresponding reference value representative of an individual having abdominal sepsis.
The method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient as described herein can be used in a decision tree process to investigate the health of a patient having or suspected of having a systemic inflammatory condition. For example, the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient can be performed before, after, or in addition to any of the other methods of the invention described herein.
In one embodiment, the method of the invention for distinguishing between abdominal sepsis and pulmonary sepsis in a patient can be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein). If the patient tests positive for a systemic inflammatory condition (using the method of the invention for diagnosing whether a patient has a systemic inflammatory condition), they may be tested using the distinguishing method described herein to determine whether they have abdominal sepsis and/or pulmonary sepsis.
In one embodiment, the method of the invention for distinguishing between abdominal sepsis and pulmonary sepsis in a patient can be performed subsequent to (or in addition to) the method for distinguishing between sepsis and SIRS in a patient (as described herein). If the patient tests positive for sepsis (using the method distinguishing between sepsis and SIRS in a patient), they may be tested using the distinguishing method described herein to determine whether they have abdominal sepsis and/or pulmonary sepsis.
In one embodiment, the method of the invention for distinguishing between abdominal sepsis and pulmonary sepsis in a patient can be performed subsequent to (or in addition to) the method for diagnosing sepsis in a patient (as described herein). If the patient tests positive for sepsis (using the method diagnosing sepsis), they may be tested using the distinguishing method described herein to determine whether they have abdominal sepsis and/or pulmonary sepsis.
In one embodiment, the method of the invention for distinguishing between abdominal sepsis and pulmonary sepsis in a patient can be performed subsequent to (or in addition to) the method for diagnosing whether a patient has a systemic inflammatory condition (as described herein), the method for distinguishing between sepsis and SIRS in a patient (as described herein), and/or the method for diagnosing sepsis (as described herein). If the patient tests positive for a systemic inflammatory condition (using the method of the invention for diagnosing whether a patient has a systemic inflammatory condition), they may be tested for sepsis using the method for distinguishing between sepsis and SIRS described herein, and/or the method for diagnosis of sepsis described herein. If the patient tests positive for sepsis, they may be tested using the distinguishing method described herein to determine whether they have abdominal sepsis and/or pulmonary sepsis.
In one embodiment, the patient may be tested for sepsis (using the method for distinguishing between sepsis and SIRS described herein, and/or the method for diagnosis of sepsis described herein). If the patient tests positive for sepsis, they may be tested using the distinguishing method described herein to determine whether they have abdominal sepsis and/or pulmonary sepsis.
The above described combination of methods may also be performed in parallel to determine the disease status of a patient by simultaneously (or substantially simultaneously) investigating the expression of all the biomarkers in a sample obtained from the patient, and determining whether the patient has or is at risk of having abdominal sepsis and/or pulmonary sepsis.
When performing these different methods in a decision tree process, the sample used in each step of the method may be the same sample obtained from the patient (as described herein). When the method comprises multiple quantification steps, these multiple steps may be performed at the same time (e.g., in parallel) and/or using the same sample. When the method comprises multiple comparison steps, these multiple steps may be performed at the same time (e.g., in parallel).
In a related aspect, the present invention also provides the use of one or more biomarker for abdominal sepsis (as described herein), and/or one or more biomarker for pulmonary sepsis (as described herein) for distinguishing between abdominal sepsis and pulmonary sepsis in a patient.
In one embodiment, the invention provides the use of one or more biomarker for abdominal sepsis selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C TNF, IFI44, IFIT1 and RPGRIP1, and/or one or more biomarker for pulmonary sepsis selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44, for distinguishing between abdominal sepsis and pulmonary sepsis in a patient.
In one embodiment, the invention provides the use of one or more biomarker for abdominal sepsis selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C and TNF, and/or one or more biomarker for pulmonary sepsis selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44, for distinguishing between abdominal sepsis and pulmonary sepsis in a patient.
In one embodiment, the invention provides the use of one or more biomarker for abdominal sepsis selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB, and/or one or more biomarker for pulmonary sepsis selected from the group consisting of: HCAR2, CXCR1, and DISC1, for distinguishing between abdominal sepsis and pulmonary sepsis in a patient. For example, the abdominal sepsis biomarkers: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB may be used in combination with the pulmonary sepsis biomarkers: HCAR2, CXCR1, and DISC1.
In one embodiment, the invention provides the use may be of one or more biomarker for abdominal sepsis selected from the group consisting of: SLC39A8, CIQC, and CIQA, and/or one or more biomarker for pulmonary sepsis selected from the group consisting of: HCAR2, CXCR1, and DISC1, for distinguishing between abdominal sepsis and pulmonary sepsis in a patient. For example, the abdominal sepsis biomarkers: SLC39A8, CIQC, and CIQA may be used in combination with the pulmonary sepsis biomarkers: HCAR2, CXCR1, and DISC1.
In one embodiment, the invention provides the use of one or more biomarker for abdominal sepsis selected from the group consisting of: SLC39A8, CIQC, and CIQA, and/or one or more biomarker for pulmonary sepsis selected from the group consisting of: HCAR2 and CXCR1, for distinguishing between abdominal sepsis and pulmonary sepsis in a patient. For example, the abdominal sepsis biomarkers: SLC39A8, CIQC, and CIQA may be used in combination with the pulmonary sepsis biomarkers: HCAR2, and CXCR1.
All embodiments described above for the “method of distinguishing between abdominal sepsis and pulmonary sepsis in a patient” apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “abdominal sepsis”, “pulmonary sepsis”, “patient”, “sample”, “the one or more biomarker for abdominal sepsis”, and “the one or more biomarker for pulmonary sepsis”. All combinations of abdominal sepsis and pulmonary sepsis biomarkers described above for the method of distinguishing between abdominal sepsis and pulmonary sepsis in a patient apply equally to the ‘use’ of the invention described herein.
Monitoring a Systemic Inflammatory Condition
The progression of a patient from normalcy (i.e., a condition characterised by not having a systemic inflammatory condition) to having a systemic inflammatory condition is characterised by changes in biomarkers, as certain biomarkers are expressed at increasingly higher levels and the expression of other biomarkers becomes down regulated. The present inventors have identified biomarkers that both increase and decrease in abundance as a physiological response to a systemic inflammatory condition is established or subsides. A feature of a patient's biomarker profile that is known to change in intensity as a physiological response to a systemic inflammatory condition becomes established may therefore be selected for monitoring of a systemic inflammatory condition in a patient. A comparison of the same feature in a profile from a subsequent biological sample from the patient can establish whether the patient is developing a more severe form of the systemic inflammatory condition or is progressing towards normalcy. The present invention therefore also provides a method of monitoring a systemic inflammatory condition in a patient.
In one embodiment, the method of monitoring a systemic inflammatory condition comprises performing any of the methods of the invention for diagnosis of a systemic inflammatory condition (including those for diagnosis of sepsis, diagnosis of abdominal sepsis, diagnosis of pulmonary sepsis, diagnosis of SIRS, for distinguishing between sepsis and SIRS, and for distinguishing between abdominal sepsis and pulmonary sepsis, or any combination of these methods described herein) at a first time point, repeating the ‘quantification’ and ‘comparison’ steps of said method at one or more later time points, and comparing the presence and/or amount of each marker determined at said one or more later time point to the presence and/or amount of each marker determined at the first time point, to monitor the systemic inflammatory condition. All embodiments of the diagnostic methods described herein apply equally to the monitoring method of the invention.
By repeating the diagnostic method at one or more later time point, the disease status of the patient can be re-classified to determine whether there has been a change or no change in the disease status of the patient. For example, when the level of the one or more biomarker returns towards (or becomes increasingly statistically similar to) the level typically observed for the reference value representative of a healthy individual, and/or increasingly statistically deviates from the level typically observed for the reference value representative of a systemic inflammatory condition, this indicates that there has been an improvement or regression of the systemic inflammatory condition in the test individual. Likewise, when the level of the one or more biomarker increasingly statistically deviates from the level typically observed for the reference value representative of a healthy individual, and/or remains statistically similar to (or becomes increasingly statistically similar to) the level typically observed for the reference value representative of a systemic inflammatory condition, this indicates that there has been a worsening or progression of the systemic inflammatory condition in the test individual.
Monitoring of a systemic inflammatory condition in a patient may be used to monitor the recovery of a patient having a systemic inflammatory condition. As used herein, the term “recovery” refers to the survival of a patient having a systemic inflammatory condition. When a patient recovers from a systemic inflammatory condition, they no longer exhibit symptoms of the condition, and return to a normal (or near normal) state of health. In contrast, non-recovery from a systemic inflammatory condition means that the patient does not survive the systemic inflammatory condition. The symptoms of the condition in the patient generally worsen, and the patient may experience multiple organ failure resulting in death.
Monitoring of a systemic inflammatory condition in a patient may be used to monitor the severity of the systemic inflammatory condition in a patient. For example, the method of the invention may comprise monitoring of the progression, regression, aggravation, alleviation, or recurrence of the condition. Monitoring of a systemic inflammatory condition in a patient may comprise determining whether the systemic inflammatory condition is progressing towards a more severe form of the condition or regressing towards normalcy. Monitoring may also comprise determining whether the systemic inflammatory condition has remained stable.
As used herein, the term “progression” refers to an increase or worsening in the symptoms of a disease or disorder, and the term “regression” refers to a decrease or improvement in the symptoms of disease or disorder.
The monitoring method of the invention may be applied in the course of a medical treatment of the patient aimed at alleviating the monitored condition. In one embodiment, the monitoring method may be used to aid determination as to the correct course of treatment, permit evaluation of the effectiveness of treatment, and/or permit determination as to whether to continue or cease treatment. In a preferred embodiment, the method is used to monitor the effectiveness of a treatment regimen for a systemic inflammatory condition. Suitable therapies are as described herein for the treatment of sepsis and/or SIRS.
The monitoring method of the invention may also be used to make decisions about a patient, such as deciding whether a patient may be discharged, needs a change in treatment or needs further hospitalisation.
The monitoring method of the invention may be used to provide a means of disease staging and/or to permit determination as to clinical outcome. In one embodiment, the method may be used to monitor a patient for prognosis of recovery.
As used herein, the terms “prognosis” or “prognosticating” refers to an anticipation on the progression of a disease or condition and the prospect of recovery. A “good prognosis” (or a “prognosis of recovery”) refers to an anticipation of a satisfactory partial or complete recovery from the disease or condition. A “poor prognosis” (or a “prognosis of non-recovery”) encompasses anticipation of a substandard recovery and/or unsatisfactory recovery, or to substantially no recovery, or even further worsening of the disease or condition.
Monitoring of a systemic inflammatory condition can also be performed without an external reference value, by obtaining samples from the patient at different time points and comparing the marker profile of these samples to one another.
In one embodiment, the method for monitoring a systemic inflammatory condition in a patient, comprises:
The “systemic inflammatory condition” monitored using the method of the invention is as described above for the diagnostic methods described herein. In one embodiment, the systemic inflammatory condition is selected from one or more (eg. both) of SIRS and sepsis. In one embodiment, the systemic inflammatory condition is selected from one or more (eg. two or more or all 3) of SIRS, abdominal sepsis and pulmonary sepsis. In one embodiment, the systemic inflammatory condition is SIRS. In one embodiment, the systemic inflammatory condition is sepsis. In one embodiment, the systemic inflammatory condition is abdominal sepsis. In one embodiment, the systemic inflammatory condition is pulmonary sepsis.
In one embodiment, steps (i) and (ii) of the method involve “determining the presence and amount of the one or more biomarker in a sample obtained from a patient”, and step (iii) involves “comparing the presence and amount of the one or more biomarker determined in step (ii) to the presence and amount of the one or more biomarker determined in step (i)”. In one embodiment, steps (i) and (ii) of the method involve “determining the amount of the one or more biomarker in a sample obtained from a patient”, and step (iii) involves “comparing the amount of the one or more biomarker determined in step (ii) to the amount of the one or more biomarker determined in step (i)”.
The “patient” for which monitoring is performed is as defined above for the diagnostic methods described herein. In one embodiment, the patient is suspected of having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition using the method described herein for diagnosing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition using the methods described herein for diagnosing SIRS, sepsis (such as abdominal sepsis, or pulmonary sepsis), or using the method described herein for distinguishing between sepsis and SIRS in a patient, or for distinguishing between abdominal sepsis and pulmonary sepsis, or any combination of these methods described herein.
In one embodiment, the patient has been diagnosed as having SIRS (e.g., using the method described herein for diagnosis of SIRS, or for distinguishing between sepsis and SIRS in a patient). In one embodiment, the patient has been diagnosed as having sepsis, such as abdominal sepsis or pulmonary sepsis (e.g., using the methods described herein for diagnosis of sepsis, abdominal sepsis or pulmonary sepsis, the method described herein for distinguishing between sepsis and SIRS in a patient, or for distinguishing between abdominal sepsis and pulmonary sepsis, or any combination of these methods described herein). The patient may be undergoing treatment for a systemic inflammatory condition.
The “sample” obtained from the patient is as defined above for the diagnostic methods.
The monitoring methods described herein allow the monitoring of a systemic inflammatory condition in a patient over time. All embodiments relating to the time point at which a sample is obtained from the patient as described above for the diagnostic methods (e.g., in the method for diagnosing a systemic inflammatory condition in a patient) apply equally to the sample obtained from the patient at “a first (or earlier) time point” in the monitoring methods described herein. For example, the sample may be obtained at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 36 hours, 48 hours, 72 hours, 96 hours, or 120 hours, after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the sample may be obtained from the patient at least 24 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The sample obtained from the patient at the “one or more later time points” may be obtained at least 6 hours (e.g., at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month) after the sample was obtained from the patient at a first (or earlier) time point.
In one embodiment, when the method is for monitoring the effectiveness of a treatment regimen for a systemic inflammatory condition in a patient, the sample obtained from the patient at a first (or earlier) time point is obtained from the patient before or during the course of treatment. For example, the sample may be obtained from the patient at least 1 hour (e.g., at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours) before treatment. The sample obtained from the patient at one or more later time points is obtained during or after a course of treatment. For example, the sample may be obtained from the patient at least 1 hour (e.g., at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours) after a treatment regimen has begun or has been completed.
The “one or more biomarker” of the invention is as described above for the diagnostic methods described herein. The one or more biomarker used in the monitoring method of the invention may include any of the biomarkers described herein (e.g., as defined in Tables 1-4), or any combination of biomarkers described herein.
In addition to the biomarkers described above, the present inventors have also identified a set of biomarkers which are particularly useful for monitoring a systemic inflammatory condition in a patient. These biomarkers include ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, MX1, NECAB1, PKHD1 and LILRB5 (see Tables 1 and 4).
Thus, in one embodiment, the one or more biomarker is selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, MX1, NECAB1, PKHD1, and LILRB5. These biomarkers were observed to change in abundance over time in the samples obtained from patients having systemic inflammatory conditions (such as sepsis or SIRS), as the patients either recovered from the condition, or did not recover and subsequently died.
The reference to the biomarker HLA-DPB1 throughout the entire description, includes the HLA-DPB1 sequence encoded by SEQ ID NO: 30 and the transcript variant X1 of HLA-DPB1 (as encoded by SEQ ID NO:31). In one embodiment, the reference to the biomarker HLA-DPB1 is a reference to sequence encoded by SEQ ID NO: 30. In one embodiment, the reference to the biomarker HLA-DPB1 is a reference to the transcript variant X1 of HLA-DPB1 (as encoded by SEQ ID NO: 31).
Each of the biomarkers may be used alone, or in combination with any of the biomarkers described herein to monitor a systemic inflammatory condition in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, up to and including all of the biomarkers may be used to monitor a systemic inflammatory condition in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or all 21) of the biomarkers selected from the group consisting of: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, MX1, NECAB1, PKHD1, and LILRB5, may be used to monitor a systemic inflammatory condition in a patient (such as abdominal sepsis and/or SIRS). For example, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, or all 5) of the biomarkers selected from the group consisting of: KLRB1, BCL11B, FCER1A, PKHD1, and LILRB5, may be used to monitor a systemic inflammatory condition in a patient (such as sepsis and/or SIRS).
A sub-set of these biomarkers is particularly useful for monitoring sepsis (e.g., abdominal sepsis), including ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, and MX1. Thus, in one embodiment, the one or more biomarker is selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, and MX1. In one embodiment, the method of the invention is for monitoring of sepsis (eg. abdominal sepsis) in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, or all 18) of the biomarkers selected from the group consisting of: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCERlA, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, and MX1, may be used to monitor a systemic inflammatory condition in a patient (such as abdominal sepsis).
Within this sub-set of biomarkers, the inventors identified markers that increase in abundance over time as the patient recovers from abdominal sepsis (returning towards the elevated level typically observed for healthy individuals) but show no increase (or a decrease) in abundance when the patient does not recover from abdominal sepsis. These include one or more biomarker selected from: ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1. The inventors also identified that the biomarker NPPC increases significantly in abundance over time when the patient does not recover from abdominal sepsis and show no increase (or a decrease) in abundance over time when the patient does recover from abdominal sepsis (thereby returning towards the reduced level typically observed for healthy individuals). These biomarkers are particularly useful for monitoring abdominal sepsis in a patient, particularly for monitoring recovery from abdominal sepsis.
In one embodiment, the one or more biomarker is selected from: ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12) of the biomarkers selected from the group consisting of: ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1, may be used to monitor a systemic inflammatory condition in a patient (such as abdominal sepsis).
In one embodiment, the one or more biomarker is selected from one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11) of: ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1, in combination with the biomarker NPPC.
The inventors also observed that a sub-set of these biomarkers is particularly useful for monitoring SIRS, including ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, CD160, KLRF1, CD2, LGALS2, MYCL, NECAB1, and PKHD1. Thus, in one embodiment, the one or more biomarker is selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, CD160, KLRF1, CD2, LGALS2, MYCL, NECAB1, and PKHD1. In one embodiment, the method of the invention is for monitoring of SIRS in a patient.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or all 16) of the biomarkers selected from the group consisting of: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, CD160, KLRF1, CD2, LGALS2, MYCL, NECAB1, and PKHD1, may be used to monitor a systemic inflammatory condition in a patient (such as SIRS).
Within this sub-set of biomarkers, the inventors further identified a sub-set of markers that increase in abundance over time as the patient recovers from SIRS (returning towards the elevated levels typically observed for healthy individuals) but show no increase (or a decrease) in abundance when the patient does not recover from SIRS. These include one or more biomarker selected from: ITM2A, CCL5, KLRK1, KLRB1, and BCL11B. The inventors also identified that the biomarkers NPPC, PKDI, CD2, LGALS2, MYCL, NECAB1, and PKHD1 increase significantly in abundance over time when the patient does not recover from SIRS and show no increase (or a decrease) in abundance over time when the patient does recover from SIRS (thereby returning towards the reduced level typically observed for healthy individuals). These biomarkers are particularly useful for monitoring SIRS in a patient, particularly for monitoring recovery from SIRS.
In one embodiment, the one or more biomarker is selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, BCL11B, CD2, LGALS2, MYCL, NECAB1, and PKHD1. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or all 12) of the biomarkers selected from the group consisting of: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, BCL11B, CD2, LGALS2, MYCL, NECAB1, and PKHD1, may be used to monitor a systemic inflammatory condition in a patient (such as SIRS).
For example, the one or more biomarker may be selected from: ITM2A, CCL5, NPPC, PKD1, LGALS2, MYCL, NECAB1, and PKHD1. For example, the one or more biomarker may be selected from: CCL5, NPPC, PKD1, LGALS2, MYCL, NECAB1, and PKHD1. For example, the one or more biomarker may be selected from: CCL5, NPPC, PKD1, LGALS2, NECAB1, and PKHD1. For example, the one or more biomarker may be selected from: CCL5, NPPC, PKDI, NECAB1, and PKHD1.
In one embodiment, the one or more biomarker is selected from one or more of: ITM2A, CCL5, KLRK1, KLRB1, and BCL11B, in combination with one or more biomarker selected from: NPPC, PKDI, CD2, LGALS2, MYCL, NECAB1, and PKHD1. For example, the one or more biomarker may be selected from one (e.g., both) or more of: ITM2A and CCL5, in combination with one or more (e.g., 2 or more, 3 or more, or all 4) biomarker selected from: NPPC, PKDI, NECAB1, and PKHD1. For example, the one or more biomarker may be CCL5 used in combination with one or more biomarker selected from: NPPC, PKD1, LGALS2, MYCL, NECAB1, and PKHD1. For example, the one or more biomarker may be CCL5 used in combination with one or more biomarker selected from: NPPC, PKD1, NECAB1, and PKHD1.
The present inventors have identified biomarkers that can be used to monitor multiple different types of systemic inflammatory condition (such SIRS and sepsis) in a single method. In one embodiment, the one or more (eg. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) biomarker is selected from: ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, and HLA-DPB1. In one embodiment, the one or more (2 or more, 3 or more, 4 or more, or all 5) biomarker is selected from: ITM2A, CCL5, NPPC, KLRK1, and BCL11B. In one embodiment, the one or more (2 or more, or all 3) biomarker is selected from: ITM2A, CCL5, and NPPC. For example, the one or more biomarker is selected from: CCL5 and NPPC. In one embodiment, the one or more biomarker may be one or more (or both) of: ITM2A and CCL5 in combination with NPPC.
In one embodiment, the one or more biomarker is FCER1A. In one embodiment, the one or more biomarker is KLRK1. In one embodiment, the one or more biomarker is KLRB1. In one embodiment, the one or more biomarker is DAAM2. In one embodiment, the one or more biomarker is HLA-DRA. In one embodiment, the one or more biomarker is BCL11B. In one embodiment, the one or more biomarker is SLAMF6. In one embodiment, the one or more biomarker is ITM2A. In one embodiment, the one or more biomarker is CD160. In one embodiment, the one or more biomarker is HLA-DPB1. In one embodiment, the one or more biomarker is KLRF1. In one embodiment, the one or more biomarker is CD2. In one embodiment, the one or more biomarker is LGALS2. In one embodiment, the one or more biomarker is NPPC. In one embodiment, the one or more biomarker is MYCL. In one embodiment, the one or more biomarker is MX1. In one embodiment, the one or more biomarker is NECAB1. In one embodiment, the one or more biomarker is NECAB2. In one embodiment, the one or more biomarker is PKHD1. In one embodiment, the one or more biomarker is PKD1. In one embodiment, the one or more biomarker is CCL5. In one embodiment, the one or more biomarker is LILRB5.
All embodiments of the ‘quantification’ and ‘comparison’ steps of the diagnostic methods described herein (such as the method for diagnosis of a systemic inflammatory condition) apply equally to the monitoring methods described herein.
The step of “comparing the presence and/or amount determined in step (ii) to the presence and/or amount determined in step (i)” involves determining whether there is a difference in the presence and/or amount of the one or more biomarkers between the samples. It is possible to monitor a systemic inflammatory condition by attributing the finding of a difference or no difference in the one or more biomarker to a change in the systemic inflammatory condition in the individual between the two or more successive time points.
A finding of “no difference” in the presence and/or amount of the one or more biomarker detected in the two or more successive time points indicates that there has been no change in the systemic inflammatory condition in the individual. In contrast, finding of a “difference” in the presence and/or amount of the one or more biomarker detected in the two or more successive time points indicates that there has been a change in the systemic inflammatory condition in the individual.
A difference in the presence and/or amount of the one or more biomarker measured by the monitoring methods of the present invention can comprise an increase or decrease in the one or more biomarkers over time. The increase or decrease in the biomarker can be, for example, at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, or at least 10) fold over time. The difference in the presence and/or amount of the biomarker is preferably statistically significant. By “statistically significant”, it is meant that the alteration is greater than what might be expected to happen by chance alone.
The increase or decrease in the one or more biomarker in the patient over time can indicate progression of the disease, the lack of efficacy of one or more treatment regimens, and/or a poor prognosis of recovery (or a prognosis of non-recovery). Alternatively, the increase or decrease in the one or more biomarker in the patient over time can indicate regression of the disease, the success of one or more treatment regimens, and/or a good prognosis of recovery (or a prognosis of recovery).
For example, an increase in any one or more of ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates regression of the systemic inflammatory condition in the patient. No increase in ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, or MX1 in the sample obtained at the later time point relative to the sample obtained from the first time indicates no regression of the systemic inflammatory condition in the patient. In one embodiment, no increase in ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, or MX1 in the sample obtained at the later time point relative to the sample obtained from the first time point may indicate progression of the systemic inflammatory condition in the patient.
For example, an increase in any one or more of ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates the success of one or more treatment regimens. No increase in ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, or MX1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates the lack of efficacy of one or more treatment regimens.
For example, an increase in any one or more of ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1 in the sample obtained at the later time v relative to the sample obtained from the first time point indicates a (good) prognosis of recovery. No increase in ITM2A, CCL5, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, or MX1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates a poor prognosis of recovery (or a prognosis of non-recovery).
In a further example, an increase in any one or more of NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, and PKHD1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates progression of the systemic inflammatory condition in the patient. No increase in NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, or PKHD1, in the sample obtained at the later time point relative to the sample obtained from the first time point indicates no progression of the systemic inflammatory condition in the patient. In one embodiment, no increase in NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, or PKHD1, in the sample obtained at the later time point relative to the sample obtained from the first time point indicates regression of the systemic inflammatory condition in the patient.
In a further example, an increase in any one or more of NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, and PKHD1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates the lack of efficacy of one or more treatment regimens. No increase in NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, or PKHD1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates the success of one or more treatment regimens.
In a further example, an increase in any one or more of NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, and PKHD1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates a poor prognosis of recovery (or a prognosis of non-recovery). No increase in NPPC, PKD1, CD2, LGALS2, MYCL, NECAB1, and PKHD1 in the sample obtained at the later time point relative to the sample obtained from the first time point indicates a (good) prognosis of recovery.
In a related aspect, the present invention also provides the use of the one or more biomarker described herein for monitoring a systemic inflammatory condition in a patient.
In one embodiment, the use is of one or more biomarker selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, MX1, NECAB1, PKHD1, and LILRB5 for monitoring a systemic inflammatory condition in a patient.
In one embodiment, the use is of one or more biomarker selected from ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, and HLA-DPB1, for monitoring a systemic inflammatory condition in a patient (such sepsis and/or SIRS). For example, the one or more biomarker may be selected from: ITM2A, CCL5, NPPC, KLRK1, and BCL11B. For example, the one or more biomarker may be selected from: ITM2A, CCL5, and NPPC.
In one embodiment, the use is of one or more biomarker selected from: ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, and MX1, for monitoring a systemic inflammatory condition in a patient (such as abdominal sepsis). For example, the one or more biomarker may be selected from: ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1.
In one embodiment, the use is of one or more biomarker selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, CD160, KLRF1, CD2, LGALS2, MYCL, NECAB1, and PKHD1 for monitoring a systemic inflammatory condition in a patient (such as SIRS). In one embodiment, the use is of one or more biomarker selected from: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, BCL11B, CD2, LGALS2, MYCL, NECAB1, and PKHD1 for monitoring a systemic inflammatory condition in a patient (such as SIRS). For example, the one or more biomarker may be selected from: CCL5, NPPC, PKD1, LGALS2, MYCL, NECAB1, and PKHD1.
All embodiments described above for the method of monitoring a systemic inflammatory condition in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “systemic inflammatory condition”, “patient”, “sample obtained a first time point”, “sample obtained at one or more later time points”, and “the one or more biomarker”.
Survival Biomarkers
A major issue facing clinicians is determining when a patient is suitable for release from medical care. In some cases, patients appear to physically recover (eg. from a systemic inflammatory condition), yet still do not survive after they are discharged from medical care. When studying the gene expression patterns of biomarkers in patients having a systemic inflammatory condition, the inventors surprisingly observed that several of the biomarkers described herein were present at much higher levels in patients that did not survive as compared to patients that made a full recovery. The inventors observed that the likelihood of survival of a patient could therefore be predicted by monitoring the levels of these “survival” biomarkers. Detection of the levels of these biomarkers in patients will therefore assist clinicians in determining whether a patient is suitable for discharge from medical care.
The present invention therefore provides a method for determining whether a patient is suitable for discharge from medical care, comprising:
“Determining whether a patient is suitable for discharge from medical care” means determining whether the patient has a good prognosis of survival and can be safely discharged from medical care. The method therefore provides a way of predicting the survival of a patient (such as a patient that has been diagnosed with a systemic inflammatory condition). The method may therefore be alternatively defined as a “method for predicting the survival of a patient”.
As used herein, “discharge from medical care” encompasses “discharge from high-dependency medical care”. For example, it may refer to the act of moving a patient from a high dependency unit (such as an intensive care unit) to a lower dependency unit (such as an outpatient unit, a hospital ward, or home care).
In one embodiment, step (i) of the method involves “determining the presence and amount of the one or more biomarker in a sample obtained from a patient”, and step (ii) involves “comparing the presence and amount of the one or more biomarker determined in said sample in (i) to a corresponding reference value”. In one embodiment, step (i) of the method involves “determining the amount of the one or more biomarker in a sample”, and step (ii) involves “comparing the amount of the one or more biomarker determined in said sample in (i) to a corresponding reference value”.
The “sample” obtained from the patient is as defined above for the diagnostic methods and monitoring methods described herein, including all embodiments relating to the time point at which the sample is obtained. In one embodiment, the sample may be obtained at least 48 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the sample may be obtained at least 72 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the sample may be obtained at least 96 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. In one embodiment, the sample may be obtained at least 120 hours after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The method of the invention is intended to be used as a point of care monitor to determine whether it is safe to discharge a patient from medical care. In one embodiment, the sample may be obtained from a patient after treatment for a systemic inflammatory condition has been completed. In one embodiment, the sample is obtained from a patient when they have been clinically diagnosed as being suitable for discharge from medical care.
The “patient” is as described above for the diagnostic methods and monitoring methods described herein. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition. In one embodiment, the patient has been diagnosed as having or being at risk of developing a systemic inflammatory condition using the method described herein for diagnosing a systemic inflammatory condition. In one embodiment, the patient may have been diagnosed as having or being at risk of developing a systemic inflammatory condition using the methods described herein for diagnosing SIRS, sepsis (such as abdominal sepsis or pulmonary sepsis), or using the method described herein for distinguishing between sepsis and SIRS in a patient, or any combination of these methods as described herein). The patient may be undergoing (or has undergone) treatment for a systemic inflammatory condition.
In one embodiment, the patient has been diagnosed as having or being at risk of developing SIRS (e.g., using any of the methods described herein for diagnosing SIRS, or for distinguishing between sepsis and SIRS in a patient). The patient may be undergoing (or has undergone) treatment for SIRS.
In one embodiment, the patient has been diagnosed as having or being at risk of developing sepsis (eg. using any of the methods described herein for diagnosing sepsis, or for distinguishing between sepsis and SIRS in a patient). The patient may be undergoing (or has undergone) treatment for sepsis.
In one embodiment, the patient has been diagnosed as having or being at risk of developing abdominal sepsis (e.g., using the method described herein for diagnosing abdominal sepsis). The patient may be undergoing (or has undergone) treatment for abdominal sepsis.
In one embodiment, the patient has been diagnosed as having or being at risk of developing pulmonary sepsis (e.g., using the method described herein for diagnosing pulmonary sepsis). The patient may be undergoing (or has undergone) treatment for pulmonary sepsis.
The “one or more biomarker” of the invention is as described above for the diagnostic methods and monitoring methods described herein. In one embodiment, the one or more biomarker may be selected from the group consisting of: NECAB1, NECAB2, PKD1, PKHD1, LILRB4, and LILRB5.
Each of the biomarkers may be used alone, or in combination with any of the survival biomarkers described herein in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more or all 6 of the biomarkers may be used in the method of the invention.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or all 6) of the biomarkers selected from the group consisting of: NECAB1, NECAB2, PKD1, PKHD1, LILRB4, and LILRB5, may be used to determine whether a patient is suitable for discharge from medical care.
A combination of the ‘survival’ biomarkers may be used in the method to determine whether a patient is suitable for discharge from medical care. In one embodiment, the method may involve determining the presence and/or amount of one or more biomarker selected from NECAB2 and PKD1, in combination with one or more biomarker selected from: NECAB1, PKDI, PKHD1, LILRB4 and LILRB5. For example, the combination of biomarkers used in the method may be NECAB2 and NECAB1. For example, the combination of biomarkers used in the method may be NECAB2 and PKHD1. For example, the combination of biomarkers used in the method may be NECAB2 and PKD1. For example, the combination of biomarkers used in the method may be NECAB2 and LILRB4. For example, the combination of biomarkers used in the method may be NECAB2 and LILRB5. For example, the combination of biomarkers used in the method may be PKD1 and PKHD1. For example, the combination of biomarkers used in the method may be PKD1 and NECAB1. For example, the combination of biomarkers used in the method may be PKD1 and LILRB4. For example, the combination of biomarkers used in the method may be PKD1 and LILRB5.
In one embodiment, the one or more biomarker is NECAB1. In one embodiment, the one or more biomarker is NECAB2. In one embodiment, the one or more biomarker is PKD1. In one embodiment, the one or more biomarker is PKHD1. In one embodiment, the one or more biomarker is LILRB4. In one embodiment, the one or more biomarker is LILRB5.
The biomarkers NECAB1 and NECAB2 are brain specific markers, and the biomarkers PKHD1 and PKD1 are kidney specific markers. These markers are not usually expressed in peripheral blood leukocytes. The high levels of these markers in the patients that did not survive indicates that these patients are suffering from kidney damage and/or brain damage. The method described herein may therefore be used to diagnose organ damage in a patient. In one embodiment, the method is for diagnosis of organ damage in a patient. For example, the method may be for diagnosis of brain damage in a patient, when the one or more biomarker is selected from NECAB1 and/or NECAB2. By performing steps (i) and (ii) of the method described herein, the method can be used to determine whether a patient has or is at risk of developing brain damage. The method may alternativelty be for diagnosis of kidney damage in a patient, when the one or more biomarker is selected from PKHD1 and/or PKD1. By performing steps (i) and (ii) of the method described herein, the method can be used to determine whether a patient has or is at risk of developing kidney damage.
The present inventors have observed that a sub-set of these biomarkers (NECAB2, PKD1, PKHD1 and LILRB5,) are particularly useful in determining whether a patient diagnosed as having or being at risk of developing sepsis (such as abdominal sepsis and/or pulmonary sepsis) is suitable for discharge from medical care. In one embodiment, the one or more biomarkers may be selected from the group consisting of NECAB2, LILRB5, PKHD1 and PKD1. The patient may be undergoing (or has undergone) treatment for sepsis (such as abdominal sepsis and/or pulmonary sepsis). Treatment for sepsis is as described herein.
A subset of the biomarkers (NECAB2 and PKD1) is particularly useful in determining whether a patient diagnosed as having or being at risk of developing abdominal sepsis is suitable for discharge from medical care. As described in Example 2, ROC analysis demonstrated that these biomarkers could be used alone or in combination to effectively distinguish between abdominal sepsis patients that died and those that survived. In one embodiment, the one or more biomarkers may be selected from the group consisting of NECAB2 and PKD1. For example, the method may be performed using the combination of biomarkers NECAB2 and PKD1. For example, the method may be performed using NECAB2. For example, the method may be performed using PKD1. The patient may be undergoing (or has undergone) treatment for abdominal sepsis.
A subset of the biomarkers (PKHD1 and LILRB5) is particularly useful in determining whether a patient diagnosed as having or being at risk of developing pulmonary sepsis is suitable for discharge from medical care. As described in Example 2, ROC analysis demonstrated that these biomarkers could be used alone or in combination to effectively distinguish between pulmonary sepsis patients that died and those that survived. In one embodiment, the one or more biomarkers may be selected from the group consisting of PKHD1 and LILRB5. For example, the method may be performed using the combination of biomarkers PKHD1 and LILRB5. For example, the method may be performed using PKHD1. For example, the method may be performed using LILRB5. The patient may be undergoing (or has undergone) treatment for pulmonary sepsis.
The present inventors have observed that a sub-set of these biomarkers (NECAB1, PKDI, PKHD1, LILRB4, and LILRB5) are particularly useful in determining whether a patient diagnosed as having or being at risk of developing SIRS is suitable for discharge from medical care. The patient may be undergoing (or has undergone) treatment for SIRS. Treatment for SIRS is as described herein. Thus, in one embodiment, the one or more biomarker is selected from the group consisting of: NECAB1, PKDI, PKHD1, LILRB4, and LILRB5. In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, 4 or more, or all 5) of the biomarkers selected from the group consisting of: NECAB1, PKD1, PKHD1, LILRB4, and LILRB5, may be used to determine whether a patient is suitable for discharge from medical care.
As described in Example 2, PKHD1 and NECAB1 were observed to provide the most accurate distinction between patients with SIRS that survived and those that died (see the ROC curve data in Example 2). Good results were observed when these markers were used on their own or in combination. In one embodiment, the one or more biomarker may be PKHD1 and/or NECAB1. For example, the markers for determining whether a patient diagnosed as having or being at risk of developing SIRS is suitable for discharge from medical care may comprise the combination of PKHD1 and NECAB1.
All embodiments of the ‘quantification’ and ‘comparison’ steps described above for the diagnostic and monitoring methods described herein apply equally to the method for determining whether a patient is suitable for discharge from medical care. This includes all embodiments relating to the “reference value”.
The one or more biomarker measured by the methods of the present invention may increase or decrease as compared to the corresponding reference value. The increase or decrease in the amount of the one or more biomarker in the patient as compared to the reference value can indicate that the patient has a good prognosis of recovery (or survival) from the systemic inflammatory condition, and thus is suitable for discharge from medical care. Alternatively, the increase or decrease in the one or more biomarker in the patient as compared to the reference can indicate that the patient has a poor prognosis of recovery (or survival) (or a prognosis of non-recovery) from the systemic inflammatory condition, and thus is not suitable for discharge from medical care.
The increase or decrease in the one or more biomarker as compared to the reference can be, for example, at least 0.1 (e.g., at least 0.2, at least 0.3, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 7 fold, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, or at least 10) fold. The difference in the amount of the biomarker is preferably statistically significant. By “statistically significant”, it is meant that the alteration is greater than what might be expected to happen by chance alone.
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By comparing the amount of markers quantified in a sample obtained from a patient to the amount of markers quantified for a reference value (such as that obtained from a healthy individual (or a population of healthy individuals), an individual (or population of individuals) that has a (good) prognosis of recovery (or survival) from a systemic inflammatory condition, and/or an individual (or population of individuals) that has a prognosis of non-recovery (or non-survival) from a systemic inflammatory condition), it is possible to determine whether a patient is suitable for discharge from medical care. The method permits classification of the patient as belonging to or not belonging to the reference population (i.e., by determining whether the amounts of marker quantified in the patient are statistically similar to the reference population or statistically deviate from the reference population). Hence, classification of the patient's marker profile (i.e., the overall pattern of change observed for the markers quantified) as corresponding to the profile derived from a particular reference population is predictive that the individual falls (or does not fall) within the reference population.
In one embodiment, a patient may be identified as being suitable for discharge from medical care, when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) that has a (good) prognosis of recovery (or survival) from a systemic inflammatory condition. In one embodiment, a patient may be identified as being suitable for discharge from medical care, when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, a patient may be identified as being unsuitable for discharge from medical care when the amount of the one or more biomarker is statistically similar to the amount determined for the corresponding reference value representative of an individual (or a population of individuals) having a prognosis of non-recovery (or non-survival) from a systemic inflammatory condition.
In one embodiment, a patient may be identified as being unsuitable for discharge from medical care, when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual (or a population of individuals) that has a (good) prognosis of recovery (or survival) from a systemic inflammatory condition. In one embodiment, a patient may be identified as being unsuitable for discharge from medical care, when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of a healthy individual (or a population of healthy individuals). In one embodiment, a patient may be identified as being suitable for discharge from medical care when the amount of the one or more biomarker statistically deviates from the amount determined for the corresponding reference value representative of an individual (or a population of individuals) that has a prognosis of non-recovery (or non-survival) from a systemic inflammatory condition.
All embodiments described above (in the context of the diagnostic methods) for classifying a patient based on their marker profile apply equally to the method for determining whether a patient is suitable for discharge from medical care. This includes all embodiments for determining whether the marker profile of the patient is “statistically similar to” or “statistically deviates from” the marker profiles observed for the corresponding reference values, and all embodiments relating to the % increase or % decrease or fold change observed in the markers as compared to the corresponding reference value.
The reference value may be as defined above for the diagnostic methods described herein. In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). In one embodiment, the reference value may be representative of an individual (or population of individuals) that has a (good) prognosis of recovery (or survival) from a systemic inflammatory condition. In one embodiment, the reference value may be representative of an individual (or population of individuals) that has a prognosis of non-recovery (or non-survival) from a systemic inflammatory condition).
The reference value that is representative of an individual (or population of individuals) having a (good) prognosis of recovery (or survival) from a systemic inflammatory condition is determined by quantifying the amount of the one or more biomarker in a sample obtained from an individual (or population of individuals) having a systemic inflammatory condition, wherein the individual (or population of individuals) goes on to make a full recovery from the systemic inflammatory condition. The sample may be obtained at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 36 hours, 48 hours, 72 hours, 96 hours, or 120 hours, after the individual presents with one or more clinical symptoms of a systemic inflammatory condition, or is admitted to a medical care facility, For example, the sample may be obtained at least 120 hours after the individual (or population of individuals) presents with one or more clinical symptoms of a systemic inflammatory condition, or is admitted to a medical care facility.
The reference value that is representative of an individual (or population of individuals) having a prognosis of non-recovery (or non-survival) from a systemic inflammatory condition, or a poor prognosis of recovery (or survival) is determined by quantifying the amount of biomarker in a sample obtained from an individual (or population of individuals) having a systemic inflammatory condition, wherein the individual (or population of individuals) does not recover from the systemic inflammatory condition. The sample may be obtained at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 36 hours, 48 hours, 72 hours, 96 hours, or 120 hours, after the individual presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility. For example, the sample may be obtained at least 120 hours after the individual (or population of individuals) presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
The present inventors observed that some of the ‘survival’ biomarkers described herein increase in abundance in non-survivors as compared to healthy individuals. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for healthy individuals can thus be used to determine whether a patient is suitable for discharge from medical care.
In one embodiment, when the reference value is representative of a healthy individual (or a population of healthy individuals), an increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. Likewise, no increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care.
For some of the ‘survival’ biomarkers identified by the present inventors, increased levels of these markers were also observed in patients that recovered from a systemic inflammatory condition as compared to healthy individuals, although much bigger increases were observed for these biomarkers in the patients that did not survive. The accuracy of determining whether a patient is suitable for discharge from medical care can thus be improved by looking for a “minimum” fold change in the levels of the one or more biomarkers as compared to the corresponding reference value that is representative of a healthy individual.
For example, an increase of at least 2.5 (e.g., at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4) fold in NECAB1 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. No increase or an increase of less than 1.5 (e.g., less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1) fold in NECAB1 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for SIRS. In one embodiment, the sample is obtained from the patient at least 72 hours (e.g., at least 96 hours, at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 3.5 (e.g., at least 3.6, at least 3.7) fold in NECAB2 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. No increase or an increase of less than 2.5 (e.g., less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1) fold in NECAB2 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for sepsis. In one embodiment, the patient is undergoing (or has undergone) treatment for abdominal sepsis and/or pulmonary sepsis. In one embodiment, the sample is obtained from the patient at least 48 hours (e.g., at least 72 hours, at least 96 hours, at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 1.5 (e.g., at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2) fold in PKD1 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. No increase or an increase of less than 1.6 (e.g., less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1) fold in PKD1 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for abdominal sepsis and/or SIRS. In one embodiment, the sample is obtained from the patient at least 72 hours (eg. at least 96 hours, at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 2.5 (e.g., at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.1, at least 5.2, or at least 5.3) fold in PKHD1 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. No increase or an increase of less than 1.5 (e.g., less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1) fold in PKHD1 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for SIRS. In one embodiment, the sample is obtained from the patient at least 72 hours (eg. at least 96 hours, at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 6.3 (e.g., at least 6.4, at least 6.5, at least 6.6, at least 6.7, at least 6.8, at least 6.9, at least 7) fold in LILRB4 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. No increase or an increase of less 5.5 (e.g., less than 5.4, less than 5.3, less than 5.2, less than 5.1, less than 5) fold in LILRB4 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for SIRS. In one embodiment, the sample is obtained from the patient at least 72 hours (e.g., at least 96 hours, at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
For example, an increase of at least 7 (e.g., at least 7.1, at least 7.2, at least 7.3 at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9, at least 8) fold in LILRB5 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. No increase or an increase of less than 4.5 (e.g., less than 5, less than 5.5, less than 6, less than 6.5, less than 7) fold in LILRB5 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for SIRS. In one embodiment, the sample is obtained from the patient at least 72 hours (e.g., at least 96 hours, at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
As described herein, LILRB5 was also observed to increase in abundance in patients that made a full recovery from pulmonary sepsis compared to patients that did not survive. Thus, in one example, an increase of at least 2.5 (e.g., at least 3, at least 3.5, at least 4, at least 4.5, at least 5) fold in LILRB5 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is suitable for discharge from medical care. No increase or an increase of less than 4.5 (e.g., less than 4, less than 3.5, less than 3, less than 2.5, less than 2) fold in LILRB5 in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual, indicates that the patient is not suitable for discharge from medical care. In one embodiment, the patient is undergoing (or has undergone) treatment for pulmonary sepsis. In one embodiment, the sample is obtained from the patient at least 72 hours (e.g., at least 96 hours or at least 120 hours) after the patient presents with one or more clinical symptoms of a systemic inflammatory condition or is admitted to a medical care facility.
As described herein, the present inventors observed that the levels of some of the one or more survival biomarkers were elevated in patients that did not recover from (or survive) a systemic inflammatory condition as compared to patients that made a full recovery. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients that recovered from (or survived) a systemic inflammatory condition can thus be used to determine whether a patient is suitable for discharge from medical care.
Thus, in one embodiment, when the reference value is representative of an individual (or population of individuals) having a (good) prognosis of recovery (or survival) from a systemic inflammatory condition, an increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient is not suitable for discharge from medical care. Likewise, no increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient is suitable for discharge from medical care.
In one embodiment, the patient may be identified as being unsuitable for discharge from medical care, when the one or more biomarker increases by at least 1 (e.g., at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value.
As described herein, the present inventors observed that the levels of some of the one or more survival biomarkers were elevated in patients that made a full recovery from a systemic inflammatory condition as compared to patients that did not recover from (or survive). Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients that recovered from (or survived) a systemic inflammatory condition can thus be used to determine whether a patient is suitable for discharge from medical care.
Thus, in one embodiment, when the reference value is representative of an individual (or population of individuals) having a (good) prognosis of recovery (or survival) from a systemic inflammatory condition, an increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient is suitable for discharge from medical care. Likewise, no increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient is not suitable for discharge from medical care.
In one embodiment, the patient may be identified as being suitable for discharge from medical care, when the one or more biomarker increases by at least 1 (e.g., at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value.
As described above for the diagnostic methods described herein, the method of the invention may involve the use of multiple separate reference values. For example, the method may involve the use of one or more (eg. two or more, or all three) reference values that are representative of an individual (or population of individuals) having a (good) prognosis of recovery (or survival) from a systemic inflammatory condition, an individual (or population of individuals) having a prognosis of non-recovery (or non-survival) (or a poor prognosis of recovery) from a systemic inflammatory condition; and a healthy individual (or a population of healthy individuals).
In a related aspect, the present invention also provides the use of one or more biomarker selected from: NECAB1, NECAB2, PKD1, PKHD1, LILRB4 and LILRB5 for determining whether a patient is suitable for discharge from medical care.
In one embodiment, the present invention provides the use of one or more biomarker selected from: PKHD1, PKDI, NECAB1, LILRB4, and LILRB5 for determining whether a patient is suitable for discharge from medical care. The patient may be undergoing (or has undergone) treatment for SIRS. In one embodiment, the one or more biomarker may be selected from: PKHD1 and NECAB1. For example, the one or more biomarker may comprise the combination of PKHD1 and NECAB1. For example, the one or more biomarker is PKHD1. For example, the one or more biomarker is NECAB1.
In one embodiment, the present invention provides the use of one or more biomarker selected from the group consisting of NECAB2, LILRB5, PKHD1 and PKD1 for determining whether a patient is suitable for discharge from medical care. The patient may be undergoing (or has undergone) treatment for sepsis (such as abdominal sepsis and/or pulmonary sepsis).
In one embodiment, the present invention provides the use of one or more biomarker selected from: NECAB2 and PKD1, for determining whether a patient is suitable for discharge from medical care. The patient may be undergoing (or has undergone) treatment for abdominal sepsis. For example, the one or more biomarker may comprise the combination of biomarkers NECAB2 and PKD1. For example, the one or more biomarker is NECAB2. For example, the one or more biomarker is PKD1.
In one embodiment, the present invention provides the use of one or more biomarker selected from: PKHD1 and LILRB5, for determining whether a patient is suitable for discharge from medical care. The patient may be undergoing (or has undergone) treatment for pulmonary sepsis. For example, the one or more biomarker may comprise the combination of biomarkers PKHD1 and LILRB5. For example, the one or more biomarker is PKHD1. For example, the one or more biomarker is LILRB5.
All embodiments described above for the method of determining whether a patient is suitable for discharge from medical care apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the terms “systemic inflammatory condition”, “patient”, “sample”, and “the one or more biomarker”.
Systemic inflammatory conditions such as SIRS and sepsis can lead to the development of multiple organ failure in patients. Early detection of organ failure in patients may improve the chances of survival in patients having a systemic inflammatory condition.
When investigating the biomarkers associated with systemic inflammatory conditions, the present inventors surprisingly observed that various organ specific biomarkers are present in high levels in peripheral blood leukocytes (PBLs) obtained from patients having systemic inflammatory conditions that did not survive. These biomarkers include the brain specific markers NECAB1 and NECAB2, and the kidney specific markers PKHD1 and PKD1. The presence of these markers in peripheral blood leukocytes indicates that the organ is damaged. Detection of these markers in samples obtained from patients therefore provides a way of diagnosing organ damage in the patient.
The present invention provides a method for diagnosing organ damage in a patient, comprising:
All embodiments described above for the method of determining whether a patient is suitable for discharge from medical care apply equally to the method for diagnosing organ damage in a patient. This includes all embodiments relating to the “patient”, “sample”, “reference value”, and the steps for “determining the presence and/or amount of the one or more biomarker” and the “comparison” for making a conclusion about the diseases state of the patient.
The term “organ damage” refers to the condition where an organ has been injured such that it does not perform its expected function. In one embodiment the organ damage is one or more of: brain damage or kidney damage.
The “patient” is as described above for the “method of determining whether a patient is suitable for discharge from medical care”. The method of the invention for diagnosing organ damage is not only applicable to such patients but may also be used to diagnose organ damage in patients having a disease or condition other than a systemic inflammatory condition. The patient may thus be an individual having any disease, condition or injury which may result in organ damage.
The “one or more biomarker” of the invention is as described above for the “method of determining whether a patient is suitable for discharge from medical care”. In one embodiment, the one or more biomarker is selected from the group consisting of: NECAB1, NECAB2, PKHD1, and PKD1.
In one embodiment, the one or more biomarker is selected from NECAB1 and/or NECAB2. These biomarkers are specific for indicating the presence of brain damage in a patient. Thus when the one or more biomarker is selected from NECAB1 and/or NECAB2, the method is for diagnosing brain damage in a patient. By performing steps (i) and (ii) of the method described herein, the method can be used to determine whether a patient has or is at risk of developing brain damage.
In one embodiment, the one or more biomarker is selected from PKHD1 and/or PKD1. These biomarkers are specific for indicating the presence of kidney damage in a patient. Thus, when the one or more biomarker is selected from PKHD1 and/or PKD1, the method is for diagnosing kidney damage in a patient. By performing steps (i) and (ii) of the method described herein, the method can be used to determine whether a patient has or is at risk of developing kidney damage.
Each of the biomarkers may be used alone, or in combination with any of the biomarkers described herein in the method of the invention. For example, any combination of 1 or more, 2 or more, 3 or more, or all 4 or more of the biomarkers may be used in the method of the invention.
In one embodiment, any combination of 1 or more (e.g., 2 or more, 3 or more, or all 4) of the biomarkers selected from the group consisting of: NECAB1, NECAB2, PKD1, and PKHD1, may be used to diagnose organ damage in a patient.
The ‘quantification’ and ‘comparison’ steps of the method, and the “reference value” used in the ‘comparison’ step are as described above for the method for determining whether a patient is suitable for discharge from medical care. This includes all embodiments described for classification of a patient based on their marker profile.
As described herein, the present inventors observed that the organ specific biomarkers described herein each increase in abundance in samples obtained from patients having a systemic inflammatory condition as compared to healthy individuals. However, much higher levels of the organ specific biomarkers were observed in patients that did not survive the systemic inflammatory condition as compared to those patients that recovered.
In one embodiment, the reference value is representative of a healthy individual (or a population of healthy individuals). The patient may be diagnosed as having organ damage or being at risk of developing organ damage when an increase is observed in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value representative of a healthy individual. More accurate diagnosis can be performed by looking for a minimum fold increase in the one or more biomarker in the patient. The minimum fold change values in the biomarkers NECAB1, NECAB2, PKD1 and PKHD1 are as defined above for the method for determining whether a patient is suitable for discharge from medical care. As described herein, the present inventors observed that the levels of the organ specific biomarkers were elevated in patients that did not recover from (or survive) a systemic inflammatory condition as compared to patients that made a full recovery. The patients that did not survive are likely to have a higher risk of organ failure as compared to patients that made a full recovery. Detection of increased levels of these biomarkers in a patient as compared to the levels detected for patients that recovered from (or survived) a systemic inflammatory condition can thus be used to determine whether a patient has organ damage.
Thus, in one embodiment, when the reference value is representative of an individual (or population of individuals) having a (good) prognosis of recovery (or survival) from a systemic inflammatory condition, an increase in the one or more biomarker in the sample obtained from the patient relative to the corresponding reference value, indicates that the patient has or is at risk of developing organ damage.
In one embodiment, the patient may be diagnosed as having or being at risk of developing organ damage, when the one or more biomarker increases by at least 1 (e.g., at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50) fold in the sample obtained from the patient relative to the corresponding reference value.
As described above for the method for determining whether a patient is suitable for discharge from medical care, multiple separate reference values may be used in the method for diagnosing organ damage. All combinations of reference values described above apply equally to the method for diagnosing organ damage.
In a related aspect, the present invention also provides the use of one or more of: NECAB1, NECAB2, PKHD1, and PKD1, as a biomarker for organ damage. In one embodiment, the use is of the one or more biomarker for diagnosis of organ damage in a patient.
In one embodiment, the use is of one or more biomarker selected from the group consisting of: NECAB1 and NECAB2, for diagnosis of brain damage in a patient. In one embodiment, the use is of one or more biomarker selected from the group consisting of: PKHD1 and PKD1, for diagnosis of kidney damage in a patient.
All embodiments described above for the method of diagnosing organ damage in a patient apply equally to the ‘use’ of the invention described herein. This includes all embodiments relating to the “patient”, “sample”, and “the one or more biomarker”.
Treatment
The methods described herein for diagnosis and/or monitoring of a systemic inflammatory condition in a patient may in certain embodiments also be applied to determine whether the patient is or is not in need of a therapeutic or prophylactic treatment of the systemic inflammatory condition. For example, a treatment may be indicated where the methods allow for a conclusion that the patient has or is at risk of having a systemic inflammatory condition, has a poor prognosis for the systemic inflammatory condition, displays a detrimental development of the condition, or has organ damage. Without limitation, a patient with the systemic inflammatory condition upon admission to or during stay in a medical care centre such as ICU may be tested as described herein for the necessity of continuing the treatment of the condition and may be discharged when such treatment is no longer needed or is needed only to a given limited extent.
In a further embodiment, any of the methods described herein may further comprise treating a systemic inflammatory condition in a patient. In one embodiment, any of the methods described herein may comprise, responsive to the diagnosis of a systemic inflammatory condition in the patient, administering to the patient a therapy for a systemic inflammatory condition. For example, the therapy may be for SIRS and/or for sepsis. The methods of the invention may therefore be for treating or preventing one or more symptoms of a systemic inflammatory condition.
For example, any of the methods described herein for diagnosis of SIRS (including the method for diagnosing SIRS in a patient, and the method for distinguishing between sepsis and SIRS in a patient) may further comprise, responsive to the diagnosis of SIRS, administering to the patient a therapy for SIRS. These methods may be for treating or preventing one or more symptoms of SIRS in a patient.
The “therapy for SIRS” may include organ support with oxygen, mechanical ventilation, circulatory support with fluid resuscitation, vasodilators, inotropes or vasopressors, renal replacement therapy.
In certain embodiments, the administering of a therapy for SIRS may comprise administering one such therapy to the patient. In certain embodiments, the administering of a therapy for SIRS may comprise administering a combination of two or more such therapies to the patient.
For example, any of the methods described herein for diagnosis of sepsis (including the method for diagnosing sepsis in a patient, the method for distinguishing between sepsis and SIRS in a patient, the method for diagnosing abdominal sepsis in a patient, the method for diagnosing pulmonary sepsis in a patient, and the method for distinguishing between abdominal sepsis and pulmonary sepsis in a patient) may further comprise, responsive to the diagnosis of sepsis, administering to the patient a therapy for sepsis. These methods may be for treating or preventing one or more symptoms of sepsis in a patient.
The “therapy for sepsis” may include anti-microbial agents (such as anti-bacterial agents e.g., antibiotics), analgesics, antipyretics, anti-inflammatory drugs (such as non-steroidal anti-inflammatory drugs), fluid resuscitation, and oxygen therapy. It may also include organ support with oxygen, mechanical ventilation, circulatory support with inotropes or vasopressors, renal replacement therapy.
In certain embodiments, the administering of a therapy for sepsis may comprise administering one such therapy to the patient. In certain embodiments, the administering of a therapy for sepsis may comprise administering a combination of two or more such therapies to the patient.
In one embodiment, the method for distinguishing between sepsis and SIRS in a patient may further comprise, responsive to the diagnosis of sepsis and/or SIRS in the patient, administering to the patient a therapy for sepsis and/or SIRS. For example, the therapy may be for SIRS as described herein. Alternatively, the therapy may be for sepsis (including abdominal sepsis and pulmonary sepsis) as described herein. The methods of the invention may therefore be for treating or preventing one or more symptoms of sepsis and/or SIRS.
In one embodiment, any of the methods described herein for diagnosis of organ damage may further comprise, responsive to the diagnosis of organ damage, administering to the patient a therapy for organ damage.
Oligonucleotide Probes and Amplification Primers
Any appropriate detection means can be used to detect or quantify the one or more biomarker in the methods and uses of the invention, as described herein.
Typically, when the one or more biomarker of the invention is a nucleic acid, the presence of the one or more biomarkers may be detected, and/or the amount of the one or more biomarker determined using an oligonucleotide probe. The methods and uses described herein may therefore use any one or more oligonucleotide probe as defined herein to detect and/or quantify the one or more biomarker of the invention. The oligonucleotide probes may be bound to a solid surface (such as a microarray). Alternatively, oligonucleotide probes may be used in quantitiative real-time PCR to detect amplified target sequence from the one or more biomarker.
An oligonucleotide probe of the invention may have at least 80% sequence identity to the one or more biomarker of the invention, or a target region within said biomarker, measured over any appropriate length of sequence. Typically, the % sequence identity is determined over a length of contiguous nucleic acid residues. An oligonucleotide probe of the invention may, for example, have at least 80% sequence identity to the one or more biomarker of the invention, or target region thereof, measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or more nucleic acid residues, up to the oligonucleotide probe having at least 80% sequence identity with the one or more biomarker of the invention, or target region thereof, over the entire length of the oligonucleotide probe.
An oligonucleotide probe of the invention may be complementary to the one or more nucleic acid biomarker of the invention, or a target region thereof. Typically, the oligonucleotide probe of the invention is complementary over a length of contiguous nucleic acid residues. An oligonucleotide probe of the invention may, for example, be complementary to the one or more biomarker of the invention, or target region thereof, measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or more nucleic acid residues, up to the oligonucleotide probe having being complementary to the one or more biomarker of the invention, or target region thereof, over the entire length of the oligonucleotide probe.
An oligonucleotide probe of the invention may be complementary to a variant of the one or more biomarker of the invention, or a variant of a target region of said biomarker. Typically, the oligonucleotide probe is complementary to a variant having at least 80% sequence identity to the one or more biomarker of the invention, or a variant having at least 80% sequence identity to the target region of said biomarker. The % sequence identity of the variant to the one or more biomarker of the invention, or a variant of a target region of said biomarker may be calculated over any appropriate length of sequence in the one or more biomarker, as described herein.
As used herein, a “sequence identity of at least 80%” includes at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity (to each and every nucleic acid sequence presented herein and/or to each and every SEQ ID NO presented herein).
Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CAB/OS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20 (9) Bioinformatics:1428-1435 (2004). Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992.
Variants of the specific sequences described herein may alternatively be defined by reciting the number of nucleotides that differ between the variant sequences and the specific reference sequences provided above. Thus, in one embodiment, the sequence may comprise (or consist of) a nucleotide sequence that differs from the specific sequences provided above at no more than 2 nucleotide positions, for example at no more than 1 nucleotide position. Conservative substitutions are preferred. The term variants as defined herein also encompasses splice variants.
An oligonucleotide probe of the invention may be at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more nucleotides in length. In a preferred embodiment, the oligonucleotide probe is 40 to 100 nucleotides in length, more preferably 50 to 100 nucleotides in length, even more preferably 50 to 80 nucleotides in length and most preferably 50 to 70 nucleotides in length. Such oligonucleotide probes are suitable for use in use in microarray analysis when bound to a solid surface. In one embodiment, the oligonucleotide probe is designed for detection of the one or more biomarker by microarray analysis.
Oligonucleotide probes may also be designed for detection of the one or more biomarker by quantitative PCR (or real-time PCR). The oligonucleotide probe may be 5-30 nucleotides long, such as at least 6, 7, 8, 9 or 10 nucleotides long. The oligonucleotide probe may be up to 25 nucleotides long, such as up to 20, 18, 16, 15, 14, 13, 12, 11 or 10 nucleotides long. The oligonucleotide probe may be 10-25 nucleotides long, such as 10-20 nucleotides long or 10-15 nucleotides long, and may be preferably about 10 nucleotides long. In this regard, the use of short probes enables faster annealing to the target nucleic acid.
The target nucleotide sequence to which the oligonucleotide probe hybridises within the amplification product may be at least 5, 6, 7, 8, 9 or 10 nucleotides long. The target sequence for the probe may be up to 30 nucleotides long, such as up to 25, 20, 18, 16, 15, 14, 13, 12, or 11 nucleotides long. The probe target sequence may be 10-25 nucleotides long or 10-15 nucleotides long and may be preferably about 10 nucleotides long.
The probes of the invention are typically designed to hybridise to their target nucleic acid sequence present in the one or more biomarker of the invention.
A probe may comprise or be complementary to a nucleic acid sequence within a target nucleic acid sequence from the one or more biomarker of the invention, or to a nucleic acid sequence having at least 80% identity to said target nucleic acid sequence. Any suitable probe which comprises or is complementary (as defined herein) to a nucleic acid sequence within a target nucleic acid sequence of one or more biomarker of the invention may be used.
In embodiments wherein the one or more biomarker is ADM, a target nucleic acid sequence may comprise bases 751 to 1590 of SEQ ID NO: 1, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CD177, a target nucleic acid sequence may comprise bases 1351 to 2220 of SEQ ID NO: 2, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is FAM20A, a target nucleic acid sequence may comprise bases 1331 to 3700 of SEQ ID NO: 3, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 1460 to 1531 of SEQ ID NO: 3 or bases 1486 to 1551 of SEQ ID NO: 3, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IL110, a target nucleic acid sequence may comprise bases 61 to 1320 of SEQ ID NO: 4, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is METTL7B, a target nucleic acid sequence may comprise bases 581 to 1340 of SEQ ID NO: 5, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MMP9, a target nucleic acid sequence may comprise bases 1511 to 2330 of SEQ ID NO: 6, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In embodiments wherein the one or more biomarker is RETN, a target nucleic acid sequence may comprise bases 81 to 478 of SEQ ID NO: 7, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is TDRD9, a target nucleic acid sequence may comprise bases 3711 to 4400 of SEQ ID NO: 8, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is ITGA7, a target nucleic acid sequence may comprise bases 3181 to 4080 of SEQ ID NO: 9, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is BMX, a target nucleic acid sequence may comprise bases 1651 to 2430 of SEQ ID NO: 10, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is HP, a target nucleic acid sequence may comprise bases 821 to 1430 of SEQ ID NO: 11, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IGFBP2, a target nucleic acid sequence may comprise bases 651 to 1430 of SEQ ID NO: 12, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is ALPL, a target nucleic acid sequence may comprise bases 1441 to 2520 of SEQ ID NO: 13, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is DACH1, a target nucleic acid sequence may comprise bases 2341 to 4990 of SEQ ID NO: 14, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IL1 R1, a target nucleic acid sequence may comprise bases 1551 to 4410 of SEQ ID NO: 15, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is OLAH, a target nucleic acid sequence may comprise bases 781 to 1480 of SEQ ID NO: 16, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 901 to 960 of SEQ ID NO: 16, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 632 to 697 of SEQ ID NO: 16, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IL1 R2, a target nucleic acid sequence may comprise bases 681 to 1310 of SEQ ID NO: 17, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CYP19A1, a target nucleic acid sequence may comprise bases 441 to 4520 of SEQ ID NO: 18, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MMP8, a target nucleic acid sequence may comprise bases 1621 to 2900 of SEQ ID NO: 19, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is TGFA, a target nucleic acid sequence may comprise bases 3321 to 4110 of SEQ ID NO: 20, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is VSTM1, a target nucleic acid sequence may comprise bases 271 to 990 of SEQ ID NO: 21, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is FCER1A, a target nucleic acid sequence may comprise bases 141 to 1110 of SEQ ID NO: 22, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 648 to 709 of SEQ ID NO: 22, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 36 to 100 of SEQ ID NO: 22, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is KLRK1, a target nucleic acid sequence may comprise bases 341 to 1590 of SEQ ID NO: 23, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is KLRB1, a target nucleic acid sequence may comprise bases 81 to 740 of SEQ ID NO: 24, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 219 to 291 or 297 to 370 of SEQ ID NO: 24, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is DAAM2, a target nucleic acid sequence may comprise bases 5131 to 6160 of SEQ ID NO: 25, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is HLA-DRA, a target nucleic acid sequence may comprise bases 561 to 1210 of SEQ ID NO: 26, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is BCL11B, a target nucleic acid sequence may comprise bases 3301 to 7670 of SEQ ID NO: 27, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 515 to 580 or 532 to 607 of SEQ ID NO: 27, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is ITM2A, a target nucleic acid sequence may comprise bases 411 to 1250 of SEQ ID NO: 28, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is SLAMF6, a target nucleic acid sequence may comprise bases 1601 to 2700 of SEQ ID NO: 29, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is HLA-DPB1, a target nucleic acid sequence may comprise bases 511 to 1090 of SEQ ID NO: 30 or bases 121 to 920 of SEQ ID NO: 31, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CD160, a target nucleic acid sequence may comprise bases 871 to 1460 of SEQ ID NO: 32, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is KLRF1, a target nucleic acid sequence may comprise bases 251 to 1240 of SEQ ID NO: 33, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CD2, a target nucleic acid sequence may comprise bases 291 to 1530 of SEQ ID NO: 34, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is LGALS2, a target nucleic acid sequence may comprise bases 101 to 520 of SEQ ID NO: 35, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is NPPC, a target nucleic acid sequence may comprise bases 261 to 640 of SEQ ID NO: 36, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MYCL, a target nucleic acid sequence may comprise bases 2931 to 3600 of SEQ ID NO: 37 or bases 781 to 1990 of SEQ ID NO: 38, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 1022 to 1113 of SEQ ID NO: 37 or bases 661 to 720 of SEQ ID NO: 38, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MX1, a target nucleic acid sequence may comprise bases 391 to 3400 of SEQ ID NO: 39, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CCL5, a target nucleic acid sequence may comprise bases 311 to 1230 of SEQ ID NO: 40, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is TGFB1, a target nucleic acid sequence may comprise bases 2091 to 2790 of SEQ ID NO: 41, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 2228 to 2090 of SEQ ID NO: 41, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is PLA2G7, a target nucleic acid sequence may comprise bases 1041 to 1810 of SEQ ID NO: 42, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 356 to 421 or 608 to 674 of SEQ ID NO: 42, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is ARHGEF10L, a target nucleic acid sequence may comprise bases 3461 to 4490 of SEQ ID NO: 43, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 2275 to 2337 of SEQ ID NO: 43, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is GPR124, a target nucleic acid sequence may comprise bases 5021 to 5870 of SEQ ID NO: 44, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IL1 RN, a target nucleic acid sequence may comprise bases 241 to 1920 of SEQ ID NO: 45, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is NLRP3, a target nucleic acid sequence may comprise bases 1921 to 4160 of SEQ ID NO: 46, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is RBP4, a target nucleic acid sequence may comprise bases 291 to 940 of SEQ ID NO: 47, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MPP3, a target nucleic acid sequence may comprise bases 531 to 2140 of SEQ ID NO: 48, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is KIF2C, a target nucleic acid sequence may comprise bases 721 to 2630 of SEQ ID NO: 49, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MAP1A, a target nucleic acid sequence may comprise bases 9521 to 10275 of SEQ ID NO: 50, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is SELP, a target nucleic acid sequence may comprise bases 1801 to 3150 of SEQ ID NO: 51, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is NEXN, a target nucleic acid sequence may comprise bases 361 to 2330 of SEQ ID NO: 52, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is ITGA2B, a target nucleic acid sequence may comprise bases 2211 to 3300 of SEQ ID NO: 53, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 2286 to 2345 of SEQ ID NO: 53, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 1480 to 1543 of SEQ ID NO: 53, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MYL9, a target nucleic acid sequence may comprise bases 221 to 1030 of SEQ ID NO: 54, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 38 to 83 or 53 to 120 of SEQ ID NO: 54, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is ITGB3, a target nucleic acid sequence may comprise bases 2611 to 4580 of SEQ ID NO: 55, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 1116 to 1182 or 1978 to 2047 of SEQ ID NO: 55, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CMTM5, a target nucleic acid sequence may comprise bases 381 to 1020 of SEQ ID NO: 56, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is LCN2, a target nucleic acid sequence may comprise bases 131 to 710 of SEQ ID NO: 57, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 532 to 603 or 632 to 689 of SEQ ID NO: 57, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is NLRC4, a target nucleic acid sequence may comprise bases 441 to 1310 of SEQ ID NO: 58, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is PPBP, a target nucleic acid sequence may comprise bases 241 to 1200 of SEQ ID NO: 59, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is TREML1, a target nucleic acid sequence may comprise bases 611 to 1340 of SEQ ID NO: 60, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 502 to 569 or 520 to 588 of SEQ ID NO: 60, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is PF4, a target nucleic acid sequence may comprise bases 261 to 850 of SEQ ID NO: 61, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CLEC1B, a target nucleic acid sequence may comprise bases 351 to 970 of SEQ ID NO: 62, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is LCN15, a target nucleic acid sequence may comprise bases 71 to 762 of SEQ ID NO: 63, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CIQC, a target nucleic acid sequence may comprise bases 501 to 1100 of SEQ ID NO: 64, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 39 to 302, 39 to 150, 61 to 150 or 61 to 302 of SEQ ID NO: 64, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CIQB, a target nucleic acid sequence may comprise bases 321 to 1020 of SEQ ID NO: 65, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 91 to 154 or 91 to 157 of SEQ ID NO: 65, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is PCOLEC2, a target nucleic acid sequence may comprise bases 1091 to 2000 of SEQ ID NO: 66, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CIQA, a target nucleic acid sequence may comprise bases 361 to 1098 of SEQ ID NO: 67, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 214 to 299 of SEQ ID NO: 67, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is TMEM37, a target nucleic acid sequence may comprise bases 471 to 1687 of SEQ ID NO: 68, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 25 to 115 of SEQ ID NO: 68, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is TNF, a target nucleic acid sequence may comprise bases 991 to 1670 of SEQ ID NO: 69, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is SLC39A8, a target nucleic acid sequence may comprise bases 2161 to 3109 of SEQ ID NO: 70, or bases 2921 to 4050 of SEQ ID NO: 71, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 1525 to 1603 or 1718 to 1787 of SEQ ID NO: 70, or bases 1360 to 1438 or 1553 to 1622 of SEQ ID NO: 71, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is MRAS, a target nucleic acid sequence may comprise bases 3581 to 4570 of SEQ ID NO: 72, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 1104 to 1167 or 1182 to 1246 of SEQ ID NO: 72, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IFIT1, a target nucleic acid sequence may comprise bases 1501 to 3960 of SEQ ID NO: 73, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is IFI44, a target nucleic acid sequence may comprise bases 901 to 1650 of SEQ ID NO: 74, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is RPGRIP1, a target nucleic acid sequence may comprise bases 2541 to 3770 of SEQ ID NO: 75, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is DISC1, a target nucleic acid sequence may comprise bases 1201 to 1707 of SEQ ID NO: 76, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is CXCR1, a target nucleic acid sequence may comprise bases 181 to 2080 of SEQ ID NO: 77, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 44 to 113 or 70 to 136 of SEQ ID NO: 77, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is HCAR2, a target nucleic acid sequence may comprise bases 21 to 1810 of SEQ ID NO: 78, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 918 to 979 or 1299 to 1356 of SEQ ID NO: 78, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is EPSTI1, a target nucleic acid sequence may comprise bases 621 to 2990 of SEQ ID NO: 79, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is LILRB4, a target nucleic acid sequence may comprise bases 1081 to 3240 of SEQ ID NO: 80, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is LILRB5, a target nucleic acid sequence may comprise bases 341 to 2120 of SEQ ID NO: 81, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. For example, a target nucleic acid sequence may comprise bases 1633 to 1697 or 1653 to 1706 of SEQ ID NO: 81, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is NECAB1, a target nucleic acid sequence may comprise bases 4231 to 5000 of SEQ ID NO: 82, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 763 to 845 or 1206 to 1285 of SEQ ID NO: 82, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is NECAB2, a target nucleic acid sequence may comprise bases 691 to 1490 of SEQ ID NO: 83, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 226 to 289 or 579 to 641 of SEQ ID NO: 83, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is PKHD1, a target nucleic acid sequence may comprise bases 10141 to 16040 of SEQ ID NO: 84, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence. In an alternative embodiment, a target nucleic acid sequence may comprise bases 9037 to 9100 or 10262 to 10335 of SEQ ID NO: 84, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
In embodiments wherein the one or more biomarker is PKD1, a target nucleic acid sequence may comprise bases 2201 to 14080 of SEQ ID NO: 85, and a probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence from this target sequence.
It is preferred that the binding conditions for a probe hybridising to its target sequence are such that a high level of specificity is provided—i.e., hybridisation of the probe occurs under “stringent conditions”. In general, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target (or complement) sequence hybridises to a perfectly matched probe. In this regard, the Tm of probes of the present invention, at a salt concentration of about 0.02M or less at pH 7, is for example above 60° C., such as about 70° C.
Premixed buffer solutions are commercially available (e.g., EXPRESSHYB Hybridisation Solution from CLONTECH Laboratories, Inc.), and hybridisation can be performed according to the manufacturer's instructions.
Probes of the present invention may be screened to minimise self-complementarity and dimer formation (probe-probe binding).
Any of the probes described herein may comprise a tag and/or label. The tag and/or label may, for example, be located (independently of one another) towards the middle or towards or at the 5′ or 3′ end of the herein described probes, for example at the 5′ end.
Hence, following hybridisation of tagged/labelled probe to target nucleic acid, the tag/label is associated with the target nucleic acid in the one or more biomarker. Alternatively, if an amplification step is employed, the probes may act as primers during the method of the invention and the tag/label may therefore become incorporated into the amplification product as the primer is extended.
Examples of suitable labels include detectable labels such as radiolabels or fluorescent or coloured molecules, enzymatic markers or chromogenic markers—e.g., dyes that produce a visible colour change upon hybridisation of the probe. By way of example, the label may be digoxygenin, fluorescein-isothiocyanate (FITC), R-phycoerythrin, Alexa 532 or Cy3. The probes preferably contain a Fam label (e.g., a 5′ Fam label), and/or a minor groove binder (MGB). The label may be a reporter molecule, which is detected directly, such as by exposure to photographic or X-ray film. Alternatively, the label is not directly detectable, but may be detected indirectly, for example, in a two-phase system. An example of indirect label detection is binding of an antibody to the label.
Examples of suitable tags include “complement/anti-complement pairs”. The term “complement/anti-complement pair” denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. Examples of suitable tags include biotin and streptavidin (or avidin). By way of example, a biotin tag may be captured using streptavidin, which may be coated onto a substrate or support such as a bead (for example a magnetic bead) or membrane. Likewise, a streptavidin tag may be captured using biotin, which may be coated onto a substrate or support such as a bead (for example a magnetic bead) or membrane. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, and the like. Another example is a nucleic acid sequence tag that binds to a complementary sequence. The latter may itself be pre-labelled, or may be attached to a surface (e.g., a bead) which is separately labelled. An example of the latter embodiment is the well-known LuminexR bead system. Other exemplary pairs of tags and capture molecules include receptor/ligand pairs and antibody/antigen (or hapten or epitope) pairs. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair has a binding affinity of, for example, less than 109 M−1. One exemplary tagged probe is a biotin-labelled probe, which may be detected using horse-radish peroxidase conjugated streptavidin.
The probes of the invention may be labelled with different labels or tags, thereby allowing separate identification of each probe when used in the method of the present invention.
Any conventional method may be employed to attach nucleic acid tags to a probe of the present invention (e.g., to the 5′ end of the defined binding region of the probe). Alternatively, nucleic acid probes of the invention (with pre-attached nucleic acid tags) may be constructed by commercial providers.
If an amplification step is employed, this step may be carried out using methods and platforms known in the art, for example PCR (for example, with the use of “Fast DNA Polymerase”, Life Technologies), such as real-time PCR, block-based PCR, ligase chain reaction, glass capillaries, isothermal amplification methods including loop-mediated isothermal amplification, rolling circle amplification transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. If employed, amplification may be carried using any amplification platform. Preferably, the amplification step may comprise quantitative PCR (real-time PCR).
A general amplification step (e.g., pre-detection) may be employed to increase the amount of the one or more biomarker of the invention present in the sample. PCR amplification primers are typically employed to amplify approximately 100-400 base pair regions of the target/complementary nucleic acid that contain the nucleotide targets of the present invention. In the presence of a suitable polymerase and DNA precursors (dATP, dCTP, dGTP and dTTP), forward and reverse primers are extended in a 5′ to 3′ direction, thereby initiating the synthesis of new nucleic acid strands that are complementary to the individual strands of the target nucleic acid. The primers thereby drive amplification of target nucleic acid sequences in the one or more biomarker, thereby generating amplification products comprising said target nucleic acid sequences.
An amplification step may be employed in which the probes of the present invention act as primers. In this embodiment, the probes (acting as primers) are extended from their 3′ ends (i.e., in a 5′-to-'3′) direction. Such an amplification step may be employed in conjunction with a general amplification step, such as the one described above.
The detection step may be carried out by any known means. In this regard, the probe or amplification product may be tagged and/or labelled, and the detection method may therefore comprise detecting said tag and/or label.
In one embodiment, the probe(s) may comprise a tag and/or label. Thus, in one embodiment, following hybridisation of tagged/labelled probe to target nucleic acid in the one or more biomarker, the tag/label becomes associated with the target nucleic acid. Thus, in one embodiment, the assay may comprise detecting the tag/label and correlating presence of tag/label with presence of the one or more nucleic acid biomarker of the invention.
In one embodiment, tag and/or label may be incorporated during extension of the probe(s). In doing so, the amplification product(s) become tagged/labelled, and the assay may therefore comprise detecting the tag/label and correlating presence of tag/label with presence of amplification product, and hence the presence of one or more nucleic acid biomarker of the invention.
By way of example, in one embodiment, the amplification product may incorporate a tag/label (e.g., via a tagged/labelled dNTP such as biotin-dNTP) as part of the amplification process, and the assay may further comprise the use of a binding partner complementary to said tag (e.g. streptavidin) that includes a detectable tag/label (e.g., a fluorescent label, such as R-phycoerythrin). In this way, the amplified product incorporates a detectable tag/label (e.g., a fluorescent label, such as R-phycoerythrin).
In one embodiment, the probe(s) and/or the amplification product(s) may include a further tag/label (as the complement component) to allow capture of the amplification product(s).
By way of example, a “complement/anti-complement” pairing may be employed in which an anti-complement capture component binds to said further tag/label (complement component) and thereby permits capture of the probe(s) and/or amplification product(s). Examples of suitable “complement/anti-complement” partners have been described earlier in this specification, such as a complementary pair of nucleic acid sequences, a complementary antibody-antigen pair, etc. The anti-complement capture component may be attached (e.g., coated) on to a substrate or solid support—examples of suitable substrates/supports include membranes and/or beads (e.g., a magnetic or fluorescent bead). Capture methods are well known in the art. For example, LuminexR beads may be employed. Alternatively, the use of magnetic beads may be advantageous because the beads (plus captured, tagged/labelled amplification product) can easily be concentrated and separated from the sample, using conventional techniques known in the art.
Immobilisation provides a physical location for the anti-complement capture component (or probes) and may serve to fix the capture component/probe at a desired location and/or facilitate recovery or separation of probe. The support may be a rigid solid support made from, for example, glass, plastic, or silica, such as a bead (for example a fluorescent or magnetic bead). Alternatively, the support may be a membrane, such as nylon or nitrocellulose membrane. 3D matrices are also suitable supports for use with the present invention—e.g., polyacrylamide or PEG gels. Immobilisation to a support/platform may be achieved by a variety of conventional means. By way of example, immobilisation onto a support such as a nylon membrane may be achieved by UV cross-linking. Alternatively, biotin-labelled molecules may be bound to streptavidin-coated substrates (and vice-versa), and molecules prepared with amino linkers may be immobilised on to silanised surfaces. Another means of immobilisation is via a poly-T tail or a poly-C tail, for example at the 3′ or 5′ end. Said immobilisation techniques apply equally to the probe component (and primer pair component, if present) of the present invention.
In one embodiment, the probes of the invention comprise a nucleic acid sequence tag/label (e.g., attached to each probe at the 5′ end of the defined sequence of the probe that binds to target/complement nucleic acid). In more detail, each of the probes is provided with a different nucleic acid sequence tag/label, wherein each of said tags/labels (specifically) binds to a complementary nucleic acid sequence present on the surface of a bead. Each of the different tags/labels binds to its complementary sequence counterpart (and not to any of the complementary sequence counterparts of the other tags), which is located on a uniquely identifiable bead. In this regard, the beads are uniquely identifiable, for example by means of fluorescence at a specific wavelength. Thus, in use, probes of the invention bind to target nucleic acid (if present in the sample). Thereafter, (only) the bound probes may be extended (in the 3′ direction) in the presence of one or more labelled dNTP (e.g., biotin labelled dNTPs, such as biotin-dCTPs).
The extended primers may be contacted with a binding partner counterpart to the labelled dNTPs (e.g., a streptavidin labelled fluorophore, such as streptavidin labelled R-phycoerythrin), which binds to those labelled dNTPs that have become incorporated into the extended primers. Thereafter, the labelled extended primers may be identified by allowing them to bind to their nucleic acid counterparts present on the uniquely identifiable beads. The latter may then be “called” (e.g., to determine the type of bead present by wavelength emission) and the nature of the primer extension (and thus the type of target/complement nucleic acid present) may be determined.
Typically, probes of the invention are oligonucleotides having sequence identity with a region of the one or more biomarker of the invention as disclosed herein. One or more probes may be immobilised on a solid support and used to interrogate mRNA obtained from a test sample. If the mRNA from the test sample contains the one or more biomarker targeted by the immobilised probe, it will bind to the probe, and may then be detected. The biomarkers of the invention may also be detected using PCR, such as real time PCR.
Any oligonucleotide with the appropriate level of sequence identity with the one or more biomarker of the invention, or with one or more target sequences within said one or more biomarker of the invention may be used as a probe in the methods and uses described herein. Any oligonucleotide with the appropriate level of complementarity with the one or more biomarker of the invention, or with one or more target sequences within said one or more biomarker of the invention may be used as a probe in the methods and uses of the invention described herein. Exemplary sequences of the one or more biomarkers of the invention are given in SEQ ID NOs: 1 to 85 (see Tables 1-4 herein). Exemplary probe nucleic acid sequences for the biomarkers disclosed herein are set out in Table 14 (SEQ ID NOs: 86-421) and are shown as underlined and bold text in the sequences of the Sequence Information section. These probes are best suited to use in microarray detection of the nucleic acid.
Further exemplary probe nucleic acid sequences are set out in Table 15 (SEQ ID NOs: 424, 427, 430, 433, 436, 439, 442, 445, 448, 451, 454, 457, 460, 463, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 506, 509, 512, 515, 518, 521, 524, 525, 528, 531, 534, 537, 540, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, 579, 582, and 585) together with the forward and reverse primers that are preferably used to amplify the target sequence prior to detection. These probes are best suited to use in quantitative PCR.
Any one or more (eg. 2 or more, 3 or more, up to an including all) of the exemplary probe sequences may be used in the methods and uses of the invention to determine the presence and/or amount of the one or more biomarker.
In embodiments wherein the one or more biomarker is ADM, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 86, 87, 88 or 89, preferably SEQ ID NO: 86.
In embodiments wherein the one or more biomarker is CD177, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 90, 91, 92, or 93, preferably SEQ ID NO: 90.
In embodiments wherein the one or more biomarker is FAM20A, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 94, 95, 96 or 97, preferably SEQ ID NO: 94 or 95. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 424 or 427.
In embodiments wherein the one or more biomarker is IL110, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 98, 99, 100 or 101, preferably SEQ ID NO: 98.
In embodiments wherein the one or more biomarker is METT7LB, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 102, 103, 104 or 105, preferably SEQ ID NO: 102.
In embodiments wherein the one or more biomarker is MMP9, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 106, 107, 108, 109, preferably SEQ ID NO:106.
In embodiments wherein the one or more biomarker is RETN, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 110, 111, 112, or 113, preferably SEQ ID NO: 110.
In embodiments wherein the one or more biomarker is TDRD9, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 114, 115, 116, or 117, preferably SEQ ID NO: 114.
In embodiments wherein the one or more biomarker is ITGA7, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 118, 119, 120, or 121, preferably SEQ ID NO: 118.
In embodiments wherein the one or more biomarker is BMX, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 122, 123, 124 or 125, preferably SEQ ID NO: 122.
In embodiments wherein the one or more biomarker is HP, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 126, 127, 128 or 129, preferably SEQ ID NO: 126.
In embodiments wherein the one or more biomarker is IGFBP2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 130, 131, 132, or 133, preferably SEQ ID NO: 130.
In embodiments wherein the one or more biomarker is ALPL, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 134, 135, 136, or 137, preferably SEQ ID NO: 134.
In embodiments wherein the one or more biomarker is DACH1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 138, 139, 140, or 141, preferably SEQ ID NO: 138 or 139.
In embodiments wherein the one or more biomarker is IL1 R1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 142, 143, 144, or 145, preferably SEQ ID NO: 142 or 143.
In embodiments wherein the one or more biomarker is OLAH, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 146, 147, 148, or 149, preferably SEQ ID NO: 146. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 430 or 433.
In embodiments wherein the one or more biomarker is IL1 R2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 150, 151, 152, or 153, preferably SEQ ID NO: 150.
In embodiments wherein the one or more biomarker is CYP19A1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 154, 155, 156 or 157, preferably SEQ ID NO: 154 or 155.
In embodiments wherein the one or more biomarker is MMP8, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 158, 159, 160, or 161, preferably SEQ ID NO: 158.
In embodiments wherein the one or more biomarker is TGFA, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 162, 163, 164, 165, preferably SEQ ID NO: 162.
In embodiments wherein the one or more biomarker is VSTM1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 166, 167, 168, or 169, preferably SEQ ID NO:166.
In embodiments wherein the one or more biomarker is FCER1A, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 170, 171, 172, or 173, preferably SEQ ID NO:170. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 528 or 531.
In embodiments wherein the one or more biomarker is KLRK1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:174, 175, 176, or 177, preferably SEQ ID NO: 174.
In embodiments wherein the one or more biomarker is KLRB1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 178, 179, 180, or 181, preferably SEQ ID NO: 178. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 546 or 549.
In embodiments wherein the one or more biomarker is DAAM2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 182, 183, 184, or 185, preferably SEQ ID NO: 182 or 183.
In embodiments wherein the one or more biomarker is HLA-DRA, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 186, 187, 188, or 189, preferably SEQ ID NO:186.
In embodiments wherein the one or more biomarker is BCL11B, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 190, 191, 192, or 193, preferably SEQ ID NO: 190 or 191.
Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 534 or 537.
In embodiments wherein the one or more biomarker is ITM2A, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 194, 195, 196, or 197, preferably SEQ ID NO: 194.
In embodiments wherein the one or more biomarker is SLAMF6, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 198, 199, 200, or 201, preferably SEQ ID NO: 198.
In embodiments wherein the one or more biomarker is HLA-DPB1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 202, 203, 204, or 205, preferably SEQ ID NO:202 or 203.
In embodiments wherein the one or more biomarker is CD160, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 206, 207, 208, or 209, preferably SEQ ID NO: 206.
In embodiments wherein the one or more biomarker is KLFF1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 210, 211, 212, or 213, preferably SEQ ID NO: 210.
In embodiments wherein the one or more biomarker is CD2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:214, 215, 216 or 217, preferably SEQ ID NO: 214.
In embodiments wherein the one or more biomarker is LGALS2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 218, 219, 220, or 221, preferably SEQ ID NO: 218.
In embodiments wherein the one or more biomarker is NPPC, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 222, 223, 224, or 225, preferably SEQ ID NO: 222.
In embodiments wherein the one or more biomarker is MYCL, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 226, 227, 228, 229, 230, 231, 232, or 233. In embodiments wherein the one or more biomarker is transcript variant 3 of MYCL, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 226, 227, 228, or 229, preferably SEQ ID NO: 226. In embodiments wherein the one or more biomarker is transcript variant 1 of MYCL, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 230, 231, 232, or 233, preferably SEQ ID NO: 230. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 481 or 484.
In embodiments wherein the one or more biomarker is MX1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 234, 235, 236, or 237, preferably SEQ ID NO: 234.
In embodiments wherein the one or more biomarker is CCL5, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 238, 239, 240, or 241, preferably SEQ ID NO: 238.
In embodiments wherein the one or more biomarker is TGFB1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 242, 243, 244, or 245, preferably SEQ ID NO: 242. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 475 or 478.
In embodiments wherein the one or more biomarker is PLA2G7, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 246, 247, 248, or 249, preferably SEQ ID NO: 246. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 466 or 469.
In embodiments wherein the one or more biomarker is ARHGEF10L, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 250, 251, 252, or 253, preferably SEQ ID NO: 250 or 251. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 472.
In embodiments wherein the one or more biomarker is GPR124, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 254, 255, 256, or 257, preferably SEQ ID NO: 254.
In embodiments wherein the one or more biomarker is IL1 RN, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 258, 259, 260, or 261, preferably SEQ ID NO: 258 or 259.
In embodiments wherein the one or more biomarker is NLRP3, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 262, 263, 264, or 265, preferably SEQ ID NO: 262.
In embodiments wherein the one or more biomarker is RBP4, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 266, 267, 268, or 269, preferably SEQ ID NO: 266.
In embodiments wherein the one or more biomarker is MPP3, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 270, 271, 272, or 273, preferably SEQ ID NO: 270.
In embodiments wherein the one or more biomarker is KIF2C, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 274, 275, 276, or 277, preferably SEQ ID NO:274.
In embodiments wherein the one or more biomarker is MAP1A, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 278, 279, 280, or 281, preferably SEQ ID NO: 278.
In embodiments wherein the one or more biomarker is SELP, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 282, 283, 284, or 285, preferably SEQ ID NO: 282.
In embodiments wherein the one or more biomarker is NEXN, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 286, 287, 288, or 289, preferably SEQ ID NO:286 or 287.
In embodiments wherein the one or more biomarker is ITGA2B, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 290, 291, 292, or 293, preferably SEQ ID NO: 290. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 460 or 463.
In embodiments wherein the one or more biomarker is MYL9, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 294, 295, 296, or 297, preferably SEQ ID NO: 294. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 448 or 451.
In embodiments wherein the one or more biomarker is ITGB3, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 298, 299, 300, or 301, preferably SEQ ID NO: 298.
In embodiments wherein the one or more biomarker is CMTM5, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 302, 303, 304 or 305, preferably SEQ ID NO: 302.
In embodiments wherein the one or more biomarker is LCN2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 306, 307, 308, or 309, preferably SEQ ID NO: 306. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 436 or 439.
In embodiments wherein the one or more biomarker is NLRC4, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 310, 311, 312, or 313, preferably SEQ ID NO: 310.
In embodiments wherein the one or more biomarker is PPBP, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 314, 315, 316, or 317, preferably SEQ ID NO: 314.
In embodiments wherein the one or more biomarker is TREML1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 318, 319, 320, 321, preferably SEQ ID NO: 318.
In embodiments wherein the one or more biomarker is PF4, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 322, 323, 324, or 325, preferably SEQ ID NO: 322.
In embodiments wherein the one or more biomarker is CLEC1B, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 326, 327, 328, or 329, preferably SEQ ID NO: 326 or 327.
In embodiments wherein the one or more biomarker is LCN15, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 330, 331, 332, or 333, preferably SEQ ID NO: 330.
In embodiments wherein the one or more biomarker is CIQC, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 334, 335, 336, or 337, preferably SEQ ID NO: 334. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 506 or 509.
In embodiments wherein the one or more biomarker is CIQB, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 338, 339, 340, or 341, preferably SEQ ID NO: 338. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 502.
In embodiments wherein the one or more biomarker is PCOLCE2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 342, 343, 344, or 345, preferably SEQ ID NO: 342.
In embodiments wherein the one or more biomarker is CIQA, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 346, 347, 348, or 349, preferably SEQ ID NO: 346. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 499.
In embodiments wherein the one or more biomarker is TMEM37, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 350, 351, 352, or 353, preferably SEQ ID NO: 350. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 524 or 525.
In embodiments wherein the one or more biomarker is TNF, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 354, 355, 356, or 357, preferably SEQ ID NO: 354.
In embodiments wherein the one or more biomarker is SLC39A8, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 358, 359, 360, 361, 362, 363, 364, or 365, preferably SEQ ID NO: 358 or 362. In embodiments wherein the one or more biomarker is transcript variant 1 of SLC39A8, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 358, 359, 360, or 361, preferably SEQ ID NO: 358. In embodiments wherein the one or more biomarker is transcript variant 3 of SLC39A8, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 362, 363, 364, or 365, preferably SEQ ID NO: 362. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 518 or 521.
In embodiments wherein the one or more biomarker is MRAS, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 366, 367, 368, or 369, preferably SEQ ID NO: 366. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 512 or 515.
In embodiments wherein the one or more biomarker is IFIT1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 370, 371, 372, or 373, preferably SEQ ID NO: 370.
In embodiments wherein the one or more biomarker is IFI44, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 374, 375, 376, or 377, preferably SEQ ID NO: 374.
In embodiments wherein the one or more biomarker is RPGRIP1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 378, 379, 380, or 381, preferably SEQ ID NO: 378.
In embodiments wherein the one or more biomarker is DISC1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 382, 383, 384, or 385, preferably SEQ ID NO: 382.
In embodiments wherein the one or more biomarker is CXCR1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 386, 387, 388, or 389, preferably SEQ ID NO: 386. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 487 or 490.
In embodiments wherein the one or more biomarker is HCAR2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 390, 391, 392, or 393, preferably SEQ ID NO: 390. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 493 or 496.
In embodiments wherein the one or more biomarker is EPST1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 394, 395, 396, or 397, preferably SEQ ID NO: 394.
In embodiments wherein the one or more biomarker is LILRB4, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 398, 399, 400, or 401, preferably SEQ ID NO: 398 and 399.
In embodiments wherein the one or more biomarker is LILRB5, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 402, 403, 404, or 405, preferably SEQ ID NO: 402. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 552 or 555.
In embodiments wherein the one or more biomarker is NECAB1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 406, 407, 408, or 409, preferably SEQ ID NO: 406. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 558 or 561.
In embodiments wherein the one or more biomarker is NECAB2, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 410, 411, 412, or 413, preferably SEQ ID NO: 410. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 564 or 567.
In embodiments wherein the one or more biomarker is PKHD1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 414, 415, 416 or 417, preferably SEQ ID NO: 414 or 415. Alternatively, the oligonucleotide probe may comprise or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 540 or 543.
In embodiments wherein the one or more biomarker is PKD1, the oligonucleotide probe typically comprises or is complementary to a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 418, 419, 420, or 421, preferably SEQ ID NO: 418.
In all of the methods and uses described herein, the presence and/or amount of the one or more biomarker is determined using an oligonucleotide probe specific for the one or more biomarker. The oligonucleotide probe used in the methods and uses of the invention may an oligonucleotide probe of the invention as described herein.
As described above, a general amplification step (e.g., pre-detection) may be employed to increase the amount of the one or more biomarker of the invention present in the sample. As well as using the oligonucleotide probes of the invention as primers, separate forward and reverse oligonucleotide primers may be used to amplify a target nucleic acid sequence. The amplified nucleic acid may be detected using an oligonucleotide probe of the invention. For example, such primers and probes may be used when the one or more biomarker is detected and/or quantified by quantitative PCR.
The present invention therefore provides a forward oligonucleotide primer and/or a reverse oligonucleotide primer for amplification of a target nucleic acid sequence in the one or more biomarker.
In one embodiment, one or more forward oligonucleotide primer and one or more reverse oligonucleotide primer may be used to amplify the one or more nucleic acid biomarker of the invention prior to detection.
In general, a reverse primer is designed to hybridise to a target nucleic acid sequence within the coding (sense) strand of a target nucleic acid, and a forward primer is designed to hybridise to a target nucleic acid sequence within the complementary (ie. anti-sense) strand of the target nucleic acid.
The term “complement of a nucleic acid sequence” refers to a nucleic acid sequence having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
The forward primer hybridises to a target nucleic acid sequence (a ‘forward primer target sequence’) located within the sequence of the nucleic acid biomarker. In one embodiment, the forward primer target sequence has a length in the range of 10-40 consecutive nucleotides, such at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides, and/or up to 38, 35, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 consecutive nucleotides.
The reverse primer hybridises to a target nucleic acid sequence (a ‘reverse primer target sequence’) located within the sequence of the nucleic acid biomarker. In one embodiment, the reverse primer target sequence has a length in the range of 10-40 consecutive nucleotides, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides, and/or up to 38, 35, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 consecutive nucleotides.
The present invention also provides oligonucleotide primers and probes for amplifying control (or reference) genes. In one embodiment, the control gene is selected from the group consisting of: ALAS1 (NM_000688 SEQ ID NO: 586, or NM_199166, SEQ ID NO: 587), GTF2D1 (NM_003194, SEQ ID NO: 588, and HMBS (NM_000190.3, SEQ ID NO: 589).
In one embodiment, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence selected from SEQ ID NOs: 1-85, or a nucleotide sequence that is at least 80% identical thereto (e.g., at least 82, 84. 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto).
In one embodiment, the reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) a nucleotide sequence that is at least 80% identical to (e.g at least 82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to) a nucleotide sequence selected from SEQ ID NOs: 1-85.
Exemplary primer and probe sequences are shown in Table 15.
In one embodiment, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence selected from SEQ ID NOs: 422, 425, 428, 431, 434, 437, 440, 443, 446, 449, 452, 455, 458, 461, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 504, 507, 510, 513, 516, 519, 522, 526, 529, 532, 535, 538, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, 577, 580, and 583 (as shown in Table 15), or a nucleotide sequence that is at least 80% identical thereto (e.g. at least 82, 84. 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto).
In one embodiment, the reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to (e.g at least 82, 84. 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to) a nucleotide sequence selected from SEQ ID NOs: 423, 426, 429, 432, 435, 438, 441, 444, 447, 450, 453, 456, 447, 450, 453, 456, 459, 462, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 503, 505, 508, 511, 514, 517, 520, 523, 527, 530, 533, 536, 539, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, 578, 581, and 584 (as shown in Table 15).
In one embodiment, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to (preferably at least 82, 84, 86, 88, 90 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to) a nucleotide sequence selected from SEQ ID NOs: 422, 425, 428, 431, 434, 437, 440, 443, 446, 449, 452, 455, 458, 461, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 504, 507, 510, 513, 516, 519, 522, 526, 529, 532, 535, 538, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, 577, 580, and 583 (as shown in Table 15). Conservative substitutions are preferred.
In one embodiment, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to (preferably at least 82, 84, 86, 88, 90 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to) a nucleotide sequence selected from SEQ ID NOs: 423, 426, 429, 432, 435, 438, 441, 444, 447, 450, 453, 456, 447, 450, 453, 456, 459, 462, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 503, 505, 508, 511, 514, 517, 520, 523, 527, 530, 533, 536, 539, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, 578, 581, and 584 (as shown in Table 15). Conservative substitutions are preferred.
In embodiments wherein the one or more biomarker is FAM20A, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 422 or 425, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 422 or 425. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 423 or 426. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 423 or 426. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 424 or 427.
In embodiments wherein the one or more biomarker is OLAH, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 428 or 431, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 428 or 431. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 429 or 432. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 429 or 432. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 430 or 433).
In embodiments wherein the one or more biomarker is LCN2, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 434 or 437, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 434 or 437. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 435 or 438. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 435 or 438. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 436 or 439).
In embodiments wherein the one or more biomarker is ITGB3, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 440 or 443, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 440 or 443. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 441 or 444. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 441 or 444. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 442 or 445).
In embodiments wherein the one or more biomarker is MYL9, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 446 or 449, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 446 or 449. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 447 or 450. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 447 or 450. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 448 or 451).
In embodiments wherein the one or more biomarker is TREML1, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 452 or 455, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 452 or 455. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 453 or 456. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 453 or 456. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 454 or 457).
In embodiments wherein the one or more biomarker is ITGA2B, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 458 or 461, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 458 or 461. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 459 or 462. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 459 or 462. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 457 or 460).
In embodiments wherein the one or more biomarker is PLA2G7, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 464 or 467, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 464 or 467. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 465 or 468. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 465 or 468. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 466 or 469).
In embodiments wherein the one or more biomarker is ARHGEF10L, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NO: 470, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 470. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 471. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 471. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NO: 472).
In embodiments wherein the one or more biomarker is TGFB1, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 473 or 476, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 473 or 476. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 474 or 477. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 474 or 477. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 475 or 478).
In embodiments wherein the one or more biomarker is MYCL, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 479 or 482, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 479 or 482. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 480 or 483. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 480 or 483. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 481 or 484).
In embodiments wherein the one or more biomarker is CXCR1, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 485 or 488, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 485 or 488. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 486 or 489. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 486 or 489. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 487 or 490).
In embodiments wherein the one or more biomarker is HCAR2, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 491 or 494, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 491 or 494. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 492 or 495. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 492 or 495. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 493 or 496).
In embodiments wherein the one or more biomarker is CIQA, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NO: 497, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 497. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 498. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 498. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NO: 499).
In embodiments wherein the one or more biomarker is CIQB, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NO: 500, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 500. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 501 or 503. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 501 or 503. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NO: 502).
In embodiments wherein the one or more biomarker is CIQC, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 504 or 507, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 504 or 507. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 505 or 508. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 505 or 508. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NO: 506 or 509).
In embodiments wherein the one or more biomarker is MRAS, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 510 or 513, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 510 or 513. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 511 or 514. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 511 or 514. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 512 or 515).
In embodiments wherein the one or more biomarker is SLC39A8, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 516 or 519, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 516 or 519. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 517 or 520. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 517 or 520. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 518 or 521).
In embodiments wherein the one or more biomarker is TMEM37, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NO: 522, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 522. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 523. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 523. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 524 or 525).
In embodiments wherein the one or more biomarker is FCER1A, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 526 or 529, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 526 or 529. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 527 or 530. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 527 or 530. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 528 or 531).
In embodiments wherein the one or more biomarker is BLC11B, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 532 or 535, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 532 or 535. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 533 or 536. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 533 or 536. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 534 or 537).
In embodiments wherein the one or more biomarker is PKHD1, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 538 or 541, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 538 or 541. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 539 or 542. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 539 or 542. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 540 or 543).
In embodiments wherein the one or more biomarker is KLRB1, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 544 or 547, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 544 or 547. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 545 or 548. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 545 or 548. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 546 or 549).
In embodiments wherein the one or more biomarker is LILRB5, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 550 or 553, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 550 or 553. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 551 or 554. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 551 or 554. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 552 or 555).
In embodiments wherein the one or more biomarker is NECAB1, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 556 or 559, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 556 or 559. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 557 or 560. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NOs: 557 or 560. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 558 or 561).
In embodiments wherein the one or more biomarker is NECAB2, the forward primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of the nucleotide sequence of SEQ ID NOs: 562 or 565, or a nucleotide sequence that is at least 80% identical thereto. For example, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 562 or 565. The reverse primer hybridises to a target nucleic acid sequence that comprises (or consists of) the complement of a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NOs: 563 or 566. For example, the reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to SEQ ID NOs: 563 or 566. The amplified target nucleic acid may be detected using an oligonucleotide probe as described herein (e.g., by reference to SEQ ID NOs: 564 or 567).
In embodiments wherein the control gene is ALAS1, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 568 or 571. The reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to SEQ ID NOs: 569 or 572. The amplified target nucleic acid may be detected using an oligonucleotide probe that comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NOs:570 or 573.
In embodiments wherein the control gene is HMBS, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 574 or 577. The reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to SEQ ID NOs: 575 or 578. The amplified target nucleic acid may be detected using an oligonucleotide probe that comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NOs: 576 or 579.
In embodiments wherein the control gene is GTF2D1, the forward primer comprises (or consists of) a nucleotide sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 580 or 583. The reverse primer comprises (or consists of) a nucleotide sequence having at least 80% identity to SEQ ID NOs: 581 or 584. The amplified target nucleic acid may be detected using an oligonucleotide probe that comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NOs: 582 or 585.
In all of the methods and uses described herein, the presence and/or amount of the one or more biomarker may be determined using a forward oligonucleotide primer and/or reverse oligonucleotide primer specific for the one or more biomarker to amplify a target nucleic acid sequence. The forward and reverse oligonucleotide primers used in the methods and uses of the invention are as described herein.
Kits and Devices
The present invention also provides kits and devices that are useful in diagnosing a systemic inflammatory condition (including diagnosing SIRS, sepsis, abdominal sepsis, and pulmonary sepsis), distinguishing between sepsis and SIRS, distinguishing between abdominal sepsis and pulmonary sepsis, monitoring of a systemic inflammatory condition (including monitoring of SIRS, sepsis, abdominal sepsis, and pulmonary sepsis), determining whether a patient is suitable for discharge from medical care, and diagnosing organ damage.
The kits and devices of the present invention comprise one or more biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more biomarker of the invention. For example, the kit and device may comprise one or more biomarker of the invention. For example, the kit and device may comprise one or more agent for the detection of or for the determination of the amount of the one or more biomarker of the invention. The “one or more agent” may comprise one or more binding agent specific for the one or more biomarker.
The “one or more biomarker of the invention” may be as described herein. Specific biomarkers and agents for the detection of said biomarkers useful in the present invention are set forth herein. The biomarkers of the kit or device can be used to generate biomarker profiles according to the present invention.
In one embodiment, the kit and device of the present invention may comprise one or more inflammation biomarker of the invention (e.g., as described herein) and/or one or more agent for the detection of or for the determination of the amount of the one or more inflammation biomarker of the invention. For example, the “one or more inflammation biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, or all 21) inflammation biomarker selected from the group consisting of: FAM20A, OLAH, CD177, ADM, I-10, METTL7B, MMP9, RETN, TDRD9, ITGA7, BMX, HP, IGFBP2, ALPL, DACH1, IL1R1, IL1R2, CYP19A1, MMP8, TGFA and VSTM1. For example, the “one or more inflammation biomarker of the invention” may comprise one of more (2 or more, or all 3) of: FAM20A, OLAH, and CD177. For example, the “one or more inflammation biomarker of the invention” may comprise FAM20A and OLAH.
In one embodiment, the kit and device of the present invention may comprise one or more sepsis biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more sepsis biomarker of the invention. For example, the “one or more sepsis biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or all 30) sepsis biomarker selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, PF4, KIF2C, MAP1A, SELP, NEXN, NLRC4, CLEC1B, SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, RPGRIP1, HCAR2, CXCR1, DISC1, and EPSTI1. For example, the “one or more sepsis biomarker of the invention” may comprise one of more (2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more or all 9) of ITGB3, ITGA2B, MYL9, LCN2, TREML1, LCN15, CMTM5, PPBP, and PF4. For example, the “one or more sepsis biomarker of the invention” may comprise one of more (2 or more, 3 or more, 4 or more, or all 5) of the sepsis biomarkers ITGB3, ITGA2B, MYL9, LCN2, and TREML1.
In one embodiment, the kit and device of the present invention may comprise one or more SIRS biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more SIRS biomarker of the invention. For example, the “one or more SIRS biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) SIRS biomarker selected from the group consisting of: of PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124, IL1RN, NLRP3, RBP4, and MPP3. For example, the “one or more SIRS biomarker of the invention” may comprise one of more (2 or more, 3 or more, 4 or more, or all 5) of PLA2G7, ARHGEF10L, MYCL, TGFBI, and GPR124. For example, the “one or more SIRS biomarker of the invention” may comprise one of more (2 or more, 3 or more, or all 4) of PLA2G7, ARHGEF10L, MYCL, and TGFBI.
In one embodiment, the kit and device of the present invention may comprise:
For example, the kit and device may comprise one or more agent for the detection of or for the determination of the amount of: (i) one or more inflammation biomarker (as described herein), (ii) one or more sepsis marker (as described herein), and/or (iii) one or more SIRS biomarker (as described herein). For example, the kit and device may comprise one or more agent for the detection of or for the determination of the amount of: (i) one of more (2 or more, or all 3) of the inflammatory biomarker selected from the group consisting of: FAM20A, OLAH, and optionally CD177; (ii) one of more (2 or more, 3 or more, 4 or more, or all 5) of the sepsis biomarker selected from the group consisting of: ITGB3, ITGA2B, MYL9, LCN2, and TREML1; and/or (iii) one of more (2 or more, 3 or more, or all 4) of the SIRS biomarker selected from the group consisting of: PLA2G7, ARHGEF10L, MYCL, and TGFBI.
In one embodiment, the kit and device of the present invention may comprise one or more abdominal sepsis biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more abdominal sepsis biomarker of the invention. For example, the “one or more abdominal sepsis biomarker of the invention” may comprise one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more or all 12) abdominal sepsis biomarker selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB, PCOLCE2, KIF2C, TNF, IFI44, IFIT1, and RPGRIP1. For example, the “one or more abdominal sepsis biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or all 6) of SLC39A8, CIQC, CIQA, MRAS, TMEM37, CIQB. For example, the “one or more abdominal sepsis biomarker of the invention” may comprise one of more (2 or more, or all 3) of SLC39A8, CIQC, CIQA.
In one embodiment, the kit and device of the present invention may comprise one or more pulmonary sepsis biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more pulmonary sepsis biomarker of the invention. For example, the “one or more pulmonary sepsis biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, or all 5) pulmonary sepsis biomarker selected from the group consisting of: HCAR2, CXCR1, DISC1, EPSTI1, and IFI44. For example, the “one or more pulmonary sepsis biomarker of the invention” may comprise one of more (2 or more, or all 3) of HCAR2, CXCR1, and DISC1.
In one embodiment, the kit and device of the present invention may comprise:
For example, the kit and device may comprise one or more agent for the detection of or for the determination of the amount of: (i) one or more abdominal sepsis biomarker (as described herein); and/or (ii) one or more pulmonary sepsis marker (as described herein). For example, the kit and device may comprise one or more agent for the detection of or for the determination of the amount of: (i) one or more (2 or more, 3 or more, 4 or more, 5 or more, or all 6) of the abdominal sepsis biomarker selected from the group consisting of: SLC39A8, CIQC, CIQA, MRAS, TMEM37, and CIQB; and/or (ii) one or more (e.g. 2 or more, or all 3) of the pulmonary sepsis biomarker selected from the group consisting of: HCAR2, CXCR1, and DISC1;
In one embodiment, the kit and device of the present invention may comprise one or more prognosis biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more recovery biomarker of the invention. For example, the “one or more prognosis biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or all 20) biomarker selected from the group consisting of: ITM2A, CCL5, NPPC, PKD1, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, FCER1A, DAAM2, SLAMF6, CD160, KLRF1, CD2, LGALS2, MYCL, MX1, NECAB1, and PKHD1. For example, the “one or more biomarker of the invention” may comprise one of more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12) of ITM2A, CCL5, NPPC, KLRK1, KLRB1, HLA-DRA, BCL11B, HLA-DPB1, SLAMF6, CD160, KLRF1, and MX1. For example, the “one or more biomarker of the invention” may comprise one of more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all 7) of CCL5, NPPC, PKD1, LGALS2, MYCL, NECAB1, and PKHD1.
In one embodiment, the kit and device of the present invention may comprise one or more survival biomarker of the invention and/or one or more agent for the detection of or for the determination of the amount of the one or more survival biomarker of the invention. For example, the “one or more survival biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or all 6) survival biomarker selected from the group consisting of: NECAB1, NECAB2, PKDI, PKHD1, LILRB4, and LILRB5. For example, the “one or more survival biomarker of the invention” may comprise one or more (e.g., 2 or more, 3 or more, of all 4) of NECAB1, PKDI, PKHD1, and LILRB5. For example, the “one or more survival biomarker of the invention” may comprise NECAB2 and/or PKD1. For example, the “one or more survival biomarker of the invention” may comprise PKHD1 and/or LILRB5.
The kit and device of the present invention may comprise:
For example, the kit and device may comprise one or more agent for the detection of or for the determination of the amount of: (i) one or more prognosis biomarker (as described herein); and/or (ii) one or more survival biomarker (as described herein).
In one embodiment, the kit and device of the present invention may comprise one or more agent for the detection of or for the determination of the amount of:
Generally, the biomarkers and agents of the kit or device will bind, with at least some specificity, to the biomarker molecules contained in the sample from which the biomarker profile is generated. In one embodiment, the kit or device of the invention comprises one or more binding agent specific for the one or more biomarker. Examples of classes of compounds of the kit or device include, but are not limited to, proteins (including antibodies), and fragments thereof, peptides, polypeptides, proteoglycans, glycoproteins, lipoproteins, carbohydrates, lipids, nucleic acids, organic and inorganic chemicals, and natural and synthetic polymers. The biomarker(s) and/or agent(s) for the detection of the one or more biomarkers may be part of an array, or the biomarker(s) and/or agent(s) may be packaged separately and/or individually. The biomarker(s) and/or agent(s) may be immobilised on an inert support. The biomarkers(s) and/or agent(s) may be immobilised on a surface to provide a binding agent array.
The device may be a lateral flow device. Lateral flow devices and methods for their construction are well known in the art, being best known as the standard pregnancy test kit. The device may be portable. The device may be disposable. The device may be suitable for use as a point of care diagnostic test. The device may be suitable for use in the home or in the clinic.
The kit or device may also comprise at least one internal standard to be used in generating the biomarker profiles of the present invention. Likewise, the internal standards can be any of the classes of compounds described above.
The kits and devices of the present invention also may contain reagents that can be used to detectably label biomarkers contained in the biological samples from which the biomarker profiles are generated. For this purpose, the kit or device may comprise a set of antibodies or functional fragments thereof that specifically bind at least two, three, four, five, 10, 20, 30, 40, 50 or more, up to all of the biomarkers set forth in any one of Tables 1 to 4 that list biomarkers for use in the invention. The antibodies themselves may be detectably labelled. The kit or device also may comprise a specific biomarker binding component, such as an aptamer.
In a preferred embodiment, a kit or device of the invention comprises (i) one or more antibody specific for the one or more biomarkers of the invention; and/or (ii) one or more oligonucleotide specific for the one or more biomarker of the invention. For example, the one or more oligonucleotide specific for the one or more biomarker is an oligonucleotide of the invention, preferably one or more of SEQ ID NOs: 86-585.
If the biomarkers comprise a nucleic acid, the kit or device may provide one or more oligonucleotide probe that is capable of forming a duplex with the one or more biomarker or with a complementary strand of said one or more biomarker. The one or more oligonucleotide probe may be detectably labelled. Typically, the one or more oligonucleotide probe used in the methods of the invention is selected from one or more of the oligonucleotide probes described herein.
In one embodiment, the one or more oligonucleotide probe is selected from an oligonucleotide probe that comprises or is complementary to at least one nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of any one or more of SEQ ID NOs: 86-421. The oligonucleotide probe(s) may be bound to a solid support (such as a microarray).
In one embodiment, the one or more oligonucleotide probe is selected from an oligonucleotide probe that comprises or is complementary to at least one nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequences of any one or more of SEQ ID NOs: 424, 427, 430, 433, 436, 439, 442, 445, 448, 451, 454, 457, 460, 463, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 506, 509, 512, 515, 518, 521, 524, 525, 528, 531, 534, 537, 540, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, 579, 582, and 585 (as described in Table 15).
The kit or device may further comprise: (i) one or more forward oligonucleotide primer; and/or (ii) one or more reverse oligonucleotide primer for amplification of the target nucleic acid sequence. The forward and reverse oligonucleotide primers may be as described herein (and exemplified in Table 15). The amplification product may be detected using the corresponding oligonucleotide probe(s) described herein (as exemplified in Table 15). The kit or device may further comprise one or more reagent for performing quantitative PCR.
The kits and devices of the present invention may also include pharmaceutical excipients, diluents and/or adjuvants when the biomarker is to be used to raise an antibody. Examples of pharmaceutical adjuvants include, but are not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
The present invention is discussed in more detail by means of the Examples described below, and by the Figures.
Patients:
Patients with severe sepsis and septic shock were recruited for the study based on the following criteria:
Patients with SIRS (Critically Ill patients without infection) were recruited based on the following criteria:
Exclusion Criteria
Volunteers above the age of 18 were recruited for use as healthy control individuals. Exclusion criteria included:
Sample Collection and Processing:
Blood samples were collected from the sepsis patients (abdominal sepsis N=54 and pulmonary sepsis patients N=76) and SIRS patients (N=38) at day 1, day 2, and day 5 of admittance to an intensive care unit (ICU) and on discharge. One blood sample was collected from healthy volunteers (N=30) similar to day 1 blood sampling of recruited patients.
5 mL of whole heparinised blood obtained from patients was mixed with Erythrocyte Lysis (EL) Buffer (Qiagen) followed by incubation on ice for 10-15 minutes. Peripheral blood leukocytes (PBLs) were recovered from erythrocyte-lysed blood by centrifugation at 400×g for 10 minutes at 4° C. and re-suspended in a further 2 mL of EL buffer. PBLs were again recovered by centrifugation as described above and processed for recovery of total RNA. RNA was then prepared from patient PBLs using a semi-automated process on the Maxwell® 16 platform using the Maxwell® 16 LEV simplyRNA Blood Kit. Concentration and purity (A260/A280 ratio≥1.8) were then assessed by spectrophotometry using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Human PBL mRNA samples were labelleled with Cy3 using the Agilent Quick Amp one colour labelling kit and then hybridised to Human SurePrint G3 Human Gene Expression v3 8×60K Microarrays according to the manufacturer's instructions. After hybridisation and wash steps the slides were scanned usning an Agilent SureScan Dx G5761AA Microarray Scanner using default settings.
Gene Expression Analysis:
Parametric; Analysis of Varience and Group T-Tests
Raw data were exported and analysed using the bioinformatics software Genespring 12.5, for differential gene expression and statistical analyses. Raw data were normalized to the 75th percentile followed by baseline transformation to the mean of all samples. Data were assessed for quality, then filtered on gene expression where entities in all samples and all conditions had normalised expression values within the cut-off −10.699 to 7.037. Statistically significant features were identified using one-way ANOVA or T-test analyses across all entities, using the Benjamini-Hochberg False Discovery Rate (BH-FDR) multiple testing correction at a cut-off p<0.05. Data were further analysed and depicted graphically using the heat map, hierarchical cluster analysis and other functions in Genespring 12.5, using default settings. To identify differentially expressed entities between patients having sepsis or SIRS and healthy individuals, fold change cut-off analysis was conduced using a default cut-off setting of >2.5.
Non Parametric; Artificial Neural Network Analyses
A stepwise Artificial Neural Network approach was used to identify an optimised gene signature panel comprising orthogonal genes from a previously established gene biomarker set for sepsis. The approach was repeated 5 times to 10 stepwise additions to assess the stability of the identified gene set given the number of cases provided. This was achieved using a stochastic data selection approach incorporating Monte Carlo cross validation.
Architecture
The ANN modelling undertaken used a supervised learning approach applied to a three-layer multi-layer perceptron architecture. The initial weights matrix was randomised with a standard deviation of 0.1 to reduce the risk of over fitting the data. The ANN architecture was initially constrained to two hidden nodes in the hidden layer also for this reason. Hidden nodes and the output node incorporated a sigmoidal transfer function. During training weights were updated by a feed forward back propagation algorithm (Rumelhart, Hinton et al. 1986). Learning rate and momentum were set at 0.1 and 0.5 respectively. The output node was coded as 0 if the patient showed no evidence of sepsis, and 1 if sepsis was evident. Similar assessments were performed for patients with SIRS.
Monte Carlo Cross Validation
Prior to ANN training, the data was randomly divided into three subsets; 60% for training, 20% for testing (to assess model performance during the training process) and 20% for validation (to independently test the model on data completely blind to the model). This process of random sample cross validation also contributed to the reduction of over-fitting to the data and assess how well the model would perform on a blind data set.
Stepwise Model Development for Consistency Analysis
The normalised intensity of each gene was used as an individual input in the ANN model, creating n individual models, where n was the number of genes in the provided panel. These n models were then split into three subsets (described above) and trained. This random resampling and training process was repeated 50 times to generate predictions and associated error values for each sample with respect to the validation (blind) data. Inputs were ranked in ascending order based on predictive error and the gene that performed with the lowest error was selected for further training. Next, each of the remaining genes were sequentially added to the previous best gene, and were used in combination in a model, creating n-1 models each containing two genes as inputs. Training was repeated and performance evaluated. The model with the highest modelling performance was again selected and the process repeated creating n-2 models each containing three inputs. This process was repeated until no significant gain was evident from the addition of further inputs. This resulted in a final model containing the expression signature that most accurately classified the patients according to development of sepsis or SIRS. A set of 85 biomarkers was identified as being useful for diagnosis and monitoring of the systemic inflammatory conditions sepsis and SIRS. The biomarkers identified as summarised below in Tables 1-4.
Table 1 lists the genes identified as biomarkers of systemic inflammatory conditions using the above methods. The identified biomarkers are useful for diagnosis of systemic inflammatory conditions (e.g., see the biomarkers of inflammation shown in the top part of the table). The identified biomarkers are also useful for monitoring of systemic inflammatory conditions (e.g., see the biomarkers of inflammation shown in bottom part of the table). The final column gives the corrected ANOVA p value illustrating the significance of the biomarkers.
Table 2 lists the genes identified as biomarkers of SIRS using the above methods. The final column gives the corrected ANOVA p value illustrating the significance of the biomarkers.
Table 3 lists the genes identified as biomarkers of sepsis using the above methods. The table also provides an indication as to whether the gene was observed to be elevated in abdominal or pulmonary sepsis. The final column gives the corrected ANOVA p value illustrating the significance of the biomarkers.
Table 4 lists the genes identified as biomarkers of patient survival using the above methods. The final column gives the corrected ANOVA p value illustrating the significance of the biomarkers.
Table 5 summarises the fold-changes observed in the amounts of the biomarkers quantified in the different patient samples as compared to the amounts quantified for the healthy control samples.
To further investigate the performance of each biomarker in the diagnosis and monitoring of systemic inflammatory disease, Receiver Operating Characteristic (ROC) analysis was used to investigate the ability of the biomarkers to discriminate between different disease conditions and/or recovery status using the gene expression expression data obtained from each group of patients.
All ROC curve analysis was performed using R software and the ROCR package using the following commands:
Each data value is assigned a predictor label where negatives (non-infected) are “0” and positives (infected) are “1” and columns are saved as .txt file to be imported into R. −(GBP <—read.table(“GBP.txt”, header=T))
To plot ROC CURVE:
−pred <—prediction(GBP$GBP1, GBP$labels)
−perf <—performance(pred, measure=“tpr”, x.measure=“fpr”)
−plot(perf); abline(0,1) (#This plots FPR (1-SPEC) on the x axis and TPR (==SENS) on the y axis.)
To measure Area Under Curve Value:
−auc.perf <—performance(pred, measure=“auc”) (#generate performance object with AUC)
−auc.perf@y.values (#and extract the AUC value thus)
To plot accuracy and predict optimal accuracy cutoff values (Accuracy is (TP+TN)/(P+N) or (TP+TN)/(TP+FN+FP+FN) which is the total number of True Positives & True Negatives over the sum of the whole population
−acc.perf <—performance(pred, measure=“acc”)
−plot(acc.perf)
−ind=which.max(slot(acc.perf, “y.values”)[[1]])
−acc=slot(acc.perf, “y.values”)[[1]][ind]
−cutoff=slot(acc.perf, “x.values”)[[1]][ind]
−print(c(accuracy=acc, cutoff=cutoff))
−abline(cutoff,1)
library(ROCR)
data(ROCR.simple)
pred <—prediction(ROCR.simple$predictions, ROCR.simple$labels)
perf <—performance(pred,“tpr”,“fpr”)
plot(perf,colorize=TRUE)
abline(0,1, col=“red”)
auc <—performance(pred, “auc”)@y.values[[1]]
legend(0.5,0.4,paste(c(“AUC=”), round(auc,2), sep=″″))
ROC analysis as described above was used to determine the sensitivity and specificity with which biomarkers of the invention discriminate between: (i) patients having a systemic inflammatory condition and healthy controls; (ii) patients having sepsis and patients having SIRS; (iii) patients having abdominal sepsis and patients having pulmonary sepsis; and (iv) healthy controls and patients that recover from a systemic inflammatory condition; and (v) patients that recover from a systemic inflammatory condition and patients that do not recover from a systemic inflammatory condition.
ROC Analysis of the Inflammatory Markers
Table 1 summarises the genes identified as general biomarkers of all systemic inflammatory conditions using gene expression analysis. To demonstrate that these biomarkers permit accurate diagnosis of systemic inflammatory conditions in patients, the “Day 1” gene expression data obtained for all patients was analysed by ROC analysis to determine how well the biomarkers discriminate between patients having a systemic inflammatory condition (including patients having sepsis and patients having SIRS) and healthy controls. ROC analysis of the Day 1 fold change gene expression data was performed as described above.
The ROC curves obtained for the best performing inflammation biomarkers (FAM20A, OLAH and CD177) are shown in
As demonstrated by the AUC and ACC values reported in the above table, FAM20A, OLAH and CD177 were observed to specifically and sensitively distinguish between healthy control patients (having no systemic inflammatory disease) and patients having a systemic inflammatory condition (i.e., those having sepsis or SIRS) when used on their own. The combination of FAM20A and OLAH and the combination of FAM20A, OLAH and CD177 also specifically and sensitively distinguished between patients having a systemic inflammatory condition and healthy controls. These biomarkers may therefore be preferably used in the methods of the invention to diagnose systemic inflammation.
ROC Analysis of the Sepsis and SIRS Markers
Tables 2 and 3 summarise the genes identified as sepsis biomarkers and SIRS biomarkers using gene expression analysis. To demonstrate that these biomarkers permit accurate diagnosis of sepsis and SIRS (and distinguish between these specific disease conditions), the “Day 1” gene expression data obtained for all disease patients was analysed by ROC analysis to determine how well the biomarkers discriminate between patients having sepsis (including patients having abdominal sepsis and patients having pulmonary sepsis) and patients having SIRS. ROC analysis of the Day 1 fold change gene expression data was performed as described above.
The ROC curves obtained for the best performing biomarkers are shown in
As demonstrated by the AUC and ACC values reported in the above table, the following biomarkers were observed to specifically and sensitively distinguish between patients having sepsis and patients having SIRS when used on their own:
The combination of all sepsis biomarkers (ITGB3, ITGA2, MYL9, LCN2 and TREML1), the combination of all SIRS biomarkers (ARGHEF10L, MYCL, TGFB1 and PLA2G7), and the combination of all sepsis and all SIRS biomarkers also specifically and sensitively distinguished between patients having sepsis and patients having SIRS. These biomarkers may therefore be preferably used on their own or in combination in the methods of the invention to diagnose sepsis or SIRS, and to distinguish between sepsis and SIRS.
ROC Analysis of Abdominal Sepsis and Pulmonary Sepsis Markers
Table 3 summarises the genes identified as biomarkers of abdominal sepsis and pulmonary sepsis using gene expression analysis. To demonstrate that these biomarkers permit accurate diagnosis of abdominal and pulmonary sepsis (and distinguish between these specific disease conditions), the “Day 1” gene expression data obtained for all sepsis patients was analysed by ROC analysis to determine how well the biomarkers discriminate between patients having abdominal sepsis and patients having pulmonary sepsis. ROC analysis of the Day 1 fold change gene expression data was performed as described above. The ROC curves obtained for the best performing biomarkers are shown in
As demonstrated by the AUC and ACC values reported in the above table, the following biomarkers were observed to specifically and sensitively distinguish between patients having abdominal sepsis and patients having pulmonary sepsis when used on their own or in combination:
These biomarkers may therefore be preferably used in the methods of the invention to diagnose and distinguish between abdominal sepsis and pulmonary sepsis.
ROC Analysis of Prognosis Biomarkers for Monitoring Disease
When investigating gene expression patterns in patients that survived a systemic inflammatory condition and were deemed suitable for discharge from a high dependency unit (e.g., into a low dependency unit), the present inventors identified various biomarkers (summarised in Table 1) that altered in abundance in disease patients compared to healthy controls, but which returned towards their normal healthy levels as the patients recovered from disease. The inventors identified that these biomarkers could be used to determine the prognosis of a patient with a systemic inflammatory condition and may be used to monitor the effectiveness of treatment in a patient or determine whether a patient is suitable for discharge.
To demonstrate that these ‘prognosis’ biomarkers can be used to monitor the recovery of a patient from a systemic inflammatory condition, ROC analysis was performed to investigate over time the ability of the biomarkers to discriminate between healthy controls and patients that survived a systemic inflammatory condition. AUC values were calculated using the “Day 1”, “Day 5” and “discharge” gene expression data and are summarised below. An AUC value close to 1 indicates that the biomarkers discriminate well between the healthy controls and disease patient populations, whilst an AUC value close to 0.5 indicates that the two populations cannot be reliably distinguished. Representative ROC curves are plotted for PKHD1 biomarker in
As demonstrated in the table above, the AUC values calculated for the biomarkers are observed to change over time as the patient recovers from the systemic inflammatory condition, shifting from ‘1’ or close to ‘1’ (for the samples taken at an early stage of the disease, such as at day 0) towards ‘0.5’ (for the samples taken at day 5 or on discharge). This indicates that the biomarkers become less able to discriminate between healthy controls and disease patients as the patient recovers (indicating that the biomarker profile of the patient becomes more representative of a healthy control). For example, when investigating the gene expression data obtained for PKDH1 in the patients with SIRS, the AUC value for the ‘day 0’ sample is 0.86 indicating that the patient with SIRS can be readily distinguished from a healthy control. By ‘day 5’ the AUC value has dropped to 0.73, indicating that there is less distinction between the patient and a healthy control. Upon discharge, the AUC value has dropped to 0.5 indicating that the biomarker profile of the patient cannot be distinguished from that observed for a healthy control. By investigating the levels of these biomarkers in patients having a systemic inflammatory condition, the disease status of the patient may be monitored to determine whether the disease is progressing towards a more severe form of the disease or regressing towards normalcy.
Surprisingly, the inventors observed that for many of the prognosis biomarkers tested, the AUC values calculated for the “discharge” samples did not drop all the way to ‘0.5’ but remained somewhere between 1 and 0.5. This indicates that patients are being discharged before they are fully immunologically recovered (i.e., before their biomarker profiles are representative of a healthy control). By monitoring a patient using the ‘prognosis’ biomarkers of the invention, it is possible to determine more accurately when a patient has recovered fully from a systemic inflammatory condition and can be safely discharged.
ROC Analysis of Survival Biomarkers
Table 4 summarises the genes identified as survival biomarkers for determining whether a patient with a systemic inflammatory condition is suitable for discharge from medical care. These markers can be used to predict whether a patient undergoing treatment for a systemic inflammatory condition is likely to survive.
To demonstrate that these biomarkers accurately predict survival, the “Day 5” gene expression data obtained for all patients was analysed by ROC analysis to determine how well the biomarkers discriminate between patients having a systemic inflammatory condition that survive and those do not survive. ROC analysis of the Day 5 fold change gene expression data was performed as described above. The ROC curves obtained for the best performing biomarkers are shown in
As demonstrated by the AUC and ACC values reported in the above tables, the following biomarkers (used alone or in combination) were observed to specifically and sensitively distinguish between patients having a systemic inflammatory condition that made a full recovery and those that did not:
These biomarkers may therefore be preferably used alone or in combination in the methods of the invention to determine whether a patient is suitable for discharge from medical care.
To further investigate the biomarkers of systemic inflammation identified by gene expression analysis, a subset of the biomarkers was selected for further analysis by ELISA. Protein quantification by ELISA was performed to investigate the abundance of specific biomarkers in whole lysed blood obtained from patients at day 1 and day 5 post admittance to an intensive care unit (as described above for Example 1).
The biomarkers chosen for further analysis were: (i) the pulmonary sepsis biomarker DISC1; (ii) the abdominal sepsis biomarker SLC39A8; (iii) the SIRS biomarker GPR124; and (iv) the survival marker NECAB1 which is used to predict survival from SIRS.
ELISA Protein Quantification:
Blood samples were collected from patients and processed as described in Example 1. 2 mL of blood sample was mixed with 8 mL of cell lysis buffer at a 1:5 dilution. The cell lysis buffer was purchased from Invitrogen (NP40 cell lysis buffer: 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% Nonidet P40 (NP40), and 0.02% NaN3, supplemented with 1 mM PMSF and protease inhibitor cocktail). The samples were incubated on ice for 60 minutes. The samples were then centrifuged at 13000 rpm for 30 minutes to pellet debris. The supernatant was removed and passed through a 0.22 μm syringe filter. The cell free supernatant was transferred to a fresh tube and stored at −80° C.
ELISA assays were performed on neat or diluted lysed blood samples using commercial ELISA kits (https://www.mybiosource.com/).
DISC1 was quantified using ELISA kit MBS9343138, NECAB1 was quantified using ELISA kit MBS9338711, and SLC39A8 was quantified using ELISA kit MBS9381303. Briefly, 50 μL of prepared blood sample was added to the sample well of a microelisa stripplate plate. In addition, 50 μL of each standard was added to the standard wells and 50 μL of sample diluent was added to each blank/control well of the plate. 100 μL of HRP-conjugate reagent was added to each well and incubated for 60 minutes at 37° C. The plate was washed 4 times and developed by adding 50 μL Chromagen Solution A and 50 μL Chromagen solution B and incubating for 15 minutes at 37° C. 50 μL stop solution was added to the wells, and the optical density at 450 nm was read.
GPR124 was quantified using ELISA kit MBS909585. Briefly, 100 μL of prepared blood sample was added to the sample well of an ELISA plate. In addition, 100 μL of each standard was added to the standard wells and 100 μL of sample diluent was added to each blank/control well of the plate. The plate was incubated for 2 hours at 37° C. 100 μL of biotin-antibody was added to each well and incubated for 60 minutes at 37° C. The plate was washed 3 times. 100 μL of HRP-avidin was added to each well and incubated for 1 hour at 37° C. The plate washed 5 times. 90 μL TMB substrate was added, and the plate was incubated for 15-30 minutes at 37° C. 50 μL stop solution was added to the wells, and the optical density at 450 nm was read.
ROC Analysis of Protein Quantification Data:
The protein quantification data obtained for the patients was analysed by ROC analysis to determine how well the markers could distinguish between different types of systemic inflammatory condition and/or between patients that survived and patients that died. The results of the analysis are presented in the table below. ROC Curves are shown in
The pulmonary sepsis marker DISC1 was tested for its ability to distinguish between patients having pulmonary sepsis and patients having another systemic inflammatory condition (such as abdominal sepsis or SIRS). DISC1 was also tested for its ability to distinguish between patients having pulmonary sepsis and healthy controls. ROC analysis revealed that this marker performed well to identify patients having pulmonary sepsis.
The abdominal sepsis biomarker SLC39A8 was tested for its ability to distinguish between patients having abdominal sepsis and patients having another systemic inflammatory condition (such as pulmonary sepsis or SIRS). DISC1 was also tested for its ability to distinguish between patients having abdominal sepsis and healthy controls. ROC analysis revealed that this marker performed well to identify patients having abdominal sepsis.
The SIRS biomarker GPR124 was tested for its ability to distinguish between patients having SIRS and patients having sepsis (including those having abdominal sepsis and pulmonary sepsis). ROC analysis revealed that this marker performed well to distinguish patients having SIRS and patients having any type of sepsis.
The survival biomarker NECAB1 was tested for its ability to distinguish between patients having SIRS that survived and patients having SIRS that died. ROC analysis revealed that this marker performed well as a survival marker for SIRS.
A patient presents at an intensive care unit (ICU) or is admitted to lower dependency hospital ward with unspecified illness. Within 6 hours of admission, a blood sample is obtained from the patient, and is tested with inflammation (as described in Table 1), SIRS (as described in Table 2) and sepsis (as described in Table 3) biomarker panels using qPCR. Raised expression of inflammation markers, as determined by a lower threshold Ct value compared with controls, indicates an ongoing systemic inflammatory condition. Raised expression of SIRS biomarkers indicates that the systemic inflammatory condition is SIRS. Raised expression of sepsis biomarkers indicates that the systemic inflammatory condition is sepsis.
A patient presents at a GP surgery, an out-of-hours clinic, or an accident and emergency department. A blood sample is obtained from the patient and tested using a rapid point of care diagnostic test for inflammation markers (as described in Table 1). The test reveals that the inflammation biomarkers are elevated in the patient. The patient is referred for further detailed investigation using a full panel of inflammation, sepsis, and SIRS biomarkers in a hospital/diagnostic laboratory setting.
A patient is undergoing treatment for a systemic inflammatory condition in an ICU or hospital. To monitor how the patient is responding to treatment, and whether continued treatment or a change in treatment is needed, a blood sample is taken and tested using a panel of prognostic recovery biomarkers (as described in Table 1).
If the level of prognosis biomarkers detected shows regression of the systemic inflammatory condition, treatment may be continued for a short while until the patient is deemed suitable for discharge. Prior to discharge, a blood sample from the patient may be taken and tested using a panel of survival biomarkers (as described in Table 4). If the levels of survival biomarkers show that the patient has a good prognosis of recovery, the patient is discharged. If the levels of survival biomarkers show that the patient has a poor prognosis of recovery, the patient is not discharged, and treatment is continued.
If the levels of prognosis biomarkers show progression or no change in the systemic inflammatory condition, the patient continues to undergo treatment and may be switched to a different treatment strategy. The patient may be monitored by testing further blood sample(s) using the panel of prognosis biomarkers to determine whether the systemic inflammatory disease is progressing or regressing towards normalcy.
After discharge, the patient may be monitored at home by community nursing staff using a prognostic marker point of care diagnostic test, to monitor ongoing response to therapy and to provide rapid indication of any relapse.
A patient presents at an intensive care unit (ICU) or is admitted to lower dependency hospital ward with unspecified illness, and is diagnosed as having a systemic inflammatory condition. To assess the severity of the disease, a blood sample is taken and tested using a panel of prognostic biomarkers (as described in Table 1). The blood sample may also be tested using a panel of survival biomarkers (as described in Table 4) to determine the degree of organ damage/failure.
The levels of prognosis biomarkers and/or survival biomarkers may be used by clinicians to inform treatment choices/tailor treatment packages and manage survival expectation in the next of kin.
GTGTAAAGTTGTTCGCCGCGTGGAATGTGAGTGTGTTTGTGTGCATGA
AAGAGAAAGACT (SEQ ID NO: 86)
CTTGGACACCAGATTCTTTCCCATTCTGTCCATGAATCATCTTCCCCAC
ACACAATCATT (SEQ ID NO: 90)
GTGGGAGGTCAATCCCCTTTACTGTGACACAGTGAAACAGATCTACCC
GTACAACAACAG (SEQ ID NO: 94)
TCGTGCGTTGCCTTGCTCCGTTTTTCCCAAAAAGCACTGGCTTCATCAA
GGCCACCGACG (SEQ ID NO: 95)
CCTAACCTCATTCCCCAACCACTTCATTCTTGAAAGCTGTGGCCAGCTT
GTTATTTATAA (SEQ ID NO: 98)
ATGGAAGCTGGGCCTTCATGTGGCAGCAAGTTTTCGAGCCCACCTGGA
AACACATTGGGG (SEQ ID NO: 102)
TGGAGGTGGGCTGGGCCCTCTCTTCTCACCTTTGTTTTTTGTTGGAGTG
TTTCTAATAAA (SEQ ID NO: 106)
CTCCAGGTCCGGAGGGGTTGCGGGGGAGCTGGAAATAAACCTGGAGA
TGATGATGATGAT (SEQ ID NO: 110)
CTCTTTGGGTGATAGTCAGAGAGTGGTGTTTTTGTTCAGGTGGGAAGG
ATTGGAAACTCT (SEQ ID NO: 114)
AGGAGGTTGTGTCACTGACTCAGGCTGCTCCTTCTCTAGTTTCCCCTCT
CATCTGACCTT (SEQ ID NO: 118)
TTATGCTGCTCCTGATATAACACTTTCCAGCCTATAGCAGAAGCACATT
TTCAGACTGCA (SEQ ID NO: 122)
GATAAGATGTGGTTTGAAGCTGATGGGTGCCAGCCCTGCATTGCTGAG
TCAATCAATAAA (SEQ ID NO: 126)
GGAGGGGGAAGAGAAATTTTTATTTTTGAACCCCTGTGTCCCTTTTGCA
TAAGATTAAAG (SEQ ID NO: 130)
GCTACAAGGTGGTGGGCGGTGAACGAGAGAATGTCTCCATGGTGGAC
TATGCTCACAACA (SEQ ID NO: 134)
GAACAAGCAGAACAGACGCTAAAACAGGCAGCTTCAACAGATAGTCT
CAGGGTCTTAAAT (SEQ ID NO: 138)
AATATTAATGTCTAGTTGTTCTATATTATAACCACATTTGCGCTCTATGC
AAGCCCTTGG (SEQ ID NO: 139)
ATAATTTTCCTCCTAAACAAAAACACATTGAGTTTAAGTCTCTGACTCTT
GCCTTTCCAC (SEQ ID NO: 142)
TGGAAAAACAGTGTGGATATAAGCTGTTCATTTATGGAAGGGATGACT
ACGTTGGGGAAG (SEQ ID NO: 143)
GGATCTGAAGACATAGCAAAGGACATGGAAGCCTGGAAAGATGTAAC
CAGTGGAAATGCT (SEQ ID NO: 146)
TGGTACAAGGATTCTCTTCTTTTGGATAAAGACAATGAGAAATTTCTAA
GTGTGAGGGGG (SEQ ID NO: 150)
GACATTGATTTGCTCTTACTACAGCTTCAGTGATTGGGGGAGGAAAAG
TCCCAACCCAAT (SEQ ID NO: 154)
GACTGAATCTCTCACCTATTCTTGCAGAAAGACATACTAATTAAACCTT
GTCAAAGTAGT (SEQ ID NO: 155)
ATCTGACTTCTAGGATTTATTGTTATATTACTTGCCTATCTGACTTCATA
CATCCCTCAG (SEQ ID NO: 158)
TTTTTTAAGCATCCTGACAGGAAATGTTTTCTTCTACATGGAAAGATAG
ACAGCAGCCAA (SEQ ID NO: 162)
TGGCCAAGGTTATCGGAAATCTGGAGATGCAGATACTGTGTTTCCTTG
CTCTTCGTCCAT (SEQ ID NO: 166)
AGCTCCGCGTGAGAAGTACTGGCTACAATTTTTTATCCCATTGTTGGTG
GTGATTCTGTT (SEQ ID NO: 170)
CCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTT
TTTGATGAGAG (SEQ ID NO: 174)
TCAACCCTTGGAATAACAGTCTAGCTGATTGTTCCACCAAAGAATCCA
GCCTGCTGCTTA (SEQ ID NO: 178)
GTCATCAACCTAACAAACACAACCTTCTCAGCAGCATTTCTCCCCTGTG
ATGGAAATAAA (SEQ ID NO: 182)
CTGGTGCCCAGTCGGGGTGGCTGAGCTGGTCCTTAATAGGTTGTTTCTT
GGTCTTGCTTT (SEQ ID NO: 183)
AGAAGATCACTGAAGAAACTTCTGCTTTAATGGCTTTACAAAGCTGGC
AATATTACAATC (SEQ ID NO: 186)
AAACCCGTGATTTTGGTGCTCCTTGTAACTCAGCCCTGCAAAGCAAAG
TCCCATTGATTT (SEQ ID NO: 190)
CTCCAACCTAACCTGTGTCTGCGAAGTCCTATGGAAACCCGAGGGTTG
ATTAAGGCAGTA (SEQ ID NO: 191)
CTAGTTGCTGTGGAGGAAATTCGTGATGTTAGTAACCTTGGCATCTTTA
TTTACCAACTT (SEQ ID NO: 194)
TGCCTAACCTTTTGGAGCCTTAGTCTCCCAGACTGAAAAAGGAAGAGG
ATGGTATTACAT (SEQ ID NO: 198)
CTGGATAGTCCTGTCACCGTGGAGTGGAAGGCACAGTCTGATTCTGCC
CGGAGTAAGACA (SEQ ID NO: 202)
TGCTTGTCTGCCACGTGACGGATTTCTACCCAGGCAGCATTCAAGTCC
GATGGTTCCTGA (SEQ ID NO: 203)
TACCAAGAATCTGTGGAAATATAAGCTGGGGCAAATCAGTGTAATCCT
TGACTTTGCTCC (SEQ ID NO: 206)
TACGTGATAGTATAAACCAATGTGACTTCATGTGATCATATCCAGGATT
TTTATTCGTCG (SEQ ID NO: 210)
GAGTTTCTTATGTGCCCTGGTGGACACTTGCCCACCATCCTGTGAGTAA
AAGT GAAATAA (SEQ ID NO: 214)
CTGAGCTACCTGAGCGTAAGGGGGGGGTTCAACATGTCCTCTTTCAAG
TTAAAAGAATAA (SEQ ID NO: 218)
TGCAAGAGCACCCCAACGCGCGCAAATACAAAGGAGCCAACAAGAAG
GGCTTGTCCAAGG (SEQ ID NO: 222)
TGGGAGCCAGCCTCCCTTTGATGATTATTGGAGCCCCAGGGGACAAGG
GATTTGAGGTGA (SEQ ID NO: 226)
GTATTTAGTTGTGATTACTGATTGCCTGATTTTAAAATGTTGCCTTCTGG
GACATCTTCT (SEQ ID NO: 230)
CAGCTTATTTCCTCATTTTTATAATGTCCCTTCACAAACCCAGTGTTTTA
GGAGCATGAG (SEQ ID NO: 234)
AGATGAGCTAGGATGGAGAGTCCTTGAACCTGAACTTACACAAATTTG
CCTGTTTCTGCT (SEQ ID NO: 238)
AGCTATGAGTTGAAATGTTCTGTCAAATGTGTCTCACATCTACACGTGG
CTTGGAGGCTT (SEQ ID NO: 242)
AAAGCATTTAGGACTTCATAAAGATTTTGATCAGTGGGACTGCTTGATT
GAAGGAGATGA (SEQ ID NO: 246)
ATAAAGTCTGTACATATTGGAGCTCTGGGAGATGCTGGAATAAAAGAC
AAGAGTTACATC (SEQ ID NO: 250)
CATCTGGAGGAAATGGCCTTCTTTTTAAAAGCAAAAAACACAAAACCT
CACAACTGCCTG (SEQ ID NO: 251)
AAAGCTTTGTATTATTCTTCCACATATGCTGGCTGCTGTTTACACACCCT
GCCAATGCCT (SEQ ID NO: 254)
CTCCAGAATGGTCTTTCTAATGTGTGAATCAGAGCACAGCAGCCCCTG
CACAAAGCCCTT (SEQ ID NO: 258)
TGCAAAGTTCCCTACTTCCTGTGACTTCAGCTCTGTTTTACAATAAAATC
TTGAAAATGC (SEQ ID NO: 259)
TTAGAAACACTTCAAGAAGAAAAGCCTGAGCTGACCGTCGTCTTTGAG
CCTTCTTGGTAG (SEQ ID NO: 262)
TCAGTTCCCATAAAACCTTCATTACACATAAAGATACACGTGGGGGTC
AGTGAATCTGCT (SEQ ID NO: 266)
TAAGTGGGAAGTCTTGTTTGTTGTTGGTTTTTGTCTGTTGTTTTTCACTG
CACCTCTTTG (SEQ ID NO: 270)
AGAGGAAGGAGCTCTTAGTTACCCTTTTGTGTTGCCCTTCTTTCCATCA
AGGGGAA (SEQ ID NO: 274)
AATGCCAGAATTCTTCCAAACTCCCTGACTCTTTGAAGTTTTTACTCAC
CCCATTTCAAT (SEQ ID NO: 278)
ATAGGTCTGATAATGGGTGGGACGCTCCTGGCTTTGCTAAGAAAGCGT
TTCAGACAAAAA (SEQ ID NO: 282)
AAATACCTCTTTATTTTTTGATTGAAGGAAGGTTTTCTCCACTTTGTTGG
AAAGCAGGTG (SEQ ID NO: 283)
TGAGCAGGATATGTTAGAAAAGAGGAAAATACAGCGTGAATTAGCAAA
AAGGG (SEQ ID NO: 286)
GCGGGGCCAAAAAAGGAAACCAGGAGTGCCACTATGCTGACTTCTTAT
TCCTTTT (SEQ ID NO: 287)
ACAACAATGGCCCTGGGACTGTGAATGGTCTTCACCTCAGCATCCACC
TTCCGGGACAGT (SEQ ID NO: 290)
TGAGGAAGTGGACGAGATGTACCGGGAGGCACCCATTGATAAGAAAG
GCAACTTCAACTA (SEQ ID NO: 294)
ATGTGTGGACACATTGGACCTTTCCTGAGGAAGAGGGACTGTTCTTTTG
TCCCAGAAAAG (SEQ ID NO: 298)
TGAGACGTCACTGGGGACTTATCTGTGGAGCCTGGTGCTCCAGGATGT
GGCTTCTCATGA (SEQ ID NO: 302)
GCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGA
TCACCCTCTAC (SEQ ID NO: 306)
TTCTTAAATCCCTTAAGGAGTGGAACTATCCTCTATTTCAGGACTTGAA
TGGACAAAGTC (SEQ ID NO: 310)
GACAGTGACTTGTATGCTGAACTCCGCTGCATGTGTATAAAGACAACC
TCTGGAATTCAT (SEQ ID NO: 314)
TCTGCTCCAAGCCTGTGACATATGCCACAGTAATCTTCCCGGGAGGGA
ACAAGGGTGGAG (SEQ ID NO: 318)
GACCTGCAAGCCCCGCTGTACAAGAAAATAATTAAGAAACTTTTGGAG
AGTTAGCTACTA (SEQ ID NO: 322)
AAACATTATTTAATGTGTGAGAGGAAGGCTGGCATGACCAAGGTGGAC
CAACTACCTTAA (SEQ ID NO: 326)
GGTGGACAGGATAACACAGATAAGGGCTTTATTGTACAATAAAAGATA
TGTATGAATGCA (SEQ ID NO: 327)
GCCTGTGGGATGCCTTGTGGGACGTCTCTTTCTATTCAATAAACAGATG
CTGCAGCCTCA (SEQ ID NO: 330)
GCAGCGGCGTCAAAGTGGTCACCTTCTGTGGCCACACGTCCAAAACCA
ATCAGGTCAACT (SEQ ID NO: 334)
CACCGACAAGAACTCACTACTGGGCATGGAGGGTGCCAACAGCATCTT
TTCCGGGTTCCT (SEQ ID NO: 338)
TATCTGATTGGAAACCTGCCGACTTAGTGCGGTGATAGGAAGCTAAAA
GTGTCAAGCGTT (SEQ ID NO: 342)
GGGTGACCAGGTCTGGGTTGAAAAAGACCCCAAAAAGGGTCACATTTA
CCAGGGCTCTGA (SEQ ID NO: 346)
AGCACGTCTGTACTTCTGTTCATTAAAGTGCTCCCTTTCTAGTCCTTTTT
CTGCCCAGAA (SEQ ID NO: 350)
GGGGTATCCTGGGGGACCCAATGTAGGAGCTGCCTTGGCTCAGACAT
GTTTTCCGTGAAA (SEQ ID NO: 354)
GGAAAATGATTGACAAAGCCCAACAATGATCTCAGGAATTACATTTTC
CAACAGACCAAA (SEQ ID NO: 358)
GGACATTAAAGGAATTTCCCTTTTGTTGACATAACAGTAGGATGGCTAT
AGCTAACAATA (SEQ ID NO: 362)
TTGTGTTTGAAATTCCATGTCGGGTTTACTTGGAATGAAAGATACTTGA
ATTATTGTGCG (SEQ ID NO: 366)
ACTTTGAGAACTCTGTGAGACAAGGTCCTTAGGCACCCAGATATCAGC
CACTTTCACATT (SEQ ID NO: 370)
AAGGATGTTCTAATTCTTTCTGCTCTGAGACGAATGCTATGGGCTGCAG
ATGACTTCTTA (SEQ ID NO: 374)
CCGAAGGCGGTTTCTGTTCGACATGCTGAATGGACAAGATCCTGATCA
AGGACATTTAAA (SEQ ID NO: 378)
GTGAATTTGAGAAATGTCAGACTGTGCCAAAGGGGGTCACATGCATCA
AATTTCCACATA (SEQ ID NO: 382)
CCGAAGGCGGTTTCTGTTCGACATGCTGAATGGACAAGATCCTGATCA
AGGACAT (SEQ ID NO: 386)
ATTGTCAAGGTGTTGCCGCCGGTGTTGGGGCTGGAGTTTATCTTCGGG
CTTCTGGGCAAT (SEQ ID NO: 390)
AGAAGAGAAGCATTTAGAGAGCATCAGCAATACAAAACCGCTGAGTTC
TTGAGCAAACTG (SEQ ID NO: 394)
TGAAGCCTTCTTCCTCCCAGGAAAGGGGACGTTCAGCTGAGCCGAGTG
TGTATACTGCTC (SEQ ID NO: 398)
AAATATTACACATCAAACCAATGACATGGGAAAATGGGAGCTTCTAAT
GAGGACAAACAA (SEQ ID NO: 399)
CTAGATTCTGCAGTCAAAGATGACTAATATCCTTGCATTTTTGAAATGA
AGCCACAGACT (SEQ ID NO: 402)
ACGGCTCATGTTTTTAAAATATATGTAACTCATTTTAAAATATATTAAAT
TGTATTCCAA (SEQ ID NO: 406)
TTTTTTCTAGACAGACACTITGGTGCAGAAGCTTCTTTTCAATCCATCCT
CCACAAGAAG (SEQ ID NO: 410)
GTACGTGTCAATAACAGTAAAGAATTTTAATTAATTCTGTCATTTAAAAT
TCATGCATTC (SEQ ID NO: 414)
ACCAGTTTCTGTATTTCCTAAAACAGAGGCAGAATGGACTGCATCCTTC
TTCAACGCAGG (SEQ ID NO: 415)
CTATGCGCTATGGAGAGAGTTCCTCTTCTCCGTTCCCGCGGGGCCCCC
CGCGCAGTACTC (SEQ ID NO: 418)
Table 15 lists various oligonucleotide primers and corresponding probes used to detect the biomarkers of the invention
acacggggct ccagcccccc cgagtggaag tgctccccac tttctttagg atttaggcgc
ccatggtaca aggaatagtc g
c
gcaagcat cccgctggtg cctcccggga cgaaggactt
cccgagcggt gtggggaccg ggctctgaca gccctgcgga gaccctgagt ccgggaggca
ccgtccggcg gcgagctctg gctttgcaag ggcccctcct tctgggggct tcgcttcctt
agccttgctc aggtgcaagt gccccagggg gcggggtgca gaagaatccg agtgtttgcc
aggcttaagg agaggagaaa ctgagaaatg aatgctgaga cccccggagc aggggtctga
gccacagccg tgctcgccca caaactgatt tctcacggcg tgtcacccca ccagggcgca
agcctcacta ttacttgaac tttccaaaac ctaaagagga aaagtgcaat gcgtgttgta
catacagagg taactatcaa tatttaagtt tgttgctgtc aagatttttt ttgtaacttc
aaatatagag atatttttgt acgttatata ttgtattaag ggcattttaa aagcaattat
attgtcctcc cctattttaa gacgtgaatg tctcagcgag gtgtaaagtt gttcgccgcg
tggaatgtga gtgtgtttgt gtgcatgaaa gagaaagact gattacctcc tgtgtggaag
aaggaaacac cgagtctctg tataatctat ttacataaaa tgggtgatat gcgaacagca
aaccaataaa ctgtctcaat gctgattcat aaaaaaaaaa aaaaaa
cccacgattc ttcaccgctg ctgaccaccc acactcaacc tccctctgac ctcataacct
aatggccttg gacaccagat tctttcccat tctgtccatg aatcatcttc cccacacaca
atcattcata tctactcacc taacagcaac actggggaga gcctggagca tccggacttg
ccctatggga gaggggacgc tggaggagtg gctgcatgta tctgataata cagaccctgt
cctttctccc agtgctggga tttctccatg tgagggggca gcaggacacc cagggatcta
gcgtggggga ggagaggagc ctaatgagaa aatgaccatc taaagcctgc ccttcattgg
tctggttcac gtctccaaac cagcttggat ggtagcagag acttcagggt gctccagcca
aacgtatttg ggcatcacca tgacctggga ggggaagatg cactgagacg tatgaggctt
ccagcctagc agccagggcc ctagcacaaa caggaggctc gccccatctg agcaactgca
ggagaggtta gtacagtcat gcattgctta acgacaggga cgtgtcgtta gaaatgtgtc
gttaggtgat tttatgacca taggaacatt gtagcgtgca cttacaccaa cccagatggt
acagcccaat acacacccag gatggacgct agagtcgact gctcctaggc tacaagcctg
cagtgcatgt tatggtgtga atactgcagg caatcttaac accacggcaa gtatttgtgc
atctacacac atctaaacat agaaaaggta cagcataaat acactattgt catctcagca
ctggcaggaa aagaggagtg ggaggtcaat cccctttact gtgacacagt gaaacagatc
tacccgtaca acaacagcca gcggctcctc aatgtcatcg acatggccat cttcgacttc
ttgataggga atatggaccg gcaccattat gagatgttca ccaagttcgg ggatgatggg
ttccttattc accttgacaa cgccagaggg ttcggacgac actcccatga tgaaatctcc
atcctctcgc ctctctccca gtgctgcatg ataaaaaaga aaacactttt gcacctgcag
ctgctggccc aagctgacta cagactcagc gatgtgatgc gagaatcact gctggaagac
cagctcagcc ctgtcctcac tgaaccccac ctccttgccc tggatcgaag gctccaaacc
atcctaagga cagtggaggg gtgcatagtg gcccatggac agcagagtgt catagtcgac
ggcccagtgg aacagttggc cccagactct ggccaggcta acttgacaag ctaagggctg
gcagagtcca gtttcagaaa atacgcctgg agccagagca gtcgactcga gtgccgaccc
tgcgtcctca ctcccacctg ttactgctgg gagtcaagtc agctaggaag gaagcaggac
attttctcaa acagcaagtg gggcccatgg aactgaatct ttactccttg gtgcaccgct
tctgtcgtgc gttgccttgc tccgtttttc ccaaaaagca ctggcttcat caaggccacc
gacgatctcc tgagtgcact gggaaatctg ggtataggtc aggcttggca gccttgatcc
caggagagta ctaatggtaa caagtcaaat aaaaggacat caagtggata cctgacttct
caggatcctt attctagcta caagtcaaag ataactcctg gtccagacaa aacacctggc
ctatcacaag ctgactaaaa atctgcactt tgggccagcg caggcaacag taactctgac
aggttcaaat tagacctcac actttctact catattctag tcactggacc catctgaatc
agtaatccct actgcccggt cctggagtaa cttcttagag atattataac aagtggcaaa
aataaaagag ggatttgcta agaatatcag aaaaggagtg ttccaatttg aagagtatta
caattgaaat aacatcaaat atgtcacact aagcagccag taacagaata aataattaca
acgaaggaaa aaaaaaggaa agtcctccaa ggtcaggatg gcatgggaac aggcctagca
gggacacaag cctggagtaa ggcaggaaaa gagccaaggc tgactacagc ccaccaacca
caatcttctt ccctaagacc ccaggattgt ccccggccca tccccaaaag aagaaagggg
ctatgtggaa aggtgaggcc ctctagatgc ccctccctgt gacggctggc tcaaaaaaga
ctaagtcagg ttttttattg aagcctccct tagaagaaag tgtagtaggg attccttgtc
tcctctacct tctccctgac ttgttgatat ctgaaactcc ttttaatagg aggctttgtt
tcttattcca taattaatga ccgaaaacac aaaggacagt aaggtcttca ttccatggag
tgactagtca gtcaacaaaa gtaaaaatgc ctcatttcat caaatcaact tttccagtaa
aggccagagt tcaaatactg taagcatgag aatagaatgg agctgtcact aaagagagta
caaggtcaag aaccagaaat gtctccaagc tttctgcagt gtgagcaaag atagcctcga
ctggacaaat aacacttctg accccgtggt acccccacat gactggtatg ctgcccctgg
actttgtgtc tctctaaaat cactaccatt cactgagatc tgaccaggtg aggcactcac
caacgctaca gaagcaacac acaggtttcc aatcctctca aacacagata caacccccat
cctccaatgt gcatggaaga actttaaatc tcagagagag taagcaaaat cacacaattt
agtggtttag acctattcca aagtcttctt ttcactgtaa cacaaaaccc aacagtatcc
cacccaattt atgaagtcaa atttgtttgc ttaaaaaaaa aatcagctgg gcatgggctc
acacctgtaa tcccagcact ttgggcctaa acaggcagat cgcttggtct caggagctcg
agaccagcct gggtaacatg acaaaaccct gtctctacaa aaaaatacaa aaaaaaaaaa
tgcacagctc agcactgctc tgttgcctgg tcctcctgac tggggtgagg gccagcccag
gccagggcac ccagtctgag aacagctgca cccacttccc aggcaacctg cctaacatgc
ttcgagatct ccgagatgcc ttcagcagag tgaagacttt ctttcaaatg aaggatcagc
tggacaactt gttgttaaag gagtccttgc tggaggactt taagggttac ctgggttgcc
aagccttgtc tgagatgatc cagttttacc tggaggaggt gatgccccaa gctgagaacc
aagacccaga catcaaggcg catgtgaact ccctggggga gaacctgaag accctcaggc
tgaggctacg gcgctgtcat cgatttcttc cctgtgaaaa caagagcaag gccgtggagc
aggtgaagaa tgcctttaat aagctccaag agaaaggcat ctacaaagcc atgagtgagt
ttgacatctt catcaactac atagaagcct acatgacaat gaagatacga aactgagaca
tcagggtggc gactctatag actctaggac ataaattaga ggtctccaaa atcggatctg
gggctctggg atagctgacc cagccccttg agaaacctta ttgtacctct cttatagaat
atttattacc tctgatacct caacccccat ttctatttat ttactgagct tctctgtgaa
cgatttagaa agaagcccaa tattataatt tttttcaata tttattattt tcacctgttt
ttaagctgtt tccatagggt gacacactat ggtatttgag tgttttaaga taaattataa
gttacataag ggaggaaaaa aaatgttctt tggggagcca acagaagctt ccattccaag
cctgaccacg ctttctagct gttgagctgt tttccctgac ctccctctaa tttatcttgt
ctctgggctt ggggcttcct aactgctaca aatactctta ggaagagaaa ccagggagcc
cctttgatga ttaattcacc ttccagtgtc tcggagggat tcccctaacc tcattcccca
accacttcat
tcttgaaagc tgtggccagc ttgttattta taacaaccta aatttggttc
taggccgggc gcggtggctc acgcctgtaa tcccagcact ttgggaggct gaggcgggtg
gatcacttga ggtcaggagt tcctaaccag cctggtcaac atggtgaaac cccgtctcta
gacagctggc tgatggctcc atggatgtgg tggtctgcac tctggtgctg tgctctgtgc
agagcccaag gaaggtcctg caggaggtcc ggagagtact gagaccggga ggtgtgctct
ttttctggga gcatgtggca gaaccatatg gaagctgggc cttcatgtgg cagcaagttt
tcgagcccac ctggaaacac attggggatg gctgctgcct caccagagag acctggaagg
atcttgagaa cgcccagttc tccgaaatcc aaatggaacg acagccccct cccttgaagt
ggctacctgt tgggccccac atcatgggaa aggctgtcaa ataatctttc ccaagctcca
aggcactcat ttgctccttc cccagcctcc aattagaaca agccacccac cagcctatct
atcttccact gagagggacc tagcagaatg agagaagaca ttcatgtacc acctactagt
ccctctctcc ccaacctctg ccagggcaat ctctaacttc aatcccgcct tcgacagtga
aaaagctcta cttctacgct gacccaggga ggaaacacta ggaccctgtt gtatcctcaa
ctgcaagttt ctggactagt ctcccaacgt ttgcctccca atgttgtccc tttccttcgt
tcccatggta aagctcctct cgctttcctc ctgaggctac acccatgcgt ctctaggaac
tggtcacaaa agtcatggtg cctgcatccc tgccaagccc ccctgaccct ctctccccac
tgcctgcaac gtgaacatct tcgacgccat cgcggagatt gggaaccagc tgtatttgtt
caaggatggg aagtactggc gattctctga gggcaggggg agccggccgc agggcccctt
ccttatcgcc gacaagtggc ccgcgctgcc ccgcaagctg gactcggtct ttgaggagcg
gctctccaag aagcttttct tcttctctgg gcgccaggtg tgggtgtaca caggcgcgtc
ggtgctgggc ccgaggcgtc tggacaagct gggcctggga gccgacgtgg cccaggtgac
cggggccctc cggagtggca gggggaagat gctgctgttc agcgggcggc gcctctggag
gttcgacgtg aaggcgcaga tggtggatcc ccggagcgcc agcgaggtgg accggatgtt
ccccggggtg cctttggaca cgcacgacgt cttccagtac cgagagaaag cctatttctg
ccaggaccgc ttctactggc gcgtgagttc ccggagtgag ttgaaccagg tggaccaagt
gggctacgtg acctatgaca tcctgcagtg ccctgaggac tagggctccc gtcctgcttt
ggcagtgcca tgtaaatccc cactgggacc aaccctgggg aaggagccag tttgccggat
acaaactggt attctgttct ggaggaaagg gaggagtgga ggtgggctgg gccctctctt
ctcacctttg ttttttgttg gagtgtttct aataaacttg gattctctaa cctttaaaaa
agaagccatc aatgagagga tccaggaggt cgccggctcc ctaatattta gggcaataag
cagcattggc ctggagtgcc agagcgtcac ctccaggggg gacctggcta cttgcccccg
aggcttcgcc gtcaccggct gcacttgtgg ctccgcctgt ggctcgtggg atgtgcgcgc
cgagaccaca tgtcactgcc agtgcgcggg catggactgg accggagcgc gctgctgtcg
tgtgcagccc tgaggtcgcg cgcagcgcgt gcacagcgcg ggcggaggcg gctccaggtc
cggaggggtt gcgggggagc tggaaataaa cctggagatg atgatgatga tgatgatg
ttattcgcac cggtgataga gttaaggatt gatcagaatg gcaagtacta tactggagtc
ctttgtggtt tggggtggaa tccagctaca ggggcttcca tactgcccga gcacgacatg
gagcttgcgt ttgacgttca attcagcgtg gaggatgtcg tcgaggttaa tattctcagg
gctgctatta acaagctagt ctgtgatgga ccaaatggat gcaagtgtct tgggccagag
agagttgcgc agcttcaaga cattgcccgt cagaagcttt taggtttgtt ctgtcagtca
aaaccaaggg agaagattgt tcccaagtgg catgaaaagc cctacgagtg gaatcaggtt
gatccaaagc tggtcatgga gcaggccgac cgtgagagca gcagagggaa gaacaccttt
ctctaccagc tccacaaact ggttgtgctc ggcacctgag catgtccaca ggtggcctcc
agcacacccc tcaggaagct gtggaggctg gattccaggc tccctccgca gactgacttt
cctctgtgtc tgggtgttac agtctgtgcc cactgcatcc taaaggcctt ttctttcttc
ttttctcttt gggtgatagt cagagagtgg tgtttttgtt caggtgggaa ggattggaaa
ctctagtctt ttctagaaac agaaaatcac tgtattaaat attttggaaa gattgttctg
gtactcagct gtgaagtccc tggaagtgat tgtccgggcc aacatcacag tgaagtcctc
cataaagaac ttgatgctcc gagatgcctc cacagtgatc ccagtgatgg tatacttgga
ccccatggct gtggtggcag aaggagtgcc ctggtgggtc atcctcctgg ctgtactggc
tgggctgctg gtgctagcac tgctggtgct gctcctgtgg aagatgggat tcttcaaacg
ggcgaagcac cccgaggcca ccgtgcccca gtaccatgcg gtgaagattc ctcgggaaga
ccgacagcag ttcaaggagg agaagacggg caccatcctg aggaacaact ggggcagccc
ccggcgggag ggcccggatg cacaccccat cctggctgct gacgggcatc ccgagctggg
ccccgatggg catccagggc caggcaccgc ctaggttccc atgtcccagc ctggcctgtg
gctgccctcc atcccttccc cagagatggc tccttgggat gaagagggta gagtgggctg
ctggtgtcgc atcaagattt ggcaggatcg gcttcctcag gggcacagac ctctcccacc
cacaagaact cctcccaccc aacttcccct tagagtgctg tgagatgaga gtgggtaaat
cagggacagg gccatggggt agggtgagaa gggcaggggt gtcctgatgc aaaggtgggg
agaagggatc ctaatccctt cctctcccat tcaccctgtg taacaggacc ccaaggacct
gcctccccgg aagtgcctta acctagaggg tcggggagga ggttgtgtca ctgactcagg
ctgctccttc tctagtttcc cctctcatct gaccttagtt tgctgccatc agtctagtgg
atggccttct tggagagtca ccaattcata caccgggact tggctgctcg taactgcttg
gtggacagag atctctgtgt gaaagtatct gactttggaa tgacaaggta tgttcttgat
gaccagtatg tcagttcagt cggaacaaag tttccagtca agtggtcagc tccagaggtg
tttcattact tcaaatacag cagcaagtca gacgtatggg catttgggat cctgatgtgg
gaggtgttca gcctggggaa gcagccctat gacttgtatg acaactccca ggtggttctg
aaggtctccc agggccacag gctttaccgg ccccacctgg catcggacac catctaccag
atcatgtaca gctgctggca cgagcttcca gaaaagcgtc ccacatttca gcaactcctg
tcttccattg aaccacttcg ggaaaaagac aagcattgaa gaagaaatta ggagtgctga
taagaatgaa tatagatgct ggccagcatt ttcattcatt ttaaggaaag tagcaaggca
taatgtaatt tagctagttt ttaatagtgt tctctgtatt gtctattatt tagaaatgaa
caaggcagga aacaaaagat tcccttgaaa tttagatcaa attagtaatt ttgtttatgc
tgctcctgat ataacacttt ccagcctata gcagaagcac attttcagac tgcaatatag
agactgtgtt catgtgtaaa gactgagcag aactgaaaaa ttacttattg gatattcatt
gagagtgatg cccatctgcc taccttcaaa ggattatgca gaagtagggc gtgtgggtta
tgtttctggc tgggggcgaa atgccaattt taaatttact gaccatctga agtatgtcat
gctgcctgtg gctgaccaag accaatgcat aaggcattat gaaggcagca cagtccccga
aaagaagaca ccgaagagcc ctgtaggggt gcagcccata ctgaatgaac acaccttctg
tgctggcatg tctaagtacc aagaagacac ctgctatggc gatgcgggca gtgcctttgc
cgttcacgac ctggaggagg acacctggta tgcgactggg atcttaagct ttgataagag
ctgtgctgtg gctgagtatg gtgtgtatgt gaaggtgact tccatccagg actgggttca
gaagaccata gctgagaact aatgcaaggc tggccggaag cccttgcctg aaagcaagat
ttcagcctgg aagagggcaa agtggacggg agtggacagg agtggatgcg ataagatgtg
gtttgaagct gatgggtgcc agccctgcat tgctgagtca atcaataaag agctttcttt
cggaagcccc tcaagtcggg tatgaaggag ctggccgtgt tccgggagaa ggtcactgag
cagcaccggc agatgggcaa gggtggcaag catcaccttg gcctggagga gcccaagaag
ctgcgaccac cccctgccag gactccctgc caacaggaac tggaccaggt cctggagcgg
atctccacca tgcgccttcc ggatgagcgg ggccctctgg agcacctcta ctccctgcac
atccccaact gtgacaagca tggcctgtac aacctcaaac agtgcaagat gtctctgaac
gggcagcgtg gggagtgctg gtgtgtgaac cccaacaccg ggaagctgat ccagggagcc
cccaccatcc ggggggaccc cgagtgtcat ctcttctaca atgagcagca ggaggctcgc
ggggtgcaca cccagcggat gcagtagacc gcagccagcc ggtgcctggc gcccctgccc
cccgcccctc tccaaacacc ggcagaaaac ggagagtgct tgggtggtgg gtgctggagg
attttccagt tctgacacac gtatttatat ttggaaagag accagcaccg agctcggcac
ctccccggcc tctctcttcc cagctgcaga tgccacacct gctccttctt gctttccccg
ggggaggaag ggggttgtgg tcggggagct ggggtacagg tttggggagg gggaagagaa
atttttattt ttgaacccct gtgtcccttt tgcataagat taaaggaagg aaaagtaaa
aactctatct ttggtctggc ccccatgctg agtgacacag acaagaagcc cttcactgcc
atcctgtatg gcaatgggcc tggctacaag gtggtgggcg gtgaacgaga gaatgtctcc
atggtggact atgctcacaa caactaccag gcgcagtctg ctgtgcccct gcgccacgag
acccacggcg gggaggacgt ggccgtcttc tccaagggcc ccatggcgca cctgctgcac
ggcgtccacg agcagaacta cgtcccccac gtgatggcgt atgcagcctg catcggggcc
aacctcggcc actgtgctcc tgccagctcg gcaggcagcc ttgctgcagg ccccctgctg
ctcgcgctgg ccctctaccc cctgagcgtc ctgttctgag ggcccagggc ccgggcaccc
acaagcccgt gacagatgcc aacttcccac acggcagccc ccccctcaag gggcagggag
gtgggggcct cctcagcctc tgcaactgca agaaagggga cccaagaaac caaagtctgc
cgcccacctc gctcccctct ggaatcttcc ccaagggcca aacccacttc tggcctccag
cctttgctcc ctccccgctg ccctttggcc aacagggtag atttctcttg ggcaggcaga
gagtacagac tgcagacatt ctcaaagcct cttatttttc tagcgaacgt atttctccag
acccagaggc cctgaagcct ccgtggaaca ttctggatct gaccctccca gtctcatctc
ctgaccctcc cactcccatc tccttacctc tggaaccccc caggccctac aatgctcatg
tccctgtccc caggcccagc cctccttcag gggagttgag gtctttctcc tcaggacaag
gccttgctca ctcactcact ccaagaccac cagggtccca ggaagccggt gcctgggtgg
ccatcctacc cagcgtggcc caggccggga agagccacct ggcagggctc acactcctgg
gctctgaaca cacacgccag ctcctctctg aagcgactct cctgtttgga acggcaaaaa
ttgcaggaag cacttgagtt tgagacgaaa cggcgtgaac aagcagaaca gacgctaaaa
caggcagctt caacagatag tctcagggtc ttaaatgact ctctgacccc agagatagag
gctgaccgca gtggcggcag aacagatgct gaaaggacaa tacaagatgg aagactgtat
ttgaaaacta ctgtcatgta ctgaatcttt cctgttgaag aaatccatgt tatagaaaag
aactttgcag tcagacattc gtcatgggaa agttcagaaa aaaataaagt ccttttaagg
gaacttcctg aattttgtgt attaatgttc tttaaaagtt taagtattct acaaaaaaaa
aaaaagtttt ctccattgat tttcacctgt ggttcatacc agagacctga gaatgtttgt
aaatgtacaa gtatcaaagt tcttacagtt aattactgca acttgctgct ggacaattgt
atacagagtt aaaggcaggt ctgaataaga cctagctttg tttttttcta atggaatgaa
ccattttcct cttctgaaaa ttctgtatct gagcacatca agagactctt gtagcagtgg
ttacccagac ttacagaatt atgtcctcca gaaaccagca agaacacttg gaatgaacga
atgaacttgt agggggcata gaggattctt gaaaaaaaaa aatgcaagag tgattttctg
ttacattcaa tttcaaactc tctaattgtg ggttttctcc tgaagaattt tttttcacat
actttccaaa agaccaacaa atggatgttg acaacaaccc aatgaaataa cattttgcat
atctgaaaag aagcattgaa tataagccaa aagctttcac tgaaggtttt tttttcttaa
aaataaaaaa aaatatataa gtgtaacatg ttttcattcc aaactggtag tggtatatag
aattaaagat aataatgttg cttcttattc aaactgttgg tcatatgtac agtatataaa
cataaaacac acaaggaagg tattatgtat gcagtagtat actagagttt aggaaaatga
aaattttaga aaatatgttt tgtcaccctg ttggtcagaa agatgtcttt ctggttttaa
cgcatgcagg catgtaaata tttgtctgga gtcacagtat taatgaatga gatcttaagc
atctggtgac atcagaactc tgtgtcagcc acttttattt gtatattgaa ccctagctag
tgccccaagc tgcactattg ggaatggatt gtggctgaac agcaaatcaa aacaccagaa
atatttttat atgttaacgt catattatgt taatgttgct gaaaacaaaa cctaacaaac
cttgatgtac cagtccaata ccatgtagcg ctgagtgata aagttaaaat gtgctgtgct
tcccaccctt gtcagaggga agggtggcta tgtgttattt tcactgtctt tttgaaagtt
acagtatgtg ttttcacttt cgtgcagata actggaagta aagcggcaaa cagtgcttat
tacatgctaa agttaccttc tctttgtttt ttgcatatct ggaattacac ctttaaagac
tgatatgaat cagtacggtc actatacatt ttatgatttt tctgtcatct taaaattgta
tgatcgtaac attatttatt accacaaaac agcaaaatct tcaatgtcta agaaaactag
cttaaaatgt ttaaatatag ttctgattgg gtattaatta cttgattaag aaaaaattaa
cattatagat actctggcat tacgcttcta taccttttag gtcttccttg caatactgga
acataattct tttgtgtagc tcactattag ccagctaagt tcatcttttt aataccataa
aaaggttata tgtacagttc ctattttagc ttgcttacaa agggagcatt atttttattt
aaagtattgc tagtaaatga tttgtagaaa cttggttttc taagcatagt tcttccataa
ccaccttttg ttgtttgagc acaagggatt cttttcctag ttctatgtgt ttgtttccct
atatgcagtc tttaaaggat tacaacactt aaaattgaat ggacttgtgt caagcttttt
gcatcataca ttttttgaaa gatttttaaa aaagcctaca acttacatat gtagtagaat
cagccattgc tctgctcctg gcatagagtc acctgtttgt tatgtggatt aaatagtttt
aaaatacata tttgaagacc tttgagaatg ctttagtgtt tgatttgaaa taaaaggaaa
ttttagcaag gattaaagaa aaaagctatc agctgtatgt taagagagac tcttactaac
atgttgtaaa tattacaatt catgaaatgt tattgtaagt ctgtaactta attttttccc
tgttttagtt atacaggttg gtttggaaat ttgtgttttg gcataaacaa gtaaaatgtg
cccattttat ggtttccatg cttttgtaat cctaaaaata ttaatgtcta gttgttctat
attataacca catttgcgct ctatgcaagc ccttggaaca gaacatactc atcttcatgt
aggacctatg aaaattgtct atttttatct atatatttaa agttttctaa aaatgataaa
gcctgaggtc ttggaaaaac agtgtggata taagctgttc atttatggaa gggatgacta
cgttggggaa gacattgttg aggtcattaa tgaaaacgta aagaaaagca gaagactgat
tatcatttta gtcagagaaa catcaggctt cagctggctg ggtggttcat ctgaagagca
aatagccatg tataatgctc ttgttcagga tggaattaaa gttgtcctgc ttgagctgga
gaaaatccaa gactatgaga aaatgccaga atcgattaaa ttcattaagc agaaacatgg
ggctatccgc tggtcagggg actttacaca gggaccacag tctgcaaaga caaggttctg
gaagaatgtc aggtaccaca tgccagtcca gcgacggtca ccttcatcta aacaccagtt
actgtcacca gccactaagg agaaactgca aagagaggct cacgtgcctc tcgggtagca
tggagaagtt gccaagagtt ctttaggtgc ctcctgtctt atggcgttgc aggccaggtt
atgcctcatg ctgacttgca gagttcatgg aatgtaacta tatcatcctt tatccctgag
gtcacctgga atcagattat taagggaata agccatgacg tcaatagcag cccagggcac
ttcagagtag agggcttggg aagatctttt aaaaaggcag taggcccggt gtggtggctc
acgcctataa tcccagcact ttgggaggct gaagtgggtg gatcaccaga ggtcaggagt
tcgagaccag cccagccaac atggcaaaac cccatctcta ctaaaaatac aaaaatgagc
taggcatggt ggcacacgcc tgtaatccca gctacacctg aggctgaggc aggagaattg
cttgaaccgg ggagacggag gttgcagtga gccgagtttg ggccactgca ctctagcctg
gcaacagagc aagactccgt ctcaaaaaaa gggcaataaa tgccctctct gaatgtttga
actgccaaga aaaggcatgg agacagcgaa ctagaagaaa gggcaagaag gaaatagcca
ccgtctacag atggcttagt taagtcatcc acagcccaag ggcggggcta tgccttgtct
ggggaccctg tagagtcact gaccctggag cggctctcct gagaggtgct gcaggcaaag
tgagactgac acctcactga ggaagggaga catattcttg gagaactttc catctgcttg
tattttccat acacatcccc agccagaagt tagtgtccga agaccgaatt ttattttaca
gagcttgaaa actcacttca atgaacaaag ggattctcca ggattccaaa gttttgaagt
catcttagct ttccacagga gggagagaac ttaaaaaagc aacagtagca gggaattgat
ccacttctta atgctttcct ccctggcatg accatcctgt cctttgttat tatcctgcat
tttacgtctt tggaggaaca gctccctagt ggcttcctcc gtctgcaatg tcccttgcac
agcccacaca tgaaccatcc ttcccatgat gccgctcttc tgtcatcccg ctcctgctga
aacacctccc aggggctcca cctgttcagg agctgaagcc catgctttcc caccagcatg
tcactcccag accacctccc tgccctgtcc tccagcttcc cctcgctgtc ctgctgtgtg
aattcccagg ttggcctggt ggccatgtcg cctgccccca gcactcctct gtctctgctc
ttgcctgcac ccttcctcct cctttgccta ggaggccttc tcgcattttc tctagctgat
cagaatttta ccaaaattca gaacatcctc caattccaca gtctctggga gactttccct
aagaggcgac ttcctctcca gccttctctc tctggtcagg cccactgcag agatggtggt
gagcacatct gggaggctgg tctccctcca gctggaattg ctgctctctg agggagaggc
tgtggtggct gtctctgtcc ctcactgcct tccaggagca atttgcacat gtaacataga
tttatgtaat gctttatgtt taaaaacatt ccccaattat cttatttaat ttttgcaatt
attctaattt tatatataga gaaagtgacc tattttttaa aaaaatcaca ctctaagttc
tattgaacct aggacttgag cctccatttc tggcttctag tctggtgttc tgagtacttg
atttcaggtc aataacggtc ccccctcact ccacactggc acgtttgtga gaagaaatga
cattttgcta ggaagtgacc gagtctagga atgcttttat tcaagacacc aaattccaaa
cttctaaatg ttggaatttt caaaaattgt gtttagattt tatgaaaaac tcttctactt
tcatctattc tttccctaga ggcaaacatt tcttaaaatg tttcattttc attaaaaatg
aaagccaaat ttatatgcca ccgattgcag gacacaagca cagttttaag agttgtatga
acatggagag gacttttggt ttttatattt ctcgtattta atatgggtga acaccaactt
ttatttggaa taataatttt cctcctaaac aaaaacacat tgagtttaag tctctgactc
ttgcctttcc acctgctttc tcctgggccc gctttgcctg cttgaaggaa cagtgctgtt
ctggagctgc tgttccaaca gacagggcct agctttcatt tgacacacag actacagcca
gaagcccatg gagcagggat gtcacgtctt gaaaagccta ttagatgttt tacaaattta
gtagtcccat cataagggca gatctgaaca ttgttagaag ttgcacctct aacgtaccat
ctaaggctgt tctttcctgt gacttgacat gttttgttgg atctgaagac atagcaaagg
acatggaagc ctggaaagat gtaaccagtg gaaatgctaa aatttaccag cttccagggg
gtcactttta tcttctggat cctgcgaacg agaaattaat caagaactac ataatcaagt
gtctagaagt atcatcgata tccaattttt agatattttc cctttcactt ttaaaataat
caaagtaata tcatactctt ctcagttatt cagatatagc tcagttttat tcagattgga
aattacacat tttctactgt cagggagatt cgttacataa atatatttac gtatctgggg
acaaaggtca agccagtaaa gaatacttct ggcagcactt tgggaggcca aggcgggcgg
atcacgaggt caggagatcg agaccgtcct ggctaacacc gtgaaacccc atctctacta
aaaatacaca aaattagccg ggcgtggtgg tgggcacctg tagtcccagc tactcgggag
gctgaggcag
gagaatggtg tgaacctggg aggtggagct tgcagtgaac cgagatcgct
ccactgcact ccagcctggg tgacagatcc agactctgtc tcaaaaaaaa aaaaaaaaaa
cgtgaagatt caatggtaca aggattctct tcttttggat aaagacaatg agaaatttct
aagtgtgagg gggaccactc acttactcgt acacgatgtg gccctggaag atgctggcta
ttaccgctgt gtcctgacat ttgcccatga aggccagcaa tacaacatca ctaggagtat
tgagctacgc atcaagaaaa aaaaagaaga gaccattcct gtgatcattt cccccctcaa
gaccatatca gcttctctgg ggtcaagact gacaatcccg tgtaaggtgt ttctgggaac
cggcacaccc ttaaccacca tgctgtggtg gacggccaat gacacccaca tagagagcgc
ctacccggga ggccgcgtga ccgaggggcc acgccaggaa tattcagaaa ataatgagaa
ctacattgaa gtgccattga tttttgatcc tgtcacaaga gaggatttgc acatggattt
taaatgtgtt gtccataata ccctgagttt tcagacacta cgcaccacag tcaaggaagc
ctcctccacg ttctcctggg gcattgtgct ggccccactt tcactggcct
tcttggtttt
acaaccgggt atatggagaa ttcatgcgag tctggatctc tggagaggaa acactcatta
tcagcaagtc ctcaagtatg ttccacataa tgaagcacaa tcattacagc tctcgattcg
gcagcaaact tgggctgcag tgcatcggta tgcatgagaa aggcatcata tttaacaaca
atccagagct ctggaaaaca actcgaccct tctttatgaa agctctgtca ggccccggcc
ttgttcgtat ggtcacagtc tgtgctgaat ccctcaaaac acatctggac aggttggagg
aggtgaccaa tgaatcgggc tatgtggacg tgttgaccct tctgcgtcgt gtcatgctgg
acacctctaa cacgctcttc ttgaggatcc ctttggacga aagtgctatc gtggttaaaa
tccaaggtta ttttgatgca tggcaagctc tcctcatcaa accagacatc ttctttaaga
tttcttggct atacaaaaag tatgagaagt ctgtcaagga tttgaaagat gccatagaag
ttctgatagc agaaaaaaga cgcaggattt ccacagaaga gaaactggaa gaatgtatgg
actttgccac tgagttgatt ttagcagaga aacgtggtga cctgacaaga gagaatgtga
accagtgcat attggaaatg ctgatcgcag ctcctgacac catgtctgtc tctttgttct
tcatgctatt tctcattgca aagcacccta atgttgaaga ggcaataata aaggaaatcc
agactgttat tggtgagaga gacataaaga ttgatgatat acaaaaatta aaagtgatgg
aaaacttcat ttatgagagc atgcggtacc agcctgtcgt ggacttggtc atgcgcaaag
ccttagaaga tgatgtaatc gatggctacc cagtgaaaaa ggggacaaac attatcctga
atattggaag gatgcacaga ctcgagtttt tccccaaacc caatgaattt actcttgaaa
attttgcaaa gaatgttcct tataggtact ttcagccatt tggctttggg ccccgtggct
gtgcaggaaa gtacatcgcc atggtgatga tgaaagccat cctcgttaca cttctgagac
gattccacgt gaagacattg caaggacagt gtgttgagag catacagaag atacacgact
tgtccttgca cccagatgag actaaaaaca tgctggaaat gatctttacc ccaagaaact
cagacaggtg tctggaacac tagagaaggc tggtcagtac ccactctgga gcatttctca
tcagtagttc acatacaaat catccatcct tgccaatagt gtcatcctca cagtgaacac
tcagtggccc atggcatttt ataggcatac ctcctatggg ttgtcaccaa gctaggtgct
atttgtcatc tgctcctgtt cacaccagag aaccaggcta caagagaaaa agcagaggcc
aagagtttga gggagaaata gtcggtgaag aaaccgtatc cataaagacc cgattccacc
aaatgtgctt tgagaaggat aggccttcat taacaaaatg tatgtctggt tccccagtag
agctctactg cctcaaccca aggggatttt tatgtctggg gcagaaacac tcaagttgat
tagaaagacc aggccaatgt cagggtacct ggggccaaac ccacctgcta gtgtgaatta
aagtacttta attttgtttt ctgtggaggt ggaaaagcaa cattcatagt ctttggagaa
atgcttagaa attcagcatt tgacccttgc tgtgaattaa gcccaattaa ttcctgtttg
tctacatatg atctgtctgt ggcaaaagtt taatcagagg aaattctttc ccagtctgtc
gatttatgcc tcagccactt gcctgtgcta caattcattg tgttacctgt agattcaggt
aatacaaact atatataatc atcaagtaat acaaactaat ttagtaatag cctgggttaa
gtattattag ggccctgtgt ctgctgtaga aaaaaaaatt cacatgatgc acttcaaatt
caaataaaaa tccttttggc atgttcccat ttttgcttag ctcaattagt gtggctaacc
aagagataac tgtaaatgtg acattgattt gctcttacta cagcttcagt gattggggga
ggaaaagtcc caacccaatg ggctcaaact tctaaggggt actcctctca tccccttatc
cttctccctc gacattttct ccctctttct tcccatgacc ccaaagccaa gggcaacaga
tcagtaaaga acgtggtcag agtagaaccc ctgaagtatt ttttaatcct acctcaaaat
ttaacagtta cctgagagat ttaacattat ctagttcatt gaatcattgt atgtggtcat
ggataaattg cacaccttgg aattcgcttt ctaaaggaaa tcaaatgaat ggaggaactt
tccaaacacc actttacttg tgttatatag ccaatataac tatctctact gaatgtcatt
gaaaaactaa aaaattaaac ttatttacaa ataggtaaat atttgtcatt gaatccattg
ccatcccatt tgactgttct tttcatccta ctgtctagta ataagctgag tataagatga
cagtgtaatc tccctgaaag caggagctac tttctttctt ttgtaatcta tttccatccc
catttccctg tcctgtctcc ctgtattcac tcccaagctc agttctgaat agacattcct
gctcagagat actcccaact gatgcagaaa ccaaataaag aggtaggtat tccaagaatt
caagaatgga cattagtaaa gaataaaaca tttatttgag cttggaatta tttggatcat
ctatatggcc taaaaatata tggactatgc ctgtgtacct gaatacgtat gtagtcaggt
caagacaatc atccaaataa cttagacccc taaaagcaag gccaggattt gcaatttaat
gtgtcccaat taattcactt gaaaattagt aacactctgt ttacgttgcc tctggctgga
gctgcatggt ggaagaagcc caactttgga tccatgtact tcacccatcc aatactcttg
ggacatttat gtgtatttta tctgtatata tgaagccaat gtctatgtct acacagtcaa
agtgaaatgc atgtttgata tagctgtaca tagatatcta ttttgcaggt acaaaaatat
cctgggggaa aactgggagt ggaagggtgg ggggtgggag tgagggacat gggggaggga
caggaagagg agaagtgttg gtttgaacga tccaagcaaa ctctcccaga atcaaattac
ctgggtagtt gttcaacttt tcactctgct tagcctgtat agacaaaccc catatatttg
tagaggcttg gccttggaat tctggaatac cattggcttt tcagtaggct gatgaacaca
ttttgaaaat tctattatct tcagaatttt gccccattgt taagtgctta accgtcactc
ttgaatgtgc aatgtgctgt ggattccatt ttcatcagtt ctgaaagaac tgcaatgtgt
aaattatcag tgaaatgcat gcatataagg gctctatcat tatcaaattg taaggacaat
tgtacccttc tatatctttg ggcatgctag acacccccat gccttcattg agatcccatt
ttccccctct caagtggaaa ataatcacat ccagcaagct ctctcattat tgagaaatac
catttggaaa ttgccacttt ttattcctaa gcagcacctt tcactgttca tgatgctaat
gttccacaaa agcatgtgcc attggcccac tgaaggatag agggaccctt ttcaatctat
atcagctggg ctctgggact gaatctctca cctattcttg cagaaagaca tactaattaa
accttgtcaa agtaaaaaaa aaaaaaaaaa a
cactgtatcc attctacctg tcctccgtga aaatatgttt ggaatattcc actatttgca
gaggcttatt cagttcttac acattccatc ttacattagt gattccatca aagagaagga
aagtaagcct ttttgtcacc tcaatattta ctatttcaat acttacatat ctgacttcta
ggatttattg ttatattact tgcctatctg acttcataca tccctcagtt tcttaaaatg
tcctatgtat atcttctaca tgcaatttag aactagattt tggttagaag taaggattat
aaacaaccta gacagtaccc ttggccttta cagaaaatat ggtgctgttt tctacccttg
gaaagaaatg tagatgatat gtttcgtggg ttgaattgtg tcccccataa aagatatgtt
gaagttctaa ccccaggtac ccatgaatgt gagcttacca gggtctttgc agatgtaatt
agttaagtta aggtgagatc acactgaatt agggtgggct ctaaatccat tatgactgtt
gttcttataa gaagaagaga ggcatagtca cctaggggag gaggccgtat gaagacagag
gcagagattg gagtgacgca tctccaagcc aaggaattcc aaggactgta agccaccagt
agaagctttg aagaggcaag gaaggattcc ctccaatagc cttcaagtgt gaccctgctg
acacctgcag aattcggact tctatcctcc aaaaccgtga gggaataaat ttcctttgtt
ttaagccacc aactttgcaa tactttgtta cagcaaccct agacatgagg tactagacac
agtacatcta cacatatgaa aatgaatcaa cacagaatgc agaagtagaa cccttgctaa
ggactactgg gcatcttccc aggacagcag ccaaaagaga accaccactt cctctcctgc
ctcctccttg ctctctccta gagtccaaac ccaaatgggc cagttggatc tgatgttcgt
cagttcttta cttctatttc ctggggtact caggagggca cacactatag ataacttggg
ttagctgcat aaaattcaat gtctcattaa gttgcattaa actgagctta gatgtgtaag
tttgctaacg gatgggtttt tttgttaaga actataggat ttatgggacc aagtctagcg
agtccagata tcaaaatcat tataatgtta tatttgctgt tattagaata taatatagct
tattatacaa taaatatgta gactgtaaaa tatatttctc actagtacct cctattttct
caggaaatgt tttcttctac atggaaagat agacagcagc caaccctgat ctggaagaca
gggccccggc tggacacacg tggaaccaag ccagggatgg gctggccatt gtgtccccgc
aggagagatg ggcagaatgg ccctagagtt cttttccctg agaaaggaga aaaagatggg
attgccactc acccacccac actggtaagg gaggagaatt tgtgcttctg gagcttctca
agggattgtg ttttgcaggt acagaaaact gcctgttatc ttcaagccag gttttcgagg
gcacatgggt caccagttgc tttttcagtc aatttggccg ggatggacta atgaggctct
aacactgctc aggagacccc tgccctctag ttggttctgg gctttgatct cttccaacct
gcccagtcac agaaggagga atgactcaaa tgcccaaaac caagaacaca ttgcagaagt
aagacaaaca tgtatatttt taaatgttct aacataagac ctgttctctc tagccattga
tttaccaggc tttctgaaag atctagtggt tcacacagag agagagagag tactgaaaaa
gcaactcctc ttcttagtct taataattta ctaaaatggt caacttttca ttatctttat
tataataaac ctgatgcttt tttttagaac tccttactct gatgtctgta tatgttgcac
tgaaaaggtt aatatttaat gttttaattt attttgtgtg gtaagttaat tttgatttct
agagcaatgt gaccctgaag tgtcaggctc attcccagaa tgtgacattt gtgctgcgca
aggtgaacga ctctgggtac aagcaggaac agagctcggc agaaaacgaa gctgaattcc
ccttcacgga cctgaagcct aaggatgctg ggaggtactt ttgtgcctac aagacaacag
cctcccatga gtggtcagaa agcagtgaac acttgcagct ggtggtcaca gataaacacg
atgaacttga agctccctca atgaaaacag acaccagaac catctttgtc gccatcttca
gctgcatctc catccttctc ctcttcctct cagtcttcat catctacaga tgcagccagc
acagttcatc atctgaggaa tccaccaaga gaaccagcca ttccaaactt ccggagcagg
aggctgccga ggcagattta tccaatatgg aaagggtatc tctctcgacg gcagaccccc
aaggagtgac ctatgctgag ctaagcacca gcgccctgtc tgaggcagct tcagacacca
cccaggagcc cccaggatct catgaatatg cggcactgaa agtgtagcaa gaagacagcc
ctggccacta aaggaggggg gatcgtgctg gccaaggtta tcggaaatct ggagatgcag
atactgtgtt tccttgctct tcgtccatat caataaaatt aagtttctcg tcttaaaaag
agtccctcag aaacctaagg tctccttgaa ccctccatgg aatagaatat ttaaaggaga
gaatgtgact cttacatgta atgggaacaa tttctttgaa gtcagttcca ccaaatggtt
ccacaatggc agcctttcag aagagacaaa ttcaagtttg aatattgtga atgccaaatt
tgaagacagt ggagaataca aatgtcagca ccaacaagtt aatgagagtg aacctgtgta
cctggaagtc ttcagtgact ggctgctcct tcaggcctct gctgaggtgg tgatggaggg
ccagcccctc ttcctcaggt gccatggttg gaggaactgg gatgtgtaca aggtgatcta
ttataaggat ggtgaagctc tcaagtactg gtatgagaac cacaacatct ccattacaaa
tgccacagtt gaagacagtg gaacctacta ctgtacgggc aaagtgtggc agctggacta
tgagtctgag cccctcaaca ttactgtaat aaaagctccg cgtgagaagt actggctaca
attttttatc ccattgttgg tggtgattct gtttgctgtg gacacaggat tatttatctc
aactcagcag caggtcacat ttctcttgaa gattaagaga accaggaaag gcttcagact
tctgaaccca catcctaagc caaaccccaa aaacaactga tataattact caagaaatat
ttgcaacatt agtttttttc cagcatcagc aattgctact caattgtcaa acacagcttg
caatatacat agaaacgtct gtgctcaagg atttatagaa atgcttcatt aaactgagtg
aaactggtta agtggcatgt aatagtaagt gctcaattaa cattggttga ataaatgaga
gaatgaatag attcatttat tagcatttgt aaaagagatg ttcaatttca ataaaataaa
gccatgggaa tccgtttcat tattatggta acaatatgga gtgctgtatt cctaaactca
ttattcaacc aagaagttca aattcccttg accgaaagtt actgtggccc atgtcctaaa
aactggatat gttacaaaaa taactgctac caattttttg atgagagtaa aaactggtat
gagagccagg cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag
gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca
acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct actaacaata
attgaaatgc agaagggaga ctgtgcactc tatgcctcga gctttaaagg ctatatagaa
aactgttcaa ctccaaatac gtacatctgc atgcaaagga ctgtgtaaag atgatcaacc
atctcaataa aagccaggaa cagagaagag attacaccag cggtaacact gccaactgag
actaaaggaa acaaacaaaa acaggacaaa atgaccaaag actgtcagat ttcttagact
ccacaggacc aaaccataga acaatttcac tgcaaacatg catgattctc caagacaaaa
gaagagagat cctaaaggca attcagatat ccccaaggct gcctctccca ccacaagccc
agagtggatg ggctggggga ggggtgctgt tttaatttct aaaggtagga ccaacaccca
ggggatcagt gaaggaagag aaggccagca gatcactgag agtgcaaccc caccctccac
aggaaattgc ctcatgggca gggccacagc agagagacac agcatgggca gtgccttccc
tgcctgtggg ggtcatgctg ccacttttaa tgggtcctcc acccaacggg gtcagggagg
tggtgctgcc ccagtgggcc atgattatct taaaggcatt attctccagc cttaagtaag
atcttaggac gtttcctttg ctatgatttg tacttgcttg agtcccatga ctgtttctct
tcctctcttt cttccttttg gaatagtaat atccatccta tgtttgtccc actattgtat
tttggaagca cataacttgt ttggtttcac aggttcacag ttaagaagga attttgcctc
tgaataaata gaatcttgag tctcatgcaa aaaaaaaaaa aaaaaa
gttcttcacc ttcatctctt cctcgggatg tctgtcaggg ttcaccttgg catcaatttg
ccctgaaact tagctgtgct gggattattc tccttgtctt ggttgttact gggttgagtg
tttcagtgac atccttaata cagaaatcat caatagaaaa atgcagtgtg gacattcaac
agagcaggaa taaaacaaca gagagaccgg gtctcttaaa ctgcccaata tattggcagc
aactccgaga gaaatgcttg ttattttctc acactgtcaa cccttggaat aacagtctag
ctgattgttc caccaaagaa tccagcctgc tgcttattcg agataaggat gaattgatac
acacacagaa cctgatacgt gacaaagcaa ttctgttttg gattggatta aatttttcat
tatcagaaaa gaactggaag tggataaacg gctctttttt aaattctaat gacttagaaa
ttagaggtga tgctaaagaa aacagctgta tttccatctc acagacatct gtgtattctg
agtactgtag tacagaaatc agatggatct gccaaaaaga actaacacct gtgagaaata
aagtgtatcc tgactcttga
tggtttgagg gggcgggcgg tgtgtgtgtg ttctggtggg agggatctga gcaagtgcaa
gcctggctga cacaggtgtg aagaggccat cctggaaccc aggtgagggc aagatgaagg
cttccaggca gaacagctgc agagagtttg gctatatgca tctgcagccc caagagctcc
cactgcaaga caagtgttgg ggaagatggg aggttgtggg tgaggcctct aaaggtcctc
tcccaaactg accaggctga tgtcaaccta accccctcag gggcagggaa caggggaggg
ctccacaagc gtgtctggca ttcccaccca ccatggaaga ctggatacgc acctggaaac
aaaaggacta tggaagctgt tcaagataca tttgatcttc agaaaagcag aatttggttc
aactgttgac agaggacaca aatacgttgt tccagagctc agccttctca ctctaaaaga
aagatatttt tctatttatt ttctacatct ggccagtggc tctggtgcta gatgccactg
tagccagatc tccaacagtg ccttggacca tggactcata ctcaactgag taagaagggg
ctggtgccca gtcggggtgg ctgagctggt ccttaatagg ttgtttcttg gtcttgcttt
cttcatgccc tccccactgc tcctgccacc tttagataag tttctctagc taattttgtg
gccaatgtaa aattcgtcat caacctaaca aacacaacct tctcagcagc atttctcccc
tgtgatggaa ataaagtgtt tagggcagtg ggaggagaaa attctccagg tgaatgggga
agggtctgtt ccagcctctc cctactccca tcccatttcc accaactggg gaactgtgac
tatctatctc ccccgacttc taccagggat gccttcacgc caaggctgtt ctcaccagct
gcctcagatg acaaatgagg ctaatggaca taatctacag tgtccttttt cacttgcacc
cagggaagac caccttttcc gcaagttcca ctatctcccc ttcctgccct caactgagga
cgtttacgac tgcagggtgg agcactgggg cttggatgag cctcttctca agcactggga
gtttgatgct ccaagccctc tcccagagac tacagagaac gtggtgtgtg ccctgggcct
gactgtgggt ctggtgggca tcattattgg gaccatcttc atcatcaagg gattgcgcaa
aagcaatgca gcagaacgca gggggcctct gtaaggcaca tggaggtgat ggtgtttctt
agagagaaga tcactgaaga aacttctgct ttaatggctt tacaaagctg gcaatattac
aatccttgac ctcagtgaaa gcagtcatct tcagcatttt ccagccctat agccacccca
agtgtggata tgcctcttcg attgctccgt actctaacat ctagctggct tccctgtcta
ttgccttttc ctgtatctat tttcctctat ttcctatcat tttattatca ccatgcaatg
cctctggaat aaaacataca ggagtctgtc tctgctatgg aatgccccat ggggcatctc
ttgtgtactt attgtttaag gtttcctcaa actgtgattt ttctgaacac aataaactat
ggaggcggca ctccaaccta acctgtgtct gcgaagtcct atggaaaccc gagggttgat
taaggcagta caaattgtgg agccttttaa ctgtgcaata atttctgtat ttattgggtt
ttgtaatttt tttggcatgt gcaggtactt tttattatta ttttttctgt ttgaattcct
ttaagagatt ttgttgggta tccatccctt ctttgttttt tttttaaccc ggtagtagcc
tgagcaatga ctcgcaagca atgttagagg ggaagcatat cttttaaatt ataatttggg
gggaggggtg gtgctgcttt tttgaaattt aagctaagca tgtgtaattt cttgtgaaga
agccaacact caaatgactt ttaaagttgt ttactttttc attccttcct tttttttgtc
ctgaaataaa aagtggcatg cagttttttt tttaattatt ttttaatttt ttttttggtt
tttgtttttg gggtgggggg tgtggatgta cagcggataa caatctttca agtcgtagca
ctttgtttca gaactggaat ggagatgtag cactcatgtc gtcccgagtc aagcggcctt
ttctgtgttg atttcggctt tcatattaca taagggaaac cttgagtggt ggtgctgggg
gaggcacccc acagactcag cgccgccaga gatagggttt ttggagggct cctctgggaa
atggcccgac agcattctga ggttgtgcat gaccagcaga tactatcctg ttggtgtgcc
ctggggtgcc atggctgcta ttcgctgtag attaggctac ataaaatggg ctgagggtac
ctttttgggg agatggggtg gcctgcagtg acacagaaag gaagaaacta gcggtgttct
tttaggcgtt ttctggcttg acggcttctc tcttttttta aatcaccccc accacataaa
tctcaaatcc tatgttgcta caaggggtca tccatcattt cccaagcaga cgaatgccct
aattaattga agttagtgtt ctctcattta atgcacactg atgatattgt agggatgggt
ggggtgggga tcttgcaaat ttctattctc ttttactgaa aaagcagggg atgagttcca
tcagaaggtg cccagcgcta cttcccaggt ttttattttt tttttcctat ctcattaggt
tggaaggtac taaatattga actgttaaga ttagacattt gaattctgtt gacccgcact
ttaaagcttt tgtttgcatt taaattaaat ggcttctaaa caagaaattg cagcatattc
ttctctttgg cccagaggtg ggttaaactg taagggacag ctgagattga gtgtcagtat
tgctaagcgt ggcattcaca atactggcac tataaagaac aaaataaaat aataatttat
aggacagttt ttctactgcc attcaatttg atgtgagtgc cttgaaaact gatcttccta
tttgagtctc ttgagacaaa tgcaaaactt tttttttgaa atgaaaagac tttttaaaaa
agtaaaacaa gaaaagtaca ttctttagaa actaacaaag ccacatttac tttaagtaaa
aaaaaaaaaa attctggttg aagatagagg atatgaaatg ccataagacc caatcaaatg
aagaaataaa cccagcacaa ccttggacat ccattagctg aattatcctc agcccctttt
gtttttggga caacgctgct tagatatgga gtggaggtga tttactgctg aattaaaact
caagtgacac aagttacaag ttgatatcgt tgaatgaaaa gcaaaacaaa aacaattcag
gaacaacggc taattttttc taaagttaaa tttagtgcac tctgtcttaa aaatacgttt
acagtattgg gtacatacaa gggtaaaaaa aaaattgtgt gtatgtgtgt tggagcgatc
tttttttttc aaagtttgct taataggtta tacaaaaatg ccacagtggc cgcgtgtata
ttgttttctt ttggtgacgg ggttttagta tatattatat atattaaaat ttcttgatta
ctgtaaaagt ggaccagtat ttgtaataat cgagaatgcc tgggcatttt acaaaacaag
aaaaaaaata cccttttctt ttccttgaaa atgttgcagt aaaatttaaa tggtgggtct
ataaatttgt tcttgttaca gtaactgtaa agtcggagtt ttagtaaatt tttttctgcc
ttgggtgttg aatttttatt tcaaaaaaaa tgtatagaaa cttgtatttg gggattcaaa
ggggattgct acaccatgta gaaaaagtat gtagaaaaaa agtgcttaat attgttattg
ctttgcagaa aaaaaaaaaa tcacatttct gacctgtact tatttttctc ttcccgcctc
cctctggaat ggatatattg gttggttcat atgatgtagg cacttgctgt atttttactg
gagctcgtaa ttttttaact gtaagcttgt ccttttaaag ggatttaatg tacctttttg
ttagtgaatt tggaaataaa aagaaaaaaa aaacaaaaac aaacaggctg ccataatata
tttttttaat ttggcaggat aaaatattgc aaaaaaaaca catttgtatg ttaagtccta
ttgtacagga gaaaaagggt tgtttgacaa cctttgagaa aaagaaacaa aaggaagtag
ttaaatgctt tggttcacaa atcatttagt tgtatatatt ttttgtcgga attggcctac
acagagaacc gttcgtgttg ggcttctctc tgaacgcccc gaaccttgca tcaaggctcc
ttggtgtggc cacagcagac cagatgggaa attatttgtg ttgagtggaa aaaaatcagt
ttttgtaaag atgtcagtaa cattccacat cgtcctccct ttctctaaga ggccatctct
aagatgtcag atgtagagga gagagagcga gagaacatct tccttctcta ccatcactcc
tgtggcggtc accaccacca cctctcccgc ccttaccagc agaaagcaat gcaaactgag
ctgctttagt ccttgagaaa ttgtgaaaca aacacaaata tcataaaagg agctggtgat
tcagctgggt ccaggtgaag tgacctgctg ttgagaccgg tacaaattgg atttcaggaa
ggagactcca tcacagccag gacctttcgt gccatggaga gtgttggcct cttgtctttc
ttccctgctt tgctgctttg ctctctgaaa cctacattcc gtcagtttcc gaatgcgagg
gcctgggatg aatttggtgc ctttccatat ctcgttctct ctccttcccc tgcgtttcct
ctccatcctt catcctccat tggtcctttt tttttctttc attttttatt taatttcttt
tcttcctgtc tcttcctccc ctaatcctct attttatttt tattttttgt aaagccaagt
agctttaaga taaagtggtg gtcttttgga tgagggaata atgcattttt aaataaaata
ccaatatcag gaagccattt tttatttcag gaaatgtaag aaaccattat ttcaggttat
gaaagtataa ccaagcatcc ttttgggcaa ttccttacca aatgcagaag cttttctgtt
cgatgcactc tttcctcctt gccacttacc tttgcaaagt taaaaaaaag gggggaggga
atgggagaga aagctgagat ttcagtttcc tactgcagtt tcctacctgc agatccaggg
gctgctgttg cctttggatg ccccactgag gtcctagagt gcctccaggg tggtcttcct
gtagtcataa cagctagcca gtgctcacca gcttaccaga ttgccaggac taagccatcc
caaagcacaa gcattgtgtg tctctgtgac tgcagagaag agagaatttt gcttctgttt
tgtgtttaaa aaaccaacac ggaagcagat gatcccgaga gagaggcctc tagcatgggt
gacccagccg acctcaggcc ggtttccgca ctgccacaac tttgttcaaa gttgccccca
attggaacct gccacttggc attagagggt ctttcatggg gagagaagga gactgaatta
ctctaagcaa aatgtgaaaa gtaaggaaat cagcctttca tcccggtcct aagtaaccgt
cagccgaagg tctcgtggaa cacaggcaaa cccgtgattt tggtgctcct tgtaactcag
ccctgcaaag caaagtccca ttgatttaag ttgtttgcat ttgtactggc aaggcaaaat
ggtcagaact cagatactga ccggcaagga gctccgagtt gccacccagg aaaaagaggg
ctcctctggg agatgtatgc ttactctctt aggcctttca ttcatcttgg caggacttat
tgttggtgga gcctgcattt acaagtactt catgcccaag agcaccattt accgtggaga
gatgtgcttt tttgattctg aggatcctgc aaattccctt cgtggaggag agcctaactt
cctgcctgtg actgaggagg ctgacattcg tgaggatgac aacattgcaa tcattgatgt
gcctgtcccc agtttctctg atagtgaccc tgcagcaatt attcatgact ttgaaaaggg
aatgactgct tacctggact tgttgctggg gaactgctat ctgatgcccc tcaatacttc
tattgttatg cctccaaaaa atctggtaga gctctttggc aaactggcga gtggcagata
tctgcctcaa acttatgtgg ttcgagaaga cctagttgct gtggaggaaa ttcgtgatgt
tagtaacctt ggcatcttta tttaccaact ttgcaataac agaaagtcct tccgccttcg
tcgcagagac ctcttgctgg gtttcaacaa acgtgccatt gataaatgct ggaagattag
acacttcccc aacgaattta ttgttgagac caagatctgt caagagtaag aggcaacaga
tagagtgtcc ttggtaataa gaagtcagag atttacaata tgactttaac attaaggttt
atgggatact caagatattt actcatgcat ttactctatt gcttatgctt
taaaaaaagg
aatccaaaat tcaaaatgct ccaataagca tttcccttga gtttcattga tgtcgatgca
gtgctcaaaa tctcagattt tggagcattt tggatattgg atttttggat ttgggatgct
caacttgtac aatgtttatt agacacatct cctgggacat actgcctaac cttttggagc
cttagtctcc cagactgaaa aaggaagagg atggtattac atcagctcca ttgtttgagc
caagaatcta agtcatccct gactccagtg tctttgtcac caggcccttt ggactctacc
tcagaaatat ttcttggacc ttccacttct cctccaactc cttgaccacc atcctgtatc
caaccatcac cacctctaac ctgaatccta ccttaagatc agaacagttg tcctcacttt
tgttcttgtc cctctccaac ccactctcca caagatggcc agagtaatgt ttttaatata
aattggatcc ttcagtttcc tgcttaaaac cctgcaggtt tcccaatgca ctcagaaaga
aatccagttt ccatggccct ggatggtctg gcccacctcc agcctcagct agcattaccc
ttctgacact ctctatgtag cctccctgat cttctttcag ctcctctatt aaaggaaaag
ttctttatgt taattattta catcttcctg caggcccttc ctctgcctgc tggggtcctc
ctattcttta ggtttaattt taaatatgtc acctcctaag agaaaccttc ccagaccact
ctttctaaaa tgaatcttct aggctgggca tggtggctca cacctgtaat ccctgtactt
tgggaggcca aggggggaga tcacttgagg tcaggagttc aagaccagcc tggccaactt
ggtgaaaccc cgtctttact aaaaatacaa aaaaattagc caggcgtggt ggtgcacccc
taaaatccca gctacttgag agactgaggc aggagaatcg cttgaaccca ggaggtggag
gttccagtga gccaaaatca tgccaatgta ttccagtctg ggtgacagag tgagactctg
ttgtctgcca cgtgacggat ttctacccag gcagcattca agtccgatgg ttcctgaatg
gacaggagga aacagctggg gtcgtgtcca ccaacctgat ccgtaatgga gactggacct
tccagatcct ggtgatgctg gaaatgaccc cccagcaggg agatgtctac acctgccaag
tggagcacac cagcctggat agtcctgtca ccgtggagtg gaaggcacag tctgattctg
cccggagtaa gacattgacg ggagctgggg gcttcgtgct ggggctcatc atctgtggag
tgggcatctt catgcacagg aggagcaaga aagttcaacg aggatctgca taaacagggt
tcctgagctc actgaaaaga ctattgtgcc ttaggaaaag catttgctgt gtttcgttag
catctggctc caggacagac cttcaacttc caaattggat actgctgcca agaagttgct
ctgaagtcag tttctatcat tctgctcttt gattcaaagc actgtttctc tcactgggcc
tccaaccatg ttcccttctt cttagcacca caaataatca aaacccaaca tgactgtttg
cagggacggc aggaatgcta cgcgtttaat gggacacagc gcttcctgga gagatacatc
tacaaccggg aggagctcgt gcgcttcgac agcgacgtgg gggagttccg ggcggtgacg
gagctggggc ggcctgaggc ggagtactgg aacagccaga aggacatcct ggaggaggag
cgggcagtgc cggacaggat gtgcagacac aactacgagc tgggcgggcc catgaccctg
cagcgccgag tccagcctag ggtgaatgtt tccccctcca agaaggggcc cttgcagcac
cacaacctgc ttgtctgcca cgtgacggat ttctacccag gcagcattca agtccgatgg
ttcctgaatg gacaggagga aacagctggg gtcgtgtcca ccaacctgat ccgtaatgga
gactggacct tccagatcct ggtgatgctg gaaatgaccc cccagcaggg agatgtctac
acctgccaag tggagcacac cagcctggat agtcctgtca ccgtggagtg gaaggcacag
tctgattctg cccggagtaa gacattgacg ggagctgggg gcttcgtgct ggggctcatc
atctgtggag tgggcatctt catgcacagg aggagcaaga aagttcaacg aggatctgca
taaacagggt tcctgagctc actgaaaaga ctattgtgcc ttaggaaaag catttgctgt
gtttcgttag catctggctc caggacagac cttcaacttc caaattggat actgctgcca
agaagttgct ctgaagtcag tttctatcat tctgctcttt gattcaaagc actgtttctc
atctttctca ttacaatagg cacagagaag aatgcaacag ggcacagggg aagagatgct
aaatatacca agaatctgtg gaaatataag ctggggcaaa tcagtgtaat ccttgacttt
gctcctcacc atcagggcaa acttgccttc ttccctccta agctccagta aataaacaga
acagctttca ccaaagtggg tagtatagtc ctcaaatatc ggataaatat atgcgttttt
gtaccccaga aaaacttttc ctccctcttc atcaacatag taaaataagt caaacaaaat
gagaacacca aattttgggg gaataaattt ttatttaaca ctgcaaagga aagagagaga
aaacaagcaa agataggtag gacagaaagg aagacagcca gatccagtga ttgacttggc
atgaaaatga gaaaatgcag acagacctca acattcaaca acatccatac agcactgctg
gaggaagagg aagatttgtg cagaccaaga gcaccacaga ctacaactgc ccagcttcat
ctaaatactt gttaacctct ttggtcattt ctctttttaa ataaatgccc atagcagtat
tcagtatgag gacactggag atctaaaagt gaataatggc acaagaagaa atataagtaa
taaggacctt tgtgcttcga gatctgcaga ccagacagta ctatgccaat cagaatggct
caaataccaa gggaagtgtt attggttctc taatgagatg aaaagctgga gtgacagtta
tgtgtattgt ttggaaagaa aatctcatct actaatcata catgaccaac ttgaaatggc
ttttatacag aaaaacctaa gacaattaaa ctacgtatgg attgggctta actttacctc
cttgaaaatg acatggactt gggtggatgg ttctccaata gattcaaaga tattcttcat
aaagggacca gctaaagaaa acagctgtgc tgccattaag gaaagcaaaa ttttctctga
aacctgcagc agtgttttca aatggatttg tcagtattag agtttgacaa aattcacagt
gaaataatca atgatcacta tttttggcct attagtttct aatattaatc tccaggtgta
agattttaaa gtgcaattaa atgccaaaat ctcttctccc ttctccctcc atcatcgaca
ctggtctagc ctcagagtaa cccctgttaa caaactaaaa tgtacacttc aaaattttta
cgtgatagta taaaccaatg tgacttcatg tgatcatatc caggattttt attcgtcgct
tattttatgc caaatgtgat caaattatgc ctgtttttct gtatcttgcg ttttaaattc
ttaataaggt cctaaacaaa atttcttata tttctaatgg ttgaattata atgtgggttt
atacattttt tacccttttg tcaaagagaa ttaactttgt ttccaggctt ttgctactct
tcactcagct acaataaaca tcctgaatgt tttcttaaaa aaaaaaaaaa aaaaaaaaaa
ctctgaaaat taagcatctg aagaccgatg atcaggatat ctacaaggta tcaatatatg
atacaaaagg aaaaaatgtg ttggaaaaaa tatttgattt gaagattcaa gagagggtct
caaaaccaaa gatctcctgg acttgtatca acacaaccct gacctgtgag gtaatgaatg
gaactgaccc cgaattaaac ctgtatcaag atgggaaaca tctaaaactt tctcagaggg
tcatcacaca caagtggacc accagcctga gtgcaaaatt caagtgcaca gcagggaaca
aagtcagcaa ggaatccagt gtcgagcctg tcagctgtcc agagaaaggt ctggacatct
atctcatcat tggcatatgt ggaggaggca gcctcttgat ggtctttgtg gcactgctcg
ttttctatat caccaaaagg aaaaaacaga ggagtcggag aaatgatgag gagctggaga
caagagccca cagagtagct actgaagaaa ggggccggaa gccccaccaa attccagctt
caacccctca gaatccagca acttcccaac atcctcctcc accacctggt catcgttccc
aggcacctag tcatcgtccc ccgcctcctg gacaccgtgt tcagcaccag cctcagaaga
ggcctcctgc tccgtcgggc acacaagttc accagcagaa aggcccgccc ctccccagac
ctcgagttca gccaaaacct ccccatgggg cagcagaaaa ctcattgtcc ccttcctcta
attaaaaaag atagaaactg tctttttcaa taaaaagcac tgtggatttc tgccctcctg
atgtgcatat ccgtacttcc atgaggtgtt ttctgtgtgc agaacattgt cacctcctga
ggctgtgggc cacagccacc tctgcatctt cgaactcagc catgtggtca acatctggag
tttttggtct cctcagagag ctccatcaca ccagtaagga gaagcaatat aagtgtgatt
gcaagaatgg tagaggaccg agcacagaaa tcttagagat ttcttgtccc ctctcaggtc
atgtgtagat gcgataaatc aagtgattgg tgtgcctggg tctcactaca agcagcctat
ctgcttaaga gactctggag tttcttatgt gccctggtgg acacttgccc accatcctgt
gagtaaaagt gaaataaaag ctttgactag aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
ggaacttgag gttaagaaca tggacatgaa gccggggtca accctgaaga tcacaggcag
catcgccgat ggcactgatg gctttgtaat taatctgggc caggggacag acaagctgaa
cctgcatttc aaccctcgct tcagcgaatc caccattgtc tgcaactcat tggacggcag
caactggggg caagaacaac gggaagatca cctgtgcttc agcccagggt cagaggtcaa
gttcacagtg acctttgaga gtgacaaatt caaggtgaag ctgccagatg ggcacgagct
gacttttccc aacaggctgg gtcacagcca cctgagctac ctgagcgtaa ggggcgggtt
caacatgtcc tctttcaagt taaaagaata aaagacttcc agccgagaaa aaaaaaaaaa
tcagaagaag ggcgacaagg ctcccggggg cgggggcgcc aatctcaagg gcgaccggtc
gcgactgctc cgggacctgc gcgtggacac caagtcgcgg gcagcgtggg ctcgccttct
gcaagagcac cccaacgcgc gcaaatacaa aggagccaac aagaagggct tgtccaaggg
ctgcttcggc ctcaagctgg accgaatcgg ctccatgagc ggcctgggat gttagtgcgg
cgccccctgg cggcggatcg ggaactggct ccgttgtgct gaggtcatct ttggtcatca
gcctccagca tctggaaaca cctccaacgc aatgtggctt ttacatttct ttctttcttt
actcggcagc tccaagtgga atccacgtgc agcttctagt ctgggaaagt cacccaacct
agcagttgtc atgtgggtaa cctcaggcac ctctaagcct gtcctggaag aaggaccagc
agcccctcca gaactctgcc caggacagca ggtgcctgct ggctctgggt ttggaagttg
gggtgggtag ggggtggtaa gtactatata tggctctgga aaaccagctg ctacttccaa
atctattgtc cataatggtt tctttctgag gttgcttctt ggcctcagag gaccccaggg
gatgtttgga aatagcctct ctacccttct ggagcatggt ttacaaaagc cagctgactt
ctggaattgt ctatggagga cagtttgggt gtaggttact gatgtctcaa ctgaatagct
tgtgttttat aagctgctgt tggctattat gctgggggag tctttttttt ttatattgta
tttttgtatg ccttttgcaa agtggtgtta actgtttttg tacaaggaaa aaaactcttg
gggcaatttc ctgttgcaag ggtctgattt attttgaaag gcaagttcac ctgaaatttt
gtatttagtt gtgattactg attgcctgat tttaaaatgt tgccttctgg gacatcttct
gaggtcaggc attggctctt ctcaagctct tgggccatct ccgcctctct ttggctgaag
ctgcccgtgt agtccccaac cgtgtctgtc tggcacgtgg gtgtgttggt aaacagtttg
gaaaagtggc gtgggagcca gcctcccttt gatgattatt ggagccccag gggacaaggg
atttgaggtg agggttggcg cttagagagg acaatactgg ggttggactg taagggattg
aagggggtac cttaagagac actccaaacc tgaagttttt ttgctgctgc ctctttccct
aggaaactca cactccccta gggggagaag aagccgagag ccttttgtgc aaagccaaaa
ccttcgtcct tttaaaaacc taggtctcca gttggcttta ctttaaaatg ccaataataa
atgccctctt ctcgtgcctc cccaccacca cttaccactc gtgcatccct gagacaggga
gggaagaatg aacactcccc attaacagat ggaaaaactg aggcttagag atagacaatc
actacaagtc agctccagct ttctgccatc tagccagccc ctcttcccca atgctccatc
ccaaccaggc acctcttcct tgatgtttgg ggtctttgtg gtagcttatc ttagaagcac
tacaccttgc cttgctgttt gtcctgagat ggaaaagtgt ccttcttgct ccccctcaat
agatctccag cgtcagctgc tccctggcat tcaacaaata ttcactggcc cctactttgt
ggcaatctgt gggctacatg ctggggtcaa ggcagtagaa ctccaggccc tcctctccca
tccttgatgc aagtgcaacc tcgctgaggg cagactgggg catcctgtgc cactaaacta
cattgttctt attctggcat cttagacctc cacacccgtg agaaatcctg gagagggtat
ttttgtagag tgtagactgt ggctagtgac aaataaatta ggaccaagaa agctcactgt
agcttttagg aataactttt acacgaccat ttgataggga actggggaat ggggtatgga
agttttccta cacttgagag aaaaaatagg ataacaaaaa ttaaaagtct tttttttcct
ggtccactgt gttaaggtca tttttaacca gcttgctttc tacaccaaga gtttatgttt
gtttaatggc tggaaagaga atcttgagat caaaaaacca ataaagatgt atctctacaa
cagagcaact ccacaccggg tgcatgccac agcaactcca tcttaaatag gagctggtaa
aacgaggctg atacctactg ggctgcattc ccagacggca tagcgaggag gtgctgaaga
gcgcaggttt ggagaatgat cacctggatt ggaaccatag ctctaccaat atggaaccca
gctccttagg cctcggtctt ctcatggaga acatggtgtg ataatcctac tcctctggga
gggtggctgt taagccttgg accgcagttg ccggccagga atcccagtgt cacggtggac
acgcctccct cgcgcccttg ccgcccacct gctcacccag ctcaggggct ttggaattct
gtggccacac tgcgaggaga tcggttctgg gtcggaggct acaggaagac tcccactccc
tgaaatctgg agtgaagaac gccgccatcc agccaccatt ccaaggaggt gcaggagaac
agctctgtga taccatttaa cttgttgaca ttacttttat ttgaaggaac gtatattaga
gcttactttg caaagaagga agatggttgt ttccgaagtg gacatcgcaa aagctgatcc
agctgctgca tcccaccctc tattactgaa tggagatgct actgtggccc agaaaaatcc
aggctcggtg gctgagaaca acctgtgcag ccagtatgag gagaaggtgc gcccctgcat
cgacctcatt gactccctgc gggctctagg tgtggagcag gacctggccc tgccagccat
cgccgtcatc ggggaccaga gctcgggcaa gagctccgtg ttggaggcac tgtcaggagt
tgcccttccc agaggcagcg ggatcgtgac cagatgcccg ctggtgctga aactgaagaa
acttgtgaac gaagataagt ggagaggcaa ggtcagttac caggactacg agattgagat
ttcggatgct tcagaggtag aaaaggaaat taataaagcc cagaatgcca tcgccgggga
aggaatggga atcagtcatg agctaatcac cctggagatc agctcccgag atgtcccgga
tctgactcta atagaccttc ctggcataac cagagtggct gtgggcaatc agcctgctga
cattgggtat aagatcaaga cactcatcaa gaagtacatc cagaggcagg agacaatcag
cctggtggtg gtccccagta atgtggacat cgccaccaca gaggctctca gcatggccca
ggaggtggac cccgagggag acaggaccat cggaatcttg acgaagcctg atctggtgga
caaaggaact gaagacaagg ttgtggacgt ggtgcggaac ctcgtgttcc acctgaagaa
gggttacatg attgtcaagt gccggggcca gcaggagatc caggaccagc tgagcctgtc
cgaagccctg cagagagaga agatcttctt tgagaaccac ccatatttca gggatctgct
ggaggaagga aaggccacgg ttccctgcct ggcagaaaaa cttaccagcg agctcatcac
acatatctgt aaatctctgc ccctgttaga aaatcaaatc aaggagactc accagagaat
aacagaggag ctacaaaagt atggtgtcga cataccggaa gacgaaaatg aaaaaatgtt
cttcctgata gataaagtta atgcctttaa tcaggacatc actgctctca tgcaaggaga
ggaaactgta ggggaggaag acattcggct gtttaccaga ctccgacacg agttccacaa
atggagtaca ataattgaaa acaattttca agaaggccat aaaattttga gtagaaaaat
ccagaaattt gaaaatcagt atcgtggtag agagctgcca ggctttgtga attacaggac
atttgagaca atcgtgaaac agcaaatcaa ggcactggaa gagccggctg tggatatgct
acacaccgtg acggatatgg tccggcttgc tttcacagat gtttcgataa aaaattttga
agagtttttt aacctccaca gaaccgccaa gtccaaaatt gaagacatta gagcagaaca
agagagagaa ggtgagaagc tgatccgcct ccacttccag atggaacaga ttgtctactg
ccaggaccag gtatacaggg gtgcattgca gaaggtcaga gagaaggagc tggaagaaga
aaagaagaag aaatcctggg attttggggc tttccagtcc agctcggcaa cagactcttc
catggaggag atctttcagc acctgatggc ctatcaccag gaggccagca agcgcatctc
cagccacatc cctttgatca tccagttctt catgctccag acgtacggcc agcagcttca
gaaggccatg ctgcagctcc tgcaggacaa ggacacctac agctggctcc tgaaggagcg
gagcgacacc agcgacaagc ggaagttcct gaaggagcgg cttgcacggc tgacgcaggc
tcggcgccgg cttgcccagt tccccggtta accacactct gtccagcccc gtagacgtgc
acgcacactg tctgcccccg ttcccgggta gccactggac tgacgacttg agtgctcagt
agtcagactg gatagtccgt ctctgcttat ccgttagccg tggtgattta gcaggaagct
gtgagagcag tttggtttct agcatgaaga cagagcccca ccctcagatg cacatgagct
ggcgggattg aaggatgctg tcttcgtact gggaaaggga ttttcagccc tcagaatcgc
tccaccttgc agctctcccc ttctctgtat tcctagaaac tgacacatgc tgaacatcac
agcttatttc ctcattttta taatgtccct tcacaaaccc agtgttttag gagcatgagt
gccgtgtgtg tgcgtcctgt cggagccctg tctcctctct ctgtaataaa ctcatttcta
acctgaactt acacaaattt gcctgtttct gcttgctctt gtcctagctt gggaggcttc
ccctcactat cctaccccac ccgctccttg aagggcccag attctaccac acagcagcag
ttacaaaaac cttccccagg ctggacgtgg tggctcacgc ctgtaatccc agcactttgg
gaggccaagg tgggtggatc acttgaggtc aggagttcga gaccagcctg gccaacatga
tgaaacccca tctctactaa aaatacaaaa aattagccgg gcgtggtagc gggcgcctgt
agtcccagct actcgggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt
gcagtgagcc gagatcgcgc cactgcactc cagcctgggc gacagagcga gactccgtct
caaaaaaaaa aaaaaaaaaa aaaatacaaa aattagccgg gcgtggtggc ccacgcctgt
aatcccagct actcgggagg ctaaggcagg aaaattgttt gaacccagga ggtggaggct
gcagtgagct gagattgtgc cacttcactc cagcctgggt gacaaagtga gactccgtca
caacaacaac aacaaaaagc ttccccaact aaagcctaga agagcttctg aggcgctgct
ttgtcaaaag gaagtctcta ggttctgagc tctggctttg ccttggcttt gccagggctc
tgtgaccagg aaggaagtca gcatgcctct agaggcaagg aggggaggaa cactgcactc
ttaagcttcc gccgtctcaa cccctcacag gagcttactg gcaaacatga aaaatcggct
taccattaaa gttctcaatg caaccataaa aaaaaaa
cagactctgc gcttgagatc ttcaaacaag catcagcgtt ttccagggct tcccagaggt
ctgtgcgact agcccctgtc tatcaaaagt tattagagag gatgaagcat tagcttgaag
cactacagga ggaatgcacc acggcagctc tccgccaatt tctctcagat ttccacagag
actgtttgaa tgttttcaaa accaagtatc acactttaat gtacatgggc cgcaccataa
tgagatgtga gccttgtgca tgtgggggag gagggagaga gatgtacttt ttaaatcatg
ttccccctaa acatggctgt taacccactg catgcagaaa cttggatgtc actgcctgac
attcacttcc agagaggacc tatcccaaat gtggaattga ctgcctatgc caagtccctg
gaaaaggagc ttcagtattg tggggctcat aaaacatgaa tcaagcaatc cagcctcatg
ggaagtcctg gcacagtttt tgtaaagccc ttgcacagct ggagaaatgg catcattata
agctatgagt tgaaatgttc tgtcaaatgt gtctcacatc tacacgtggc ttggaggctt
ttatggggcc ctgtccaggt agaaaagaaa tggtatgtag agcttagatt tccctattgt
gacagagcca tggtgtgttt gtaataataa aaccaaagaa acata
gacattcttt tggtggagca acggttattc agactcttag tgaagatcag agattcagat
gtggtattgc cctggatgca tggatgtttc cactgggtga tgaagtatat tccagaattc
ctcagcccct cttttttatc aactctgaat atttccaata tcctgctaat atcataaaaa
tgaaaaaatg ctactcacct gataaagaaa gaaagatgat tacaatcagg ggttcagtcc
accagaattt tgctgacttc acttttgcaa ctggcaaaat aattggacac atgctcaaat
taaagggaga catagattca aatgtagcta ttgatcttag caacaaagct tcattagcat
tcttacaaaa gcatttagga cttcataaag attttgatca gtgggactgc ttgattgaag
gagatgatga gaatcttatt ccagggacca acattaacac aaccaatcaa cacatcatgt
tacagaactc ttcaggaata gagaaataca attaggatta aaataggttt tttaaaagtc
ttgtttcaaa actgtctaaa attatgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgagag
agagagagag agagagagag agagagagag agaattttaa tgtattttcc caaaggactc
atattttaaa atgtaggcta tactgtaatc gtgattgaag cttggactaa gaattttttc
cctttagatg taaagaaaga atacagtata caatattcaa aaaaaaaaaa aaaaaaaaaa
tcactcaacg ggcactgtgg gcctgtggcc ttcctggctg tggctaccag catcctggcc
cctgacatcc tgcggagtga ccaggaggag gctgaggggc cccgggctga ggaggacaag
ccagacgggc aggcacacga gcccatgccc gatagccacg tgggccgaga gctgacccgc
aagaagggca tcctcttgca gtaccgcctg cgctccaccg cacacctccc gggcccgctg
ctctccatgc gggagccggc gcctgctgat ggcgcagctt tggagcacag cgaggaggac
ggctccattt acgagatggc cgacgacccc gacatctggg tgcgcagccg gccctgcgcc
cgcgacgccc accgcaagga gatttgctct gtggccatca tctccggcgg gcagggctac
cgcaactttg gcagcgctct gggcagcagt gggaggcagg ccccgtgtgg ggagacggac
agcaccctcc tcatctggca ggtgcccttg atgctatagc gcctcccctc tcccctcaga
gggcacagct gcaggcctga ccaaggccac gcccggctct cgtgctctag gacctgcacg
ggacttgtgg atgggcctgg actctccaga aactacttgg gcagagcaaa ggaaaacctc
ttgttttaaa aaaatttttt tcagagtgtt ttggggagga gttttagggc ttggggagag
ggaggacaca tctggaggaa atggccttct ttttaaaagc aaaaaacaca aaacctcaca
actgcctggc aagcccagta tcacttgttt gggccctagc gggactccaa ggcagccaca
cgcccctcct ggaagggtgt gtgcgtgtga gtgtgtgcga gtgtgtgggc tggtgtgtga
atatctataa ataagtatat atggtgtata ttatatgtgt ataaataaag tctgtacata
ttggagctct gggagatgct ggaataaaag acaagagtta catctggact tggaaaaaaa
aacaatgcca ccattcccac aggagtggta cttaaaccag acagcagggt tcagaggtgg
cacaccggga caaagctgag gccctgcacc tcaacagctg actgccaggt gcctgtgggt
gaactgaggg gagtagaggg agagggcagg tggaactggg gcagaatcta gtcatgccct
aaagctagtc ctgtaaacaa tggtgcccca gaaagctgca ggtggtgttt ggagaagcag
ttacttttca gttacaagac ccatctccct agtctcagcc ttacaacacc acgggactaa
ggaagagcac ttccttgcct ccgtaaggcc agaggaagaa ccatcccaat catttgatct
ccagctccac agtagagaga aacctacaaa atgtcaaacc agcttcccga ctcccaggag
ctcaagccaa gcccagaggc agtggctggg gtccctgcag gtcatgaggg gcctatgcct
ttactccttt taaacaccag cacccgtctt ttccccaacc taaaaccaac caccagcatt
tcactacagg accaaatgga aaccgaggga accctgggtc ttgggaagaa caacaggaaa
ccaaggtctg acctagggtt ccctcccagt cttcacatca ctctggcctc atcaccaagg
tgacagagga cacaggggag ggggaaaacc cacacacact ccttggaatg ggtcctgtta
tttatgcttg ctgcacagac atattagaag aaaaaaaaaa gctttgtatt attcttccac
atatgctggc tgctgtttac acaccctgcc aatgccttag cactggagag ctttttgcaa
agctaaccca taaactgctg gggcaaaaat catcttggaa ggctctgaac ctcagaaagg
attcacaaga cgatctgccg accctctggg agaaaatcca gcaagatgca agccttcaga
atctgggatg ttaaccagaa gaccttctat ctgaggaaca accaactagt tgctggatac
ttgcaaggac caaatgtcaa tttagaagaa aagatagatg tggtacccat tgagcctcat
gctctgttct tgggaatcca tggagggaag atgtgcctgt cctgtgtcaa gtctggtgat
gagaccagac tccagctgga ggcagttaac atcactgacc tgagcgagaa cagaaagcag
gacaagcgct tcgccttcat ccgctcagac agtggcccca ccaccagttt tgagtctgcc
gcctgccccg gttggttcct ctgcacagcg atggaagctg accagcccgt cagcctcacc
aatatgcctg acgaaggcgt catggtcacc aaattctact tccaggagga cgagtagtac
tgcccaggcc tgcctgttcc cattcttgca tggcaaggac tgcagggact gccagtcccc
ctgccccagg gctcccggct atgggggcac tgaggaccag ccattgaggg gtggaccctc
agaaggcgtc acaacaacct ggtcacagga ctctgcctcc tcttcaactg accagcctcc
atgctgcctc cagaatggtc tttctaatgt gtgaatcaga gcacagcagc ccctgcacaa
agcccttcca tgtcgcctct gcattcagga tcaaaccccg accacctgcc caacctgctc
tcctcttgcc actgcctctt cctccctcat tccaccttcc catgccctgg atccatcagg
ccacttgatg acccccaacc aagtggctcc cacaccctgt tttacaaaaa agaaaagacc
agtccatgag ggaggttttt aagggtttgt ggaaaatgaa aattaggatt tcatgatttt
tttttttcag tccccgtgaa ggagagccct tcatttggag attatgttct ttcggggaga
ggctgaggac ttaaaatatt cctgcatttg tgaaatgatg gtgaaagtaa gtggtagctt
ttcccttctt tttcttcttt ttttgtgatg tcccaacttg taaaaattaa aagttatggt
actatgttag ccccataatt ttttttttcc ttttaaaaca cttccataat ctggactcct
ctgtccaggc actgctgccc agcctccaag ctccatctcc actccagatt ttttacagct
gcctgcagta ctttacctcc tatcagaagt ttctcagctc ccaaggctct gagcaaatgt
ggctcctggg ggttctttct tcctctgctg aaggaataaa ttgctccttg acattgtaga
gcttctggca cttggagact tgtatgaaag atggctgtgc ctctgcctgt ctcccccacc
gggctgggag ctctgcagag caggaaacat gactcgtata tgtctcaggt ccctgcaggg
ccaagcacct agcctcgctc ttggcaggta ctcagcgaat gaatgctgta tatgttgggt
gcaaagttcc ctacttcctg tgacttcagc tctgttttac aataaaatct tgaaaatgcc
ccagggcagc cttcagtctg attcaggaga acgaggtcct cttcaccatg tgcttcatcc
ccctggtctg ctggatcgtg tgcactggac tgaaacagca gatggagagt ggcaagagcc
ttgcccagac atccaagacc accaccgcgg tgtacgtctt cttcctttcc agtttgctgc
agccccgggg agggagccag gagcacggcc tctgcgccca cctctggggg ctctgctctt
tggctgcaga tggaatctgg aaccagaaaa tcctgtttga ggagtccgac ctcaggaatc
atggactgca gaaggcggat gtgtctgctt tcctgaggat gaacctgttc caaaaggaag
tggactgcga gaagttctac agcttcatcc acatgacttt ccaggagttc tttgccgcca
tgtactacct gctggaagag gaaaaggaag gaaggacgaa cgttccaggg agtcgtttga
agcttcccag ccgagacgtg acagtccttc tggaaaacta tggcaaattc gaaaaggggt
atttgatttt tgttgtacgt ttcctctttg gcctggtaaa ccaggagagg acctcctact
tggagaagaa attaagttgc aagatctctc agcaaatcag gctggagctg ctgaaatgga
ttgaagtgaa agccaaagct aaaaagctgc agatccagcc cagccagctg gaattgttct
actgtttgta cgagatgcag gaggaggact tcgtgcaaag ggccatggac tatttcccca
agattgagat caatctctcc accagaatgg accacatggt ttcttccttt tgcattgaga
actgtcatcg ggtggagtca ctgtccctgg ggtttctcca taacatgccc aaggaggaag
aggaggagga aaaggaaggc cgacaccttg atatggtgca gtgtgtcctc ccaagctcct
ctcatgctgc ctgttctcat ggattggtga acagccacct cacttccagt ttttgccggg
gcctcttttc agttctgagc accagccaga gtctaactga attggacctc agtgacaatt
ctctggggga cccagggatg agagtgttgt gtgaaacgct ccagcatcct ggctgtaaca
ttcggagatt gtggttgggg cgctgtggcc tctcgcatga gtgctgcttc gacatctcct
tggtcctcag cagcaaccag aagctggtgg agctggacct gagtgacaac gccctcggtg
acttcggaat cagacttctg tgtgtgggac tgaagcacct gttgtgcaat ctgaagaagc
tctggttggt cagctgctgc ctcacatcag catgttgtca ggatcttgca tcagtattga
gcaccagcca ttccctgacc agactctatg tgggggagaa tgccttggga gactcaggag
tcgcaatttt atgtgaaaaa gccaagaatc cacagtgtaa cctgcagaaa ctggggttgg
tgaattctgg ccttacgtca gtctgttgtt cagctttgtc ctcggtactc agcactaatc
agaatctcac gcacctttac ctgcgaggca acactctcgg agacaagggg atcaaactac
tctgtgaggg actcttgcac cccgactgca agcttcaggt gttggaatta gacaactgca
acctcacgtc acactgctgc tgggatcttt ccacacttct gacctccagc cagagcctgc
gaaagctgag cctgggcaac aatgacctgg gcgacctggg ggtcatgatg ttctgtgaag
tgctgaaaca gcagagctgc ctcctgcaga acctggggtt gtctgaaatg tatttcaatt
atgagacaaa aagtgcgtta gaaacacttc aagaagaaaa gcctgagctg accgtcgtct
ttgagccttc ttggtaggag tggaaacggg gctgccagac gccagtgttc tccggtccct
ccagctgggg gccctcaggt ggagagagct gcgatccatc caggccaaga ccacagctct
gtgatccttc cggtggagtg tcggagaaga gagcttgccg acgatgcctt cctgtgcaga
gcttgggcat ctcctttacg ccagggtgag gaagacacca ggacaatgac agcatcgggt
gttgttgtca tcacagcgcc tcagttagag gatgttcctc ttggtgacct catgtaatta
gctcattcaa taaagcactt tctttatttt tctcttctct gtctaacttt ctttttccta
ccatggccaa gaaggacccc gagggcctct ttctgcagga caacatcgtc gcggagttct
ccgtggacga gaccggccag atgagcgcca cagccaaggg ccgagtccgt cttttgaata
actgggacgt gtgcgcagac atggtgggca ccttcacaga caccgaggac cctgccaagt
tcaagatgaa gtactggggc gtagcctcct ttctccagaa aggaaatgat gaccactgga
tcgtcgacac agactacgac acgtatgccg tgcagtactc ctgccgcctc ctgaacctcg
atggcacctg tgctgacagc tactccttcg tgttttcccg ggaccccaac ggcctgcccc
cagaagcgca gaagattgta aggcagcggc aggaggagct gtgcctggcc aggcagtaca
ggctgatcgt ccacaacggt tactgcgatg gcagatcaga aagaaacctt ttgtagcaat
atcaagaatc tagtttcatc tgagaacttc tgattagctc tcagtcttca gctctattta
tcttaggagt ttaatttgcc cttctctccc catcttccct cagttcccat aaaaccttca
ttacacataa agatacacgt gggggtcagt gaatctgctt gcctttcctg aaagtttctg
gcctgacaat atcgatgagg attttgatga ggaatcggtg aagatcgtcc gcttggtgaa
gaacaaggaa cccctgggtg ccaccatccg gcgggacgag cactcagggg ctgttgtggt
ggccaggatc atgcgaggag gcgcagcaga caggagcggc ctggtccacg ttggagatga
gctccgagaa gtgaacggga tcgcagtcct gcacaagcgg cccgacgaga tcagccagat
tctggcccag tcccagggat ccatcaccct aaaaatcatc ccagccaccc aggaggaaga
tcgcttaaag gagagcaagg tgttcatgcg cgccctcttc cactacaacc ctcgggagga
ccgggccatc ccttgccagg aggcgggcct gcccttccag cgcaggcagg tcctggaggt
ggtgagccag gacgacccca cgtggtggca ggccaagcga gtcggggaca ccaaccttcg
agccggcctc atcccctcca aggggttcca ggagagacga ctaagctacc ggagagccgc
gggcaccctg ccgagccccc agagcctcag gaagcccccc tatgatcagc cttgtgacaa
agagacctgt gactgtgagg gctacctcaa agggcactat gtggctggtc ttcggaggag
cttccggctg ggctgtaggg agagactggg tggctcgcag gaaggaaaga tgtcctccgg
agctgagtct ccggagctgc tgacttacga agaggtggcc aggtaccaac accagcccgg
agagcggccc cgcctggtgg ttctgatcgg gtctctggga gcccgactgc acgagctgaa
gcaaaaggtg gtggctgaga acccacagca ctttggcgtc gctgttccac ataccaccag
gccccgaaag agccatgaga aggaaggagt ggaatatcac tttgtgtcta agcaagcatt
tgaggccgac ttacatcaca acaagttcct ggaacatggt gaatataagg aaaatctgta
tggaaccagc ctggaggcca ttcaggctgt tatggccaaa aacaaagttt gtttggtgga
tgtggagcca gaagcactga aacaactgag gacctcagaa tttaaaccct atattatatt
tgtaaagcct gcaattcagg aaaaaagaaa aacgccacct atgtccccag cttgtgagga
cacagcagcc ccatttgatg agcagcagca agagatggcc gcttctgccg ccttcataga
ccggcattac gggcacctgg tagacgccgt gctggtgaag gaggatctcc agggtgccta
cagccagctc aaagtggtct tagagaagct gagcaaggac actcactggg tacctgttag
ttgggtcagg taactttatc ccagaacatc caagctggac gggaccttga agatcatcta
gtccagactc cctcatttta ccatcaagga atctcaagcg cagagaggga gagaattctc
cacaaattcc atcatcgaga agagtataag tgggaagtct tgtttgttgt tggtttttgt
ctgttgtttt tcactgcacc tctttggatc atgatttgaa aggggcatat cagaaaacaa
gaacaagcga gaagagaaga aggcccagaa ctctgaaatg agaatgaaga gagctcagga
gtatgacagt agttttccaa actgggaatt tgcccgaatg attaaagaat ttcgggctac
tttggaatgt catccactta ctatgactga tcctatcgaa gagcacagaa tatgtgtctg
tgttaggaaa cgcccactga ataagcaaga attggccaag aaagaaattg atgtgatttc
cattcctagc aagtgtctcc tcttggtaca tgaacccaag ttgaaagtgg acttaacaaa
gtatctggag aaccaagcat tctgctttga ctttgcattt gatgaaacag cttcgaatga
agttgtctac aggttcacag caaggccact ggtacagaca atctttgaag gtggaaaagc
aacttgtttt gcatatggcc agacaggaag tggcaagaca catactatgg gcggagacct
ctctgggaaa gcccagaatg catccaaagg gatctatgcc atggcctccc gggacgtctt
cctcctgaag aatcaaccct gctaccggaa gttgggcctg gaagtctatg tgacattctt
cgagatctac aatgggaagc tgtttgacct gctcaacaag aaggccaagc tgcgcgtgct
ggaggacggc aagcaacagg tgcaagtggt ggggctgcag gagcatctgg ttaactctgc
tgatgatgtc atcaagatga tcgacatggg cagcgcctgc agaacctctg ggcagacatt
tgccaactcc aattcctccc gctcccacgc gtgcttccaa attattcttc gagctaaagg
gagaatgcat ggcaagttct ctttggtaga tctggcaggg aatgagcgag gcgcggacac
ttccagtgct gaccggcaga cccgcatgga gggcgcagaa atcaacaaga gtctcttagc
cctgaaggag tgcatcaggg ccctgggaca gaacaaggct cacaccccgt tccgtgagag
caagctgaca caggtgctga gggactcctt cattggggag aactctagga cttgcatgat
tgccacgatc tcaccaggca taagctcctg tgaatatact ttaaacaccc tgagatatgc
agacagggtc aaggagctga gcccccacag tgggcccagt ggagagcagt tgattcaaat
ggaaacagaa gagatggaag cctgctctaa cggggcgctg attccaggca atttatccaa
ggaagaggag gaactgtctt cccagatgtc cagctttaac gaagccatga ctcagatcag
ggagctggag gagaaggcta tggaagagct caaggagatc atacagcaag gaccagactg
gcttgagctc tctgagatga ccgagcagcc agactatgac ctggagacct ttgtgaacaa
agcggaatct gctctggccc agcaagccaa gcatttctca gccctgcgag atgtcatcaa
ggccttgcgc ctggccatgc agctggaaga gcaggctagc agacaaataa gcagcaagaa
acggccccag tgacgactgc aaataaaaat ctgtttggtt tgacacccag cctcttccct
ggccctcccc agagaacttt gggtacctgg tgggtctagg cagggtctga gctgggacag
gttctggtaa atgccaagta tgggggcatc tgggcccagg gcagctgggg agggggtcag
agtgacatgg gacactcctt ttctgttcct cagttgtcgc cctcacgaga ggaaggagct
cttagttacc cttttgtgtt gcccttcttt ccatcaaggg gaatgttctc agcatagagc
tttctccgca gcatcctgcc tgcgtggact ggctgctaat ggagagctcc ctggggttgt
gctttatacc attcacatcc cagggctgtg tccagacagc acaaaacggc aaggagagcc
caagccccaa tgccagaatt cttccaaact ccctgactct ttgaagtttt tactcacccc
atttcaatta tcctgatccc ttctcatccc ctgcttggct tctctgcatg tggtcatctg
ctgtggcttg gtgtttaatg ggttaaaaat aagccactgc ctgacatccc aacatttgac
accccagcaa tgtgtgactc ccccaacatt ccactatgcc atcctgcagc tgaaatggga
acactggctg cctctccaaa cccgctcttg gacagaggat ctgggaggtg gaagccaggc
cagaggactt ggggaaaatg agatggagga aggaaaaagg gagaagctga gccacagctt
aactcctaca gagtgaaatg aaaacgggct gaaaatacca ccccaggaga ggacctcgcc
ccaagcaagc cagtgagcag ccctgccaga ctactgccag actgagaaac ccagaagctg
gtagtcatgt gggcttgcct tctctgccaa acgactggga aaccaaaatg agcccacctt
gtgttcttcc tagctccacc ctccccgtgc tgctgtgttc tgctcctccc cacgcttccc
tgctatagtt cccagctgct gtaacggagc cacctccaac tctaacaata aaccaagttc
attgcagata gtgta
agcagggcag cctggattgt tctgacactc gtggagaatt caatgttggc tccacctgcc
atttctcttg tgacaacggc tttaagctgg aggggcccaa taatgtggaa tgcacaactt
ctggaagatg gtcagctact ccaccaacct gcaaaggcat agcatcactt cctactccag
gggtgcaatg tccagccctc accactcctg ggcagggaac catgtactgt aggcatcatc
cgggaacctt tggttttaat accacttgtt actttggctg caacgctgga ttcacactca
taggagacag cactctcagc tgcagacctt caggacaatg gacagcagta actccagcat
gcagagctgt gaaatgctca gaactacatg ttaataagcc aatagcgatg aactgctcca
acctctgggg aaacttcagt tatggatcaa tctgctcttt ccattgtcta gagggccagt
tacttaatgg ctctgcacaa acagcatgcc aagagaatgg ccactggtca actaccgtgc
caacctgcca agcaggacca ttgactatcc aggaagccct gacttacttt ggtggagcgg
tggcttctac gataggtctg ataatgggtg ggacgctcct ggctttgcta agaaagcgtt
tcagacaaaa agatgatggg aaatgcccct tgaatcctca cagccaccta ggaacatatg
gagtttttac aaacgctgca tttgacccga gtccttaagg tttccataaa cacccatgaa
tcaaagacat ggaattacct tagattagct ctggaccagc ctgttggacc cgctctggac
caaccctgtt tcctgagttt gggattgtgg tacaatctca aattctcaac ctaccacccc
ttcctgtccc acctcttctc ttcctgtaac acaagccaca gaagccagga gcaaatgttt
ctgcagtagt ctctgtgctt tgactcacct gttacttgaa ataccagtga accaaagaga
ctggagcatc tgactcacaa gaagaccaga ctgtggagaa ataaaaatac ctctttattt
tttgattgaa ggaaggtttt ctccactttg ttggaaagca ggtggcatct ctaattggaa
gaaattcctg tagcatcttc tggagtctcc agtggttgct gttgatgagg cctcttggac
ctctgctctg aggcttccag agagtcctct ggatggcacc agaggctgca gaaggccaag
aatcaagcta gaaggccaca tgtcaccgtg gaccttcctg ccaccagtca ctgtccctca
aatgacccaa agaccaatat tcaaatgcgt aattaaaaga attttcccca aaaaaaaaaa
tatgtaccaa aacttggcaa gggtgatgta aaggataagt ttgaagccat gcagagagcc
agggaagaaa gaaatcaaag gagatctaga gacgaaaaac aaagaagaaa agaacaatat
attagagaga gagaatggaa caggagaaag caggagatta aagaaatgct tgcttctgat
gatgaggaag atgtatcttc taaagtagaa aaggcttatg ttccaaaatt aacaggaact
gtgaagggta gatttgctga aatggagaaa caaagacaag aggaacaaag gaagagaacg
gaggaggaac gaaaacgcag aattgagcag gatatgttag aaaagaggaa aatacagcgt
gaattagcaa aaagggctga acagattgag gacataaaca atacgggaac tgaatcagca
tcagaggaag gagatgattc actacttata actgtggtac ctgtcaaatc atataaaaca
tctggaaaaa tgaaaaagaa ttttgaggat ctagaaaaag aacgtgaaga gaaagaaagg
atcaagtacg aggaagataa aagaataaga tatgaagaac aacgaccatc tctcaaggaa
gcaaagtgtc tttcattagt tatggatgat gaaatagaaa gtgaagcaaa aaaagaatca
ctttctcccg gaaaattgaa actaactttt gaagaactgg agcgacaaag acaagaaaac
cgaaagaagc aagctgaaga ggaagcaaga aaacgtttag aagaagagaa gcgtgctttt
gaagaagcaa ggcggcaaat ggtaaatgaa gatgaggaaa accaagacac agcaaaaatt
tttaaagggt accgccctgg taaactcaaa ctcagttttg aagaaatgga aaggcaaaga
agagaagatg aaaaaaggaa agcagaagaa gaagccagaa ggagaataga ggaagaaaag
aaggcgtttg ctgaagcaag gagaaatatg gtagtagatg atgactcccc agagatgtat
aagacaatct ctcaagaatt tcttacaccg ggaaaactgg aaattaattt tgaagaatta
ttaaaacaaa aaatggaaga agaaaaacga cgaacagagg aggaacggaa gcataagcta
gaaatggaga aacaagaatt tgaacaactg agacaggaaa tgggagagga agaggaagaa
aatgaaacct ttggattgag cagagaatat gaagaactga tcaaattaaa aaggagtggc
tctattcaag ctaaaaacct aaaaagcaag tttgaaaaaa ttggacagtt gtctgaaaaa
gaaatacaga aaaaaataga agaagagcga gcaagaagga gagcaattga ccttgaaatt
aaagagcgag aagctgaaaa ttttcatgag gaagatgatg ttgatgttag gcctgcaaga
aaaagcgagg ctccatttac tcacaaagtg aatatgaaag ctagatttga acaaatggct
aaggcaagag aagaagaaga acaaagaaga attgaagaac aaaagttact acgcatgcag
tttgaacaaa gggaaattga tgcagcacta caaaagaaaa gagaagagga ggaggaggaa
gaaggtagca tcatgaatgg ctccactgct gaagatgaag agcaaaccag atcaggagct
ccatggttca agaagcctct taaaaacaca tcagttgtag acagtgagcc agtcagattt
acggttaaag taacaggaga acccaaacca gaaattacat ggtggtttga aggagaaata
ctgcaggatg gagaagacta tcaatatatt gaaaggggag aaacttactg cctttactta
ccagaaactt tcccagaaga tggaggagag tatatgtgta aagcagtcaa caataaagga
tctgcagcta gtacctgtat tcttaccatt gaaagtaaga attaatcact ctttttatct
agggctttga gagactcatc tgtaatcaga agaaggagaa tgagaccagg gtggtgctgt
gtgagctggg caaccccatg aagaagaacg cccagatagg aatcgcgatg ttggtgagcg
tggggaatct ggaagaggct ggggagtctg tgtccttcca gctgcagata cggagcaaga
acagccagaa tccaaacagc aagattgtgc tgctggacgt gccggtccgg gcagaggccc
aagtggagct gcgagggaac tcctttccag cctccctggt ggtggcagca gaagaaggtg
agagggagca gaacagcttg gacagctggg gacccaaagt ggagcacacc tatgagctcc
acaacaatgg ccctgggact gtgaatggtc ttcacctcag catccacctt ccgggacagt
cccagccctc cgacctgctc tacatcctgg atatacagcc ccaggggggc cttcagtgct
tcccacagcc tcctgtcaac cctctcaagg tggactgggg gctgcccatc cccagcccct
cccccattca cccggcccat cacaagcggg atcgcagaca gatcttcctg ccagagcccg
agcagccctc gaggcttcag gatccagttc tcgtaagctg cgactcggcg ccctgtactg
tggtgcagtg tgacctgcag gagatggcgc gcgggcagcg ggccatggtc acggtgctgg
ccttcctgtg gctgcccagc ctctaccaga ggcctctgga tcagtttgtg ctgcagtcgc
acgcatggtt caacgtgtcc tccctcccct atgcggtgcc cccgctcagc ctgccccgag
gggaagctca ggtgtggaca cagctgctcc gggccttgga ggagagggcc attccaatct
ggtgggtgct ggtgggtgtg ctgggtggcc tgctgctgct caccatcctg gtcctggcca
tgtggaaggt cggcttcttc aagcggaacc ggccacccct ggaagaagat gatgaagagg
gggagtgatg gtgcagccta cactattcta gcaggagggt tgggcgtgct acctgcaccg
atggcttcat tgacaaggag gacctgcacg acatgctggc ctcgctgggt ttcatccatg
aggaccacct ccgggagctg ctcaccacca tgggtgaccg cttcacagat gaggaagtgg
acgagatgta ccgggaggca cccattgata agaaaggcaa cttcaactac gtggagttca
cccgcatcct caaacatggc gccaaggata aagacgacta ggccacccca gccccctgac
accccagccc ccgccagtca cccctccccg cacacacccg tccataccag ctccctgccc
atgaccctcg ctcagggatc cccctttgag gggttagggt cccagttccc agtggaagaa
acaggccagg agaagtgcgt gccgagctga ggcagatgtt cccacagtga ccccagagcc
ctgggctata gtctctgacc cctccaagga aagaccacct tctggggaca tgggctggag
ggcaggacct agaggcacca agggaaggcc ccattccggg gctgttcccc gaggaggaag
ggaaggggct ctgtgtgccc cccaggagga agaggccctg agtcctggga tcagacaccc
cttcacgtgt atccccacac aaatgcaagc tcaccaaggt cccctctcag tccccttccc
tacaccctga ccggccactg ccgcacaccc acccagagca cgccacccgc catgggagtg
tgctcaggag tcgcgggcag cgtggacatc tgtcccagag ggggcagaat ctccaataga
ggactgagca ctgctaaaaa aaaaaaaaaa aaaa
gcctttgtcc cagaatgcct cctgcaggga ttcttcctgc ttagcttgag ggtgactatg
gagctgagca ggtgttcttc attacctcag tgagaagcca gctttcctca tcaggccatt
gtccctgaag agaagggcag ggctgaggcc tctcattcca gaggaaggga caccaagcct
tggctctacc ctgagttcat aaatttatgg ttctcaggcc tgactctcag cagctatggt
aggaactgct gggcttggca gcccgggtca tctgtacctc tgcctccttt cccctccctc
aggccgaagg aggagtcagg gagagctgaa ctattagagc tgcctgtgcc ttttgccatc
ccctcaaccc agctatggtt ctctcgcaag ggaagtcctt gcaagctaat tctttgacct
gttgggagtg aggatgtctg ggccactcag gggtcattca tggcctgggg gatgtaccag
catctcccag ttcataatca caacccttca gatttgcctt attggcagct ctactctgga
ggtttgttta gaagaagtgt gtcaccctta ggccagcacc atctctttac ctcctaattc
cacaccctca ctgctgtaga catttgctat gagctgggga tgtctctcat gaccaaatgc
ttttcctcaa agggagagag tgctattgta gagccagagg tctggcccta tgcttccggc
ctcctgtccc tcatccatag cacctccaca tacctggccc tgtgccttgg tgtgctgtat
ccatccatgg ggctgattgt atttaccttc tacctcttgg ctgccttgtg aaggaattat
tcccatgagt tggctgggaa taagtgccag gatggaatga tgggtcagtt gtatcagcac
gtgtggcctg ttcttctatg ggttggacaa cctcatttta actcagtctt taatctgaga
ggccacagtg caattttatt ttatttttct catgatgagg ttttcttaac ttaaaagaac
atgtatataa acatgcttgc attatatttg taaatttatg tgatggcaaa gaaggagagc
ataggaaacc acacagactt gggcagggta cagacactcc cacttggcat cattcacagc
aagtcactgg ccagtggctg gatctgtgag gggctctctc atgatagaag gctatgggga
tagatgtgtg gacacattgg acctttcctg aggaagaggg actgttcttt tgtcccagaa
aagcagtggc tccattggtg ttgacataca tccaacatta aaagccaccc ccaaatgccc
aagaaaaaaa gaaagactta tcaacatttg ttccatgagc agaaaactgg agctctggcc
tcagtgttac agctaaataa tctttaatta aggcaagtca ctttcttctt cttaaagctg
ttttctagtt tgagaaatga tgggatttta gcagccagtc ttgaaggtct ctttcagtat
caacattcta agatgctggg acttactgtg tcatcaaatg tgcggttaag attctctggg
atattgatac tgtttgtgtt tttagttggg agatctgaga gacctggctt tggcaagagc
agatgtcatt ccatatcacc tttctcaatg aaagtctcat tctatcctct ctccaaaccc
gttttccaac atttgttaat agttacgtct ctcctgatgt agcacttaag cttcatttag
ttattatttc tttcttcact ttgcacacat ttgcatccac atattaggga agaggaatcc
ataagtagct gaaatatcta ttctgtatta ttgtgttaac attgagaata agccttggaa
ttagatatgg ggcaatgact gagccctgtc tcacccatgg attactcctt actgtaggga
atggcagtat ggtagaggga taaatagggg gcggggaggg atagtcatgg atccaagaag
gctgtgttgg tgggccctac gaagatgctc agtgctcgag atcgccggga ccggcaccct
gaggaggggg tagttgcaga gctccagggc ttcgcggtgg acaaggcctt cctcacctcc
cacaagggca tcctgctgga aaccgagctg gccctgaccc tcatcatctt catctgcttc
acggcctcca tctctgccta catggccgcg gcgctactgg agttcttcat cacacttgcc
ttcctcttcc tctatgccac ccagtactac cagcgcttcg accgaattaa ctggccctgt
ctggtttttg gcatcatcct ggtttccatc tttgcctatg atgccttcaa gatctaccgg
actgagatgg cacccggggc cagccagggg gaccagcagt gactctgggg ctacctggct
cctaggccca gccagccaga gaggacagtg gagcccagac acgtctcctt gggattcact
agcccccagc ccgccaaacc ccaccccagc cctacacagc agtctggcct gagacgtcac
tggggactta tctgtggagc ctggtgctcc aggatgtggc ttctcatgaa gctctggcca
ccagccccac ctctgagcaa ggtccctctg cagcagaact tccaggacaa ccaattccag
gggaagtggt atgtggtagg cctggcaggg aatgcaattc tcagagaaga caaagacccg
caaaagatgt atgccaccat ctatgagctg aaagaagaca agagctacaa tgtcacctcc
gtcctgttta ggaaaaagaa gtgtgactac tggatcagga cttttgttcc aggttgccag
cccggcgagt tcacgctggg caacattaag agttaccctg gattaacgag ttacctcgtc
cgagtggtga gcaccaacta caaccagcat gctatggtgt tcttcaagaa agtttctcaa
aacagggagt acttcaagat caccctctac gggagaacca aggagctgac ttcggaacta
aaggagaact tcatccgctt ctccaaatct ctgggcctcc ctgaaaacca catcgtcttc
cctgtcccaa tcgaccagtg tatcgacggc tgagtgcaca ggtgccgcca gctgccgcac
cctctatttc aggacttgaa tggacaaagt ctttttcatc agacatcaga aggagacttg
gacgatttgg ctcaggattt aaaggacttg taccataccc catcttttct gaacttttat
ccccttggtg aagatattga cattattttt aacttgaaaa gcaccttcac agaacctgtc
ctgtggagga aggaccaaca ccatcaccgc gtggagcagc tgaccctgaa tggcctcctg
caggctcttc agagcccctg catcattgaa ggggaatctg gcaaaggcaa gtccactctg
ctgcagcgaa ttgccatgct ctggggctcc ggaaagtgca aggctctgac caagttcaaa
ttcgtcttct tcctccgtct cagcagggcc cagggtggac tttttgaaac cctctgtgat
caactcctgg atatacctgg cacaatcagg aagcagacat tcatggccat gctgctgaag
ctgcggcaga gggttctttt ccttcttgat ggctacaatg aattcaagcc ccagaactgc
ccagaaatcg aagccctgat aaaggaaaac caccgcttca agaacatggt catcgtcacc
actaccactg agtgcctgag gcacatacgg cagtttggtg ccctgactgc tgaggtgggg
gatatgacag aagacagcgc ccaggctctc atccgagaag tgctgatcaa ggagcttgct
gaaggcttgt tgctccaaat tcagaaatcc aggtgcttga ggaatctcat gaagacccct
ctctttgtgg tcatcacttg tgcaatccag atgggtgaaa gtgagttcca ctctcacaca
agtctagaca gtgacttgta tgctgaactc cgctgcatgt gtataaagac aacctctgga
attcatccca aaaacatcca aagtttggaa gtgatcggga aaggaaccca ttgcaaccaa
gtcgaagtga tagccacact gaaggatggg aggaaaatct gcctggaccc agatgctccc
agaatcaaga aaattgtaca gaaaaaattg gcaggtgatg aatctgctga ttaatttgtt
ctgtttctgc caaacttctt taactcccag gaagggtaga attttgaaac cttgattttc
tagagttctc atttattcag gatacctatt cttactgtat taaaatttgg atatgtgttt
cattctgtct caaaaatcac attttattct gagaaggttg gttaaaagat ggcagaaaga
agatgaaaat aaataagcct ggtttcaacc ctctaattct tgcctaaaca ttggactgta
ctttgcattt ttttctttaa aaatttctat tctaacacaa cttggttgat ttttcctggt
ctactttatg gttattagac atactcatgg gtattattag atttcataat ggtcaatgat
aataggaatt acatggagcc caacagagaa tatttgctca atacattttt gttaatatat
ttaggaactt aatggagtct ctcagtgtct tagtcctagg atgtcttatt taaaatactc
cctgaaagtt tattctgatg tttattttag ccatcaaaca ctaaaataat aaattggtga
atatgaatct tataaactgt ggttagctgg tttaaagtga atatatttgc cactagtaga
acaaaaatag atgatgaaaa tgaattaaca tatctacata gttataattc tatcattaga
atgagcctta taaataagta caatatagga cttcaacctt actagactcc taattctaaa
gagcagcaga gtttcaggca tgaatccctc ctcagtggtc caccacgtca gtgactctgg
accggctgct gaattgcctt tggatgtacc acacattagg cttgactcac caccttcatt
tgacaatacc acctacacca gcctacctct tgattcccca tcaggaaaac cttcactccc
agctccatcc tcattgcccc ctctacctcc taaggtcctg gtctgctcca agcctgtgac
atatgccaca gtaatcttcc cgggagggaa caagggtgga gggacctcgt gtgggccagc
ccagaatcca cctaacaatc agactccatc cagctaagct gctcatcaca ctttaaactc
atgaggacca tccctagggg ttctgtgcat ccatccagcc agctcatgcc ctaggatcct
taggatatct gagcaaccag ggactttaag atctaatcca atgtcctaac tttactaggg
aaagtgacgc tcagacatga ctgagatgtc ttggggaaga cctccctgca cccaactccc
ccactggttc ttctaccatt acacactggg ctaaataaac cctaataatg atgtgcaaac
tcttaatggc tgaatgggaa aggaaactgc ccaagtttga ctaattgctt ggcctgtgaa
tggaaaagac tctggtctaa aaaaaaaaaa aaaaaa
gtgaagacca cctcccaggt ccgtcccagg cacatcacca gcctggaggt gatcaaggcc
ggaccccact gccccactgc ccaactgata gccacgctga agaatggaag gaaaatttgc
ttggacctgc aagccccgct gtacaagaaa ataattaaga aacttttgga gagttagcta
ctagctgcct acgtgtgtgc atttgctata tagcatactt cttttttcca gtttcaatct
aactgtgaaa gaacttctga tatttgtgtt atccttatga ttttaaataa acaaaataaa
tcaagttgta gtatagtcaa aatacttctt aataatagtg caaaaattgt gttgacacat
aacaatttca tggaagaaaa aaattccggt attttaagca aaaagtattt tgaaggaagg
tgtgaatact ggttatgctt ggtgttacat gttggctgat acatattcat gcatttacat
gattgcagta ctttatagct acatatttac cttgaccatt attattacct ttgccaataa
atatcagtaa cacagaaaaa aaaaaaaaaa aaaaaaaa
ctttgattct gctgatcctg tgcgtgggga tggttgtcgg gctggtggct ctggggattt
ggtctgtcat gcagcgcaat tacctacaag gtgagaatga aaatcgcaca ggaactctgc
aacaattagc aaagcgcttc tgtcaatatg tggtaaaaca atcagaacta aagggcactt
tcaaaggtca taaatgcagc ccctgtgaca caaactggag atattatgga gatagctgct
atgggttctt caggcacaac ttaacatggg aagagagtaa gcagtactgc actgacatga
atgctactct cctgaagatt gacaaccgga acattgtgga gtacatcaaa gccaggactc
atttaattcg ttgggtcgga ttatctcgcc agaagtcgaa tgaggtctgg aagtgggagg
atggctcggt tatctcagaa aatatgtttg agtttttgga agatggaaaa ggaaatatga
attgtgctta ttttcataat gggaaaatgc accctacctt ctgtgagaac aaacattatt
taatgtgtga gaggaaggct ggcatgacca aggtggacca actaccttaa tgcaaagagg
tggacaggat aacacagata agggctttat tgtacaataa aagatatgta tgaatgcatc
agtagctgaa aaaaaaaaaa aa
gttctcaggc ctctggtacg tggtctccat ggcatctgac tgcagggtct tcctgggcaa
gaaggaccac ctgtccatgt ccaccagggc catcaggccc acagaggagg gcggcctcca
cgtccacatg gagttcccgg gggcggacgg ctgtaaccag gtggatgccg agtacctgaa
ggtgggctcc gagggacact tcagagtccc ggccttgggc tacctggacg tgcgcatcgt
ggacacagac tacagctcct tcgccgtcct ttacatctac aaggagctgg agggggccct
cagcaccatg gtgcagctct acagccggac ccaggatgtg agtccccagg ctctgaagtc
cttccaggac ttctacccga ccctggggct ccccaaggac atgatggtca tgctgcccca
gtcagatgca tgcaaccctg agagcaagga ggcgccctga cacctccgga gccccacccc
cgcccttccc aggtggagcc aaagcagcag gcgcctttgc ccctggagtc aagacccaca
gccctcgggg accacctgga gtctctccat cctccacccc ccgcctgtgg gatgccttgt
gggacgtctc tttctattca ataaacagat gctgcagcct ca
cccgcaggga gattatgaca cgagcactgg caagttcacc tgcaaagtcc ccggcctcta
ctactttgtc taccacgcgt cgcatacagc caacctgtgc gtgctgctgt accgcagcgg
cgtcaaagtg gtcaccttct gtggccacac gtccaaaacc aatcaggtca actcgggcgg
tgtgctgctg aggttgcagg tgggcgagga ggtgtggctg gctgtcaatg actactacga
catggtgggc atccagggct ctgacagcgt cttctccggc ttcctgctct tccccgacta
gggcgggcag atgcgctcga gccccacggg ccttccacct ccctcagctt cctgcatgga
cccaccttac tggccagtct gcatccttgc ctagaccatt ctccccacca gatggacttc
tcctccaggg agcccaccct gacccacccc cactgcaccc cctccccatg ggttctctcc
ttcctctgaa cttctttagg agtcactgct tgtgtggttc ctgggacact taaccaatgc
cttctggtac tgccattctt
tttttttttt ttttcaagta ttggaagggg tggggagata
aagggagacc cagggattcc tgggaatcca ggaaaagtcg gccccaaggg ccccatgggc
cctaaaggtg gcccaggggc ccctggagcc ccaggcccca aaggtgaatc gggagactac
aaggccaccc agaaaatcgc cttctctgcc acaagaacca tcaacgtccc cctgcgccgg
gaccagacca tccgcttcga ccacgtgatc accaacatga acaacaatta tgagccccgc
agtggcaagt tcacctgcaa ggtgcccggt ctctactact tcacctacca cgccagctct
cgagggaacc tgtgcgtgaa cctcatgcgt ggccgggagc gtgcacagaa ggtggtcacc
ttctgtgact atgcctacaa caccttccag gtcaccaccg gtggcatggt cctcaagctg
gagcaggggg agaacgtctt cctgcaggcc accgacaaga actcactact gggcatggag
ggtgccaaca gcatcttttc cgggttcctg ctctttccag atatggaggc ctgacctgtg
ggctgcttca catccacccc ggctccccct gccagcaacg ctcactctac ccccaacacc
accccttgcc caaccaatgc acacagtagg
gcttggtgaa tgctgctgag tgaatgagta
gcctgtcacc accacattcc ctgtaaccac gggtttaaaa cccaccgtgg ccttgtgtca
acaaaagtgt agacggacgg ggactctgga gggcaattat tgttcaagtg actttgtatt
agccggcact gttatcacaa ccatcactcg cgatgggagt ttgcacgcca cagtctcgat
catcaacatc tacaaagagg gaaatttggc gattcagcag gcgggcaaga acatgagtgc
caggctgact gtcgtctgca agcagtgccc tctcctcaga agaggtctaa attacattat
tatgggccaa gtaggtgaag atgggcgagg caaaatcatg ccaaacagct ttatcatgat
gttcaagacc aagaatcaga agctcctgga tgccttaaaa aataagcaat gttaacagtg
aactgtgtcc atttaagctg tattctgcca ttgcctttga aagatctatg ttctctcagt
agaaaaaaaa atacttataa aattacatat tctgaaagag gattccgaaa gatgggactg
gttgactctt cacatgatgg aggtatgagg cctccgagat agctgaggga agttctttgc
ctgctgtcag aggagcagct atctgattgg aaacctgccg acttagtgcg gtgataggaa
gctaaaagtg tcaagcgttg acagcttgga agcgtttatt tatacatctc tgtaaaagga
tattttagaa ttgagttgtg tgaagatgtc aaaaaaagat tttagaagtg caatatttat
agtgttattt gtttcacctt caagcctttg ccctgaggtg ttacaatctt gtcttgcgtt
ttctaaatca atgcttaata aaatattttt aaaggattac agaacaacca aaaaaaaaaa
agcccgtggc atcccgggaa ttaaaggcac caagggcagc ccaggaaaca tcaaggacca
gccgaggcca gccttctccg ccattcggcg gaacccccca atggggggca acgtggtcat
cttcgacacg gtcatcacca accaggaaga accgtaccag aaccactccg gccgattcgt
ctgcactgta cccggctact actacttcac cttccaggtg ctgtcccagt gggaaatctg
cctgtccatc gtctcctcct caaggggcca ggtccgacgc tccctgggct tctgtgacac
caccaacaag gggctcttcc aggtggtgtc agggggcatg gtgcttcagc tgcagcaggg
tgaccaggtc tgggttgaaa aagaccccaa aaagggtcac atttaccagg gctctgaggc
cgacagcgtc ttcagcggct tcctcatctt cccatctgcc tgagccaggg aaggaccccc
tcccccaccc acctctctgg cttccatgct ccgcctgtaa aatgggggcg ctattgcttc
agctgctgaa gggagggggc tggctctgag agccccagga ctggctgccc cgtgacacat
gctctaagaa gctcgtttct tagacctctt cctggaataa acatctgtgt ctgtgtctgc
tgaacatgag cttcagttgc tactcggagc attgagaggg aggcctaaga ataataacaa
tccagtgctt aagagtca
caggaaccaa gtcacactca tcggcttcac cctaatgttt tggtgcgaat tcactgcctc
cttcctcctc ttcctgaacg ccatcagcgg ccttcacatc aacagcatca cccatccctg
ggaatgaccg tggaaatttt aggccccctc cagggacatc agattccaca agaaaatatg
gtcaaaatgg gacttttcca gcatgtggcc tctggtgggg ctgggttgga caagggcctt
gaaacggctg cctgtttgcc gataacttgt gggtggtcag ccagaaatgg cccgggggcc
tctgcacctg gtctgcaggg ccagaggcca ggagggtgcc tcagtgccac caactgcaca
ggcttagcca gatgttgatt ttagaggaag aaaaaaacat tttaaaactc cttcttgaat
tttcttccct ggactggaat acagttggaa gcacaggggt aactggtacc tgagctagct
gcacagccaa ggatagttca tgcctgtttc attgacacgt gctgggatag gggctgcaga
atccctgggg ctcccagggt tgttaagaat ggatcattct tccagctaag ggtccaatca
gtgcctagga ctttcttcca ccagctcaaa gggccttcgt atgtatgtcc ctggcttcag
ctttggtcat gccaaagagg cagagttcag gattccctca gaatgccctg cacacagtag
gtttccaaac catttgactc ggtttgcctc cctgcccgtt gtttaaacct tacaaaccct
ggataacccc atcttctagc agctggctgt gcctctggga gctctgccta tcagaaccct
accttaaggt gggtttcctt ccgagaagag ttcttgagca agctctccca ggagggccca
cctgactgct aatacacagc cctccccaag gcccgtgtgt gcatgtgtct gtcttttgtg
agggttagac agcctcaggg caccattttt aatcccagaa cacatttcaa agagcacgta
tctagacctg ctggactctg cagggggtga gggggaacag cgagagcttg ggtaatgatt
aacacccatg ctggggatgc atggaggtga agggggccag gaaccagtgg agatttccat
ccttgccagc acgtctgtac ttctgttcat taaagtgctc cctttctagt cctttttctg
cccagaa
acaagaccac cacttcgaaa cctgggattc aggaatgtgt ggcctgcaca gtgaagtgct
ggcaaccact aagaattcaa actggggcct ccagaactca ctggggccta cagctttgat
ccctgacatc tggaatctgg agaccaggga gcctttggtt ctggccagaa tgctgcagga
cttgagaaga cctcacctag aaattgacac aagtggacct taggccttcc tctctccaga
tgtttccaga cttccttgag acacggagcc cagccctccc catggagcca gctccctcta
tttatgtttg cacttgtgat tatttattat ttatttatta tttatttatt tacagatgaa
tgtatttatt tgggagaccg gggtatcctg ggggacccaa tgtaggagct gccttggctc
agacatgttt tccgtgaaaa cggagctgaa caataggctg ttcccatgta gccccctggc
ctctgtgcct tcttttgatt atgtttttta aaatatttat ctgattaagt tgtctaaaca
atgctgattt ggtgaccaac tgtcactcat tgctgagcct ctgctcccca ggggagttgt
gtctgtaatc gccctactat tcagtggcga gaaataaagt ttgcttagaa aagaaaaaaa
agtattccat gcatattaga tatcaccatg aagcaagaga catgcattct ataatcatgt
agacactcag actcagggga aaatacaagt tatatcctga aagcctttaa aactctatgg
taggatcaaa gattcaaatg gtttcagaga ggttttattt caattaattt gttctagtgc
tttcaagagc aagtacatca aaatgtagaa ggtaaaatgt atgcaacact aatataaatt
attccaagtc tttaaggagc caaagaaaaa aaagatttct cacagctttt tgttctgttt
tgtatttcaa ttaggaactt gcagtattat tttgaaaacc attctaaaat aataggagtt
aggaaataaa taaagttttg ctagccctgc taagttcagg cttagaggct tatcgctaag
tataaacttc accagattcc acgaaaagct ggatagcttt ttttctgact tatgttgtgg
ttgcacccct cacaaatggc agaacagtat gtaaagctgg taacacctcg gtttcagtgc
accatgtgtt tgctttgtga aggtgaagaa tatgttggtt tagagaaaga aattggatgt
aattttatgc aatttacttt taaagacaaa cataactatt tagcagagaa tattttaata
aatgcaaaac aacagctgga ctgctgtaca tcaaggacag attaactgga aaacatatgt
tccttatgtg tgatcgagag ccattcagaa aagacttcct ttgtgttcag cctatacttt
tccatatggt ataccttgaa aaaaattagc acaccatggt tatttttcta ccttttataa
aagacagagc ctgtttactc atttagaaga tagagaaaat tggtctaaaa ttgaacatcc
tagattcaca ctcccaagtc acttaaggtg atttgatggt gaggaaaatg attgacaaag
cccaacaatg atctcaggaa ttacattttc caacagacca aaaaatgttt tcatgtagca
gcaatgcaga tttggtgaat atttaatata tattttagta tgtatttcac tttatgactg
ttgattattg caggggagat ggaaaagaag caggttctgg cctatgctct agttcctcac
tttctggagt tatcttttgg gcctggagtc tcttggagtg aggaaaggaa gccattttta
tactttactt atggtagagt taaacttcgt actcttagga ggctacatgc aatctccaag
aggaagagag tgaagaggtt aagaattatg ccatcaaaat gtgctgctct ggcatattga
ctatttgaat tacagatatt ttaaaaacat cagatatgaa gaggtcactc tgatcttaat
tctgtgtctt aacagcagga gatgaaattc ccacatggga gatggcctcc ctgtcacaga
aggaatacca ttctccctat caatgatgag aaattggaac tgagagaatt ctgtatagac
cttgttcaag taattcttct attttaaacc tccccacata atttagctgc cttttcaaaa
ctactgtcct ttgtgcaacc agtatagaag cgattgactc tcactgcatc cttgggtctt
cttttcccta tatgggctct tttcccatat gggctcttat gtcctgtaaa acatgtatta
aataaatttg tatgcttttc tcccgttaac ctgtcttaca tcaatttaat tcttgggccc
aggtaggacc ctaagaggac agaggtcgag ttttgccacc tctgcaagag aaaagagaga
tgcccagacc accaggcctg ggaaatcttt gatttcccag gctttgaagt gggcactgtg
tctgaacctg gccctctgaa acttcctgtt cctggacatc ccagctgcta atgtgagttg
cccttgcact ggctcagggc tgggacatta aaggaatttc ccttttgttg acataacagt
aggatggcta tagctaacaa taatatatta tttagtttca agtagctaga aggaacatat
gaatgttccc aacacaaaga aatgacaact atttgagatg atggatatgc taataacctt
aatctgattc ctatacatta tatgcatcac atatacatac attatatgta taacatcact
atgtacccca taaatatgtg caattattgt caattaaaca aataaattta aacaaacatt
cacttggccc acgttgaact ccatggtgcc tgagagagac tagttaaggg ttggtcttct
gtatcctctg ctgttgagcc tctggtaagc tttcatctcc catgaactca tttccccata
aatgaaatgg gtaaataatg ccccatttgt agaagtgggc cctcatgact gaggtagctt
ccagataggc cagagtagag tgtagagtgt gccccgtgac atccctccat cttctcctcc
attatcatct agcagggtca gactgggaaa cctggttggc cacgccacac catgaccgag
gagccaactg ggacttctgg ctgtttgaca tcctcatgtt cccgttggtc ttccggagaa
tagtgctacc ctcacatccc ctggagcaca gccttcctga aatgccctca ccccatgcct
ttgccattgt gtgctctcag atttcttcca ctgtttgaca ccctccttag agggctgctc
ttttttttcc agagataatc ctagccatcc tctccactcc cacggctggg gacaatggcc
acttactacc tgtgcacttt gccactcggg acacctggat ggtttctctt aggactttgc
ccacctcctt ctcatggcac ttgctgtgga aaatgcctgg ctggcctcgt ggggcctgtc
tcacttttcc aggagacatg acccactaac gtggcaactt taacccaaag gcccctcaga
catgttacag caaatctgga gccacagaca ggttccctcc attggcagcc cattgtgttt
gaaattccat gtcgggttta cttggaatga aagatacttg aattattgtg cgcctgtgag
cgcccagctt ctgtttcata gtcttaacag gtggccattg tcgtgaaacg agtgatgcct
gaagatctca gtgatgtttg aaccttctgt gtaacttttt attaagtctt tgtatctctc
gactgattaa taaagaagag aaacacgtaa aaaaaaaaaa aaaaaaaaa
tcgtctacaa attggaagga aatatgaatg aagccctgga gtactatgag cgggccctga
gactggctgc tgactttgag aactctgtga gacaaggtcc ttaggcaccc agatatcagc
cactttcaca tttcatttca ttttatgcta acatttacta atcatctttt ctgcttactg
ttttcagaaa cattataatt cactgtaatg atgtaattct tgaataataa atctgacaaa
atattagttg tgttcaacaa ttagtgaaac agaatgtgtg tatgcatgta agaaagagaa
atcatttgta tgagtgctat gtagtagaga aaaaatgtta gttaactttg taggaaataa
aacattggac ttacactaaa tgtttaattc attcatttta ttgtgaaata aaaataaaat
ccttagctcc tccaccaact gaacagaccc tcttggccaa ggagacccca gaaaccttaa
aaactaagtt tcccaaccat gacaagatga gagatcattc acacctcatt atattccctc
ccttgctaac tgccattgga ctttttccac tgagttaaac agaaacccat ggaaaacaaa
gaacagaaga ctcactcctt ggctgacttc acctagctca ctccacgtag cgccacagcc
agactcccct cccctcttgc ggtttccaca tgacaactga tcagccttcc ctcctgataa
gtgaccactg cccacagact ggttctggcc agtccatgga ggctgcacac agggtgcctc
tatgtccttt gtttcacctt ttgatataga aaggctaatt ttgctgtatt ttaatgttaa
gtctccacca cagagtgaac acagaatgca tgtgacatac atgtttacat accactattg
tgtgactgcc cctcatgaat attcatagcc ccccataacc tgttaactat gtgtgtctag
ccaatccacc aaccataaaa cttctgtaat accctccctt cctccaagag cctgcttttg
gttgctgtgg taggctctgc ttcccaggct gcaggttgca ggagaggagg ctgcagtggc
tcacgcctgt aatctcagca cttcgatggg acgaggcagg cagatcacct gaacccagga
gttcgagagc agccttggca atggcaaaac caaccgtctc tacaaaaaat gcaaaaactt
agctgggtgt ggtggcatgc acctgtagct tcagttccag ctactcagga ggctgaggtg
agtggactgc tggagccagg gagttcgagg ctgcagtgtc gagatcttgc cactgcactc
cattctggat gatagaacga gaccccatct caaaaaaaaa aaaagttctc tccaattgta
tatagcttgt gattttatgt caacactatc aataaatagc tttcagtgca agaaaccaaa
aatactgtaa taaacaggca catattcttc ccaaacctca tgcagtttac aatctagtga
gagacacaga tagcagtaca gagtcaatta aaggttagtt ttcttcatga agatgtttta
attttaattc aatgtgaaag ggttccaagg agtttatctt gttttatgcc attttatttg
aagcactact tactaagtca tttgctgata ttaatctagt taaatcaaga aatattacat
gaaaatgttg ctaaatcaga gatcatgggt aacaatcacc tttgattatg aataatcata
ttttattgaa aggcaaggca caacaaataa taagaaggaa aaaataaata agcaatgtta
ttgatctttc attctgtata tgttttgggg ggaatatact agtttctttt agtggctgta
acaaattacc acaaacttgg tgacttaaaa tttcacagat ttactctttc ttacagttct
ggaggtcaga agtctgaaat gggtttcaat gagccaaagt caaggtattg atgacgctac
actcctccgg aggctctagg cagatagcct tttccagctt ccagaggctg cctgaattct
ttcatccatc ttaaaaacca acagtgtagt agcctcaaat ctctctctct gcttccttct
tcacatctcc ttctctcctc tgactctttt gcctctttct tctaaggacg caccaggtcc
acctgcataa tccagaataa ttgccccatc cgcaaatcct taatttaata acatctgcaa
agtccctttt gctatgtaaa gtagcatgtt cacaggttct ggagacttgg ccatggatac
gattgcgggg ggggcattat tcttaccaca gagcacccca agaaaatctc caaattttgg
gcttccaatc cattttgctt caattattta atatttttac tccttccagt agatactgat
ttcatccatt gcccttaaga aggtaggaca gagattatgg cacatctcac attaaatgct
aggcggcctg tgcagggatg acatattcta tatcttgaac ggtaacattc gtgatagata
ccagtttaat cccatggaat caatcaaatt aaatcatcat gactacattg attccccatc
gctgaaggac agaattcatt gtgtggcatt tgtatttgat gccagctcta ttcaatactt
ctcctctcag atgatagtaa agatcaaaag aattcgaagg gagttggtaa acgctggtgt
ggtacatgtg gctttgctca ctcatgtgga tagcatggat ttgattacaa aaggtgacct
tatagaaata gagagatgtg agcctgtgag gtccaagcta gaggaagtcc aaagaaaact
tggatttgct ctttctgaca tctcggtggt tagcaattat tcctctgagt gggagctgga
ccctgtaaag gatgttctaa ttctttctgc tctgagacga atgctatggg ctgcagatga
cttcttagag gatttgcctt ttgagcaaat agggaatcta agggaggaaa ttatcaactg
tgcacaagga aaaaaataga tatgtgaaag gttcacgtaa atttcctcac atcacagaag
attaaaattc agaaaggaga aaacacagac caaagagaag tatctaagac caaagggatg
tgttttatta atgtctagga tgaagaaatg catagaacat tgtagtactt gtaaataact
agaaataaca tgatttagtc ataattgtga aaaataataa taatttttct tggatttatg
ctggaccatt atctgagacg ggaggccttg tctatacatg tttttgatga tgaagactta
gagcctggct cgtatcttgg ccgagcccga gtgcctttac tgcctcttgc aaaaaatgaa
tctatcaaag gtgattttaa cctcactgac cctgcagaga aacccaacgg atctattcaa
gtgcaactgg attggaagtt tccctacata ccccctgaga gcttcctgaa accagaagct
cagactaagg ggaaggatac caaggacagt tcaaagatct catctgaaga ggaaaaggct
tcatttcctt cccaggatca gatggcatct cctgaggttc ccattgaagc tggccagtat
cgatctaaga gaaaacctcc tcatggggga gaaagaaagg agaaggagca ccaggttgtg
agctactcaa gaagaaaaca tggcaaaaga ataggtgttc aaggaaagaa tagaatggag
tatcttagcc ttaacatctt aaatggaaat acaccagagc aggtgaatta cactgagtgg
aagttctcag agactaacag cttcataggt gatggcttta aaaatcagca cgaggaagag
gaaatgacat tatcccattc agcactgaaa cagaaggaac ctctacatcc tgtaaatgac
aaagaatcct ctgaacaagg ttctgaagtc agtgaagcac aaactaccga cagtgatgat
gtcatagtgc cacccatgtc tcagaaatat cctaaggcag attcagagaa gatgtgcatt
gaaattgtct ccctggcctt ctacccagag gcagaagtga tgtctgatga gaacataaaa
caggtgtatg tggagtacaa attctacgac ctacccttgt cggagacaga gactccagtg
tccctaagga agcctagggc aggagaagaa atccactttc actttagcaa ggtaatagac
ctggacccac aggagcagca aggccgaagg cggtttctgt tcgacatgct gaatggacaa
gatcctgatc aaggacattt aaagtttaca gtggtaagtg atcctctgga tgaagaaaag
aaagaatgtg aagaagtggg atatgcatat cttcaactgt ggcagatcct ggagtcagga
agagatattc tagagcaaga gctagacatt gttagccctg aagatctggc taccccaata
gtcatcatgt cccaattttc tttccatctt tactcatatc taccttttga atcccaaaag
aattgtacaa tctgttcctc tgatcatctc taccagggaa tagttgactc ttttacagca
ttattgtttt gtagatttca aagtacttca tgaacattaa tcctttgggt tataaatata
accttatcaa ttgtccagaa acacttagca tactcacaca ataaaaatta tattagcttg
ccacctgtct gcccatggtg tgatgtatct ataatccact tgttcataaa aaatattcgt
ctgacaccta tgatatgcta gggaatatgg gagacaatag gaaagtaaga cagacatggt
atctgccctt gtgatgccag taaacttaag ccttcatggg gctctctgac ttcataattt
ccagaaccag agataggaaa tgaggtgaat ttgagaaatg tcagactgtg ccaaaggggg
tcacatgcat caaatttcca catacat
cacctgcaga tgaagattac agcccctgta tgctagaaac tgagacactc aacaagtatg
ttgtgatcat cgcctatgcc ctagtgttcc tgctgagcct gctgggaaac tccctggtga
tgctggtcat cttatacagc agggtcggcc gctccgtcac tgatgtctac ctgctgaacc
tggccttggc cgacctactc tttgccctga ccttgcccat ctgggccgcc tccaaggtga
atggctggat ttttggcaca ttcctgtgca aggtggtctc actcctgaag gaagtcaact
tctacagtgg catcctgctg ttggcctgca tcagtgtgga ccgttacctg gccattgtcc
atgccacacg cacactgacc cagaagcgtc acttggtcaa gtttgtttgt cttggctgct
ggggactgtc tatgaatctg tccctgccct tcttcctttt ccgccaggct taccatccaa
acaattccag tccagtttgc tatgaggtcc tgggaaatga cacagcaaaa tggcggatgg
tgttgcggat cctgcctcac acctttggct tcatcgtgcc gctgtttgtc atgctgttct
gctatggatt caccctgcgt acactgttta aggcccacat ggggcagaag caccgagcca
tgagggtcat ctttgctgtc gtcctcatct tcctgctttg ctggctgccc tacaacctgg
tcctgctggc agacaccctc atgaggaccc aggtgatcca ggagagctgt gagcgccgca
acaacatcgg ccgggccctg gatgccactg agattctggg atttctccat agctgcctca
accccatcat ctacgccttc atcggccaaa attttcgcca tggattcctc aagatcctgg
ctatgcatgg cctggtcagc aaggagttct tggcacgtca tcgtgttacc tcctacactt
cttcgtctgt caatgtctct tccaacctct gaaaaccatc gatgaaggaa tatctcttct
cagaaggaaa gaataaccaa caccctgagg ttgtgtgtgg aaggtgatct ggctctggac
aggcactatc tgggttttgg ggggacgcta taggatgtgg ggaagttagg aactggtgtc
ttcaggggcc acaccaacct tctgaggagc tgttgaggta cctccaagga ccggcctttg
cacctccatg gaaacgaagc accatcattc ccgttgaacg tcacatcttt aacccactaa
ctggctaatt agcatggcca catctgagcc ccgaatctga cattagatga gagaacaggg
ctgaagctgt gtcctcatga gggctggatg ctctcgttga ccctcacagg agcatctcct
caactctgag tgttaagcgt tgagccacca agctggtggc tctgtgtgct ctgatccgag
ctcagggggg tggttttccc atctcaggtg tgttgcagtg tctgctggag acattgaggc
aggcactgcc aaaacatcaa cctgccagct ggccttgtga ggagctggaa acacatgttc
cccttggggg tggtggatga acaaagagaa agagggtttg gaagccagat ctatgccaca
agaaccccct ttacccccat gaccaacatc gcagacacat gtgctggcca cctgctgagc
cccaagtgga acgagacaag cagcccttag cccttcccct ctgcagcttc caggctggcg
tgcagcatca gcatccctag aaagccatgt gcagccacca gtccattggg caggcagatg
ttcctaataa agcttctgtt ccgtgcttgt ccctgtggaa gtatcttggt tgtgacagag
tcaagggtgt gtgcagcatt gttggctgtt cctgcagtag aatgggggca gcacctccta
cactagccgc actcatgaat cggcaccatc tgcaggatca ctttctggaa atagacaaga
agaactgctg tgtgttccga gatgacttca ttgtcaaggt gttgccgccg gtgttggggc
tggagtttat cttcgggctt ctgggcaatg gccttgccct gtggattttc tgtttccacc
tcaagtcctg gaaatccagc cggattttcc tgttcaacct ggcagtggct gactttctac
tgatcatctg cctgcccttc ctgatggaca actatgtgag gcgttgggac tggaagtttg
gggacatccc ttgccggctg atgctcttca tgttggctat gaaccgccag ggcagcatca
tcttcctcac ggtggtggcg gtagacaggt atttccgggt ggtccatccc caccacgccc
tgaacaagat ctccaatcgg acagcagcca tcatctcttg ccttctgtgg ggcatcacta
ttggcctgac agtccacctc ctgaagaaga agatgccgat ccagaatggc ggtgcaaatt
tgtgcagcag cttcagcatc tgccatacct tccagtggca cgaagccatg ttcctcctgg
agttcttcct gcccctgggc atcatcctgt tctgctcagc cagaattatc tggagcctgc
ggcagagaca aatggaccgg catgccaaga tcaagagagc catcaccttc atcatggtgg
tggccatcgt ctttgtcatc tgcttccttc ccagcgtggt tgtgcggatc cgcatcttct
ggctcctgca cacttcgggc acgcagaatt gtgaagtgta ccgctcggtg gacctggcgt
tctttatcac tctcagcttc acctacatga acagcatgct ggaccccgtg gtgtactact
tctccagccc atcctttccc aacttcttct ccactttgat caaccgctgc ctccagagga
agatgacagg tgagccagat aataaccgca gcacgagcgt cgagctcaca ggggacccca
acaaaaccag aggcgctcca gaggcgttaa tggccaactc cggtgagcca tggagcccct
cttatctggg cccaacctct ccttaaataa ccatgccaag aagggacatt gtcaccaaga
accaggatct ctggagaaac agttgggctg ttgcatcgag taatgtcact ggactcggcc
taaggtttcc tggaacttcc agattcagag aatgcgattt agggaaacgg tggcagatga
gtgggagact ggttgcaagg tgtgaccgca ggaatcctgg aggaatagag agtaaagctt
ctaggcatct gaaacttttg cttcatctct gacgctcgca ggactgaaga tgggcaaatt
gtaggcattt ctgctgagca gagttggagc cagagatcta cttgtgactt gttggccttc
ttcccacatc tgcctcagac tggagggggc tcagctcctg gggtgatatc tagcctgctt
gtgagctcta gcagggataa ggagagctga gattggaggg aattgtgttg ctcctggagg
gagcccaggc atcattaaac aagccagtag gtcacctggc ttccgtggac caattcatct
ttcagacaag ctttagcaga aatggactca gggaagagac tcacacgctt tggttaatat
ctgtgtttcc ggtgggtgta ataggggatt agccccagaa gggactgagc taaacagtgt
tattatggga aaggaaatgg cattgctgct ttcaaccagc gactaatgca atccattcct
tcagcaatac aaaaccgctg agttcttgag caaactgaac acagaatcgc cagacagaag
tgcctgtcaa agtgctgttt gtggcccaca atcctcaaca tgggccagaa gctgggctta
cagagattct ctaaaggcag aagaaaacag aaaattgcaa aagatgaagg atgaacaaca
tcaaaagagt gaattactgg aactgaaacg gcagcagcaa gagcaagaaa gagccaaaat
ccaccagact gaacacagga gggtaaataa tgcttttctg gaccgactcc aaggcaaaag
tcaaccaggt ggcctcgagc aatctggagg ctgttggaat atgaatagcg gtaacagctg
gggtatatga gaaaatattg actcctatct ggccttcatc aactgacctc gaaaagcctc
atgagatgct ttttcttaat gtgattttgt tcagcctcac tgtttttacc ttaatttcaa
ctgcccacac acttgaccgt gcagtcagga gtgactggct tctccttgtc ctcatttatg
catgtttgga ggagctgatt cctgaactca tatttaatct ctactgccag ggaaatgcta
cattattttt ctaattggaa gtataattag agtgatgttg gtagggtaga aaaagaggga
gtcacttgat gctttcaggt taatcagagc tatgggtgct acaggcttgt ctttctaagt
gacatattct tatctaattc tcagatcagg ttttgaaagc tttgggggtc tttttagatt
ttaatcccta ctttctttat ggtacaaata tgtacaaaag aaaaaggtct tatattcttt
tacacaaatt tataaataaa ttttgaactc cttctgtata aatgggtcat ttttattttt
aatgaaaagt tattggggtt ttctctcttg aagggtctca ttttaattcc cttttccagg
ccgtatagat caaatatagt actgtcatta ctgttggctc ttgttttggt cttgacttac
taatagtgtt accctgattt tcagaggggg acagtttatc tccagaaagg ccaatgtttg
tatacacatc agctagacac aaatatagac atcatatgta gtttgtacat gtttcagaaa
cttgtttttt ctttgctctg tgtaacctat ttcctattgc tagttcagtt ggctttctta
ttcacttctg tgaccctgaa ccagttctca gaccctagag tgtaagagca ttgattttct
acgctgtgta atctagctca atccctctgt cccctccgcc tcaccgtccc ccagccacca
cattgtatag caaaagcatt acattcaatc ctagaataaa ggtaaataca acaaatcatc
tttgcagctg gacaactaat aatactttgc agcattaaga gatcttctgt gttaccagtc
actctgttga aatgaacttt ccgaatctct ttattcagga aaacatgggg ttttgaaatt
cttgggccaa gagacataac tgaggggttc gcagagctag gcaagggtgc actaggaaag
ggccacattg gtgggtgggg ggtaacagag aacagatggt gtcaggaagt ttctctggag
taaataatgt ggatattctt ggtttccctc tcctccgcca gctgaagctg tgttagtgct
gttgacacta atataaaatg tttggtccat ttgaaatcct tgtcattgcc ttatatgggg
gaaactcaat cccccagcct gtgttggaaa tatcaccaaa ctgattgtaa atgtgcggct
gtagcagaca ttttagtgtg gtggtgtgca gccatttcgg ccctacacct gccagcctgg
ctaccttaca gttgtgttcc gatttttgcg tctatgcttg gtgtgcctca cttgctgcat
tttccagcat gcaaccagga gttgacgtag gaaaaaggga tgctttctta ctttggaagc
tctcagggaa gttggtgtca atttctcctc cactgcctgg cctaccctgc actcccaaag
attttgtgca gatgggtagt tccatttttt aaaaattgtg cagatatgga aaattgtgac
ttacttcatg accagaacta tctagaatat gtgtgggggt ataaacatct tgcttaacca
aatatctatg taggcagagg taaccaggag agaagcaaga cttgctgcct aaaggagccc
accattttac ttttcacatt taatctgcca cgttgaatca attggaataa aacctgactc
gcaggtgact ggacaggaaa tcccaaagtt ccaccatttc tatgcttaat tttaatgtcc
ggacattggc ccagagacag gctgatttcc aacgtcctcc aggggctgcc gagccagagc
ccaaggacgg gggcctacag aggaggtcca gcccagctgc tgacgtccag ggagaaaact
tctgtgctgc cgtgaagaac acacagcctg aggacggggt ggaaatggac actcggcaga
gcccacacga tgaagacccc caggcagtga cgtatgccaa ggtgaaacac tccagaccta
ggagagaaat ggcctctcct ccctccccac tgtctgggga attcctggac acaaaggaca
gacaggcaga agaggacaga cagatggaca ctgaggctgc tgcatctgaa gccccccagg
atgtgaccta cgcccggctg cacagcttta ccctcagaca gaaggcaact gagcctcctc
catcccagga aggggcctct ccagctgagc ccagtgtcta tgccactctg gccatccact
aatccagggg ggacccagac cccacaagcc atggagactc aggaccccag aaggcatgga
agctgcctcc agtagacatc actgaacccc agccagccca gacccctgac acagaccact
agaagattcc gggaacgttg ggagtcacct gattctgcaa agataaataa tatccctgca
ttatcaaaat aaagtagcag acctctcaat tcacaatgag ttaactgata aaacaaaaca
gaagtcagac aatgttttaa attgaatgat catgtaaata ttacacatca aaccaatgac
atgggaaaat gggagcttct aatgaggaca aacaaaaaat agagaaaaat taataaagtc
aaaatgttta ttcttgaaaa cattaatgat acatgaatct tggccacaat gagaaaaata
aaaatgaaaa aagagcaggc atccatttcc atacaggaac aaaataggag gcagcactac
agaccctaca cacagcttta cagaggtgaa agaaaactgt cagcaattct atgctgacat
aacagaaaat gtagatgaga tagatgaaat acgaaaaatt acagtttact taatgaacat
aaggataaat agaaaaactg aatcatcata cataaacata tataaaatgc attgatcctg
taatcaaaaa tgttcccaca aagtaaatgc cacttcagca aggtttgttg gtggtttttt
caaactctta tgcactcatg aaacacacag acacacacac acacaaactt gcataaattt
tccctgagaa tattttgtat atatttacac aaatacattt gatcagacta ggaacaagtt
gataccaaaa cctgaaaagg aaactacaga atgggaaagt catagaagat ctctcacaga
aatataaatc ccttaacaaa tattaacaag taagattcat gtctctataa aatagacagt
atatcatgac cacactggtt ttttgttatc ctttgatttt gtttatgaaa agcaaggata
gcttaatttt caaaaactca atcaatgtaa ttcagtattt taacaaaagg aatgaaaaat
tatcatctca atagacaaag cttttgtctg agcacctttt catatagctg ctgaccattt
gtatgtcttc ttttgagaaa tgcctgttca gctactttgc ccatgtttca agtagttttt
ggtttcttgc tgttgctttg ttttagttcc ttacatattt ttgcatatta accctttatc
aggtatacag cttgcaacta ttttctccca tttctgagtt gtctcttcat tctgtttgca
gaagctgttt agaagccaca ccttttgtct atttttgctt ttgttgcttg tgttttcagg
gccatatcca aaaaaacctt gcccggacca acgtcttgaa gcttttctcc cacccatttt
tgtatatggg ataagggttc aatttcattc ttcttcatat gaatatcccc aggatgtgtc
ctatgcccag ctgcacagct taccctcaaa cagaaaataa tgaagccttc ttcctcccag
gaaaggggac gttcagctga gccgagtgtg tatactgctc tggccatcca ctagcccagg
gaggacccag acctccacac tccatggaga ctcagttctc ctaggaccat ttattcaaaa
gttccacatt ccatccacgg tgtatgacag tgcagggcga taccgctgct actatgagac
ccctgcaggc tggtcagagc ccagtgaccc cctggagctg gtggcgacag gattctatgc
agaacccact cttttagccc tgccgagtcc tgtggtggcc tcaggaggaa atgtgaccct
ccagtgtgat acactggacg gacttctcac gtttgttctt gttgaggaag aacagaagct
ccccaggacc ctgtactcac agaagctccc caaagggcca tcccaggccc tgttccctgt
gggtcccgtg acccccagct gcaggtggag gttcagatgc tattactatt acaggaaaaa
ccctcaggtg tggtcgaacc ccagtgacct cctggagatt ctggtcccag gcgtgtctag
gaagccctcc ctcctgatcc cgcagggctc tgtcgtggcc cgcggaggca gcctgaccct
gcagtgtcgc tctgatgtcg gctatgacat attcgttctg tacaaggagg gggaacatga
cctcgtccag ggctctggcc agcagcccca ggctgggctc tcccaggcca acttcaccct
gggccctgtg agccgctccc acgggggcca gtacagatgc tacggtgcac acaacctctc
ccctaggtgg tcggccccca gcgaccccct ggacatcctg atcgcaggac tgatccctga
catacccgcc ctctcggtgc agccgggccc caaggtggcc tcaggagaga acgtgaccct
gctgtgtcag tcatggcatc agatagacac tttctttttg accaaggagg gggcagccca
tcccccgctg tgtctaaagt caaagtacca gtcttataga caccaggctg aattctccat
gagtcctgtg acctcagccc agggtggaac ctaccgatgc tacagcgcaa tcaggtccta
cccctacctg ctgtccagcc ctagttaccc ccaggagctc gtggtctcag gaccctctgg
ggatcccagc ctctcaccta caggctccac ccccacacct ggccctgagg accagcccct
cacccccacg gggttggatc cccagagtgg tctgggaagg cacctggggg ttgtgactgg
ggtctcagtg gccttcgtcc tgctgctgtt cctcctcctc ttcctcctcc tccgacatcg
gcatcagagc aaacacagga catcggccca tttctaccgt cctgcagggg ctgcggggcc
agagcccaag gaccagggcc tgcagaagag ggccagccca gttgctgaca tccaggagga
aattctcaat gctgccgtga aggacacaca gcccaaggac ggggtggaga tggatgctcg
ggctgctgca tctgaagccc cccaggatgt gacctacgcc cagctgcaca gcttgaccct
cagacgggag gcaactgagc ctcctccatc ccaggaaagg gaacctccag ctgaacccag
catctacgcc cccctggcca tccactagcc cacgggggac ccagatctca tactcaacag
aaggagactc agagactcca gaaggcacag gagctgcccc cagtggacac caatgaaccc
cagccagcct ggacccctaa caaagaccac caggacatcc tgggaactct gggactcact
agattctgca gtcaaagatg actaatatcc ttgcattttt gaaatgaagc cacagacttc
tcaataaatc aatgagctga gaaaactgaa acagaaatta gagcatggta taaatttgga
cattctaagc ctctggagag actgtaatga tccattcatg agctggtatg aaatcagtgg
gaagcagaac aacagaagga actttaaaag caacaagcaa ttatgtacca tatatacact
gtagcaaata ttttatattt gagagtcttt acagcttaca tttccattcc attattacaa
gtgatgaaaa acaaaaaatt gcaagtagta tgcaaattat aaatatacag tcttcccact
tcactaacca aattcctact ttccagtgtt acttcccaat ttatgcagga aacctcctgc
aaagctgaaa ctgattagaa aattctttat attttaaaat agctctttct catttttaga
gaagtcaaat agccaaccat caaaattaag aataaattga attgtcacag tccattacag
ttattgttgc tagatccacc tcatttgcag atgtccaaac ttaaattcat ctgttcttaa
aatgctactt aaaactttgg ttgttttcct gtaatataaa agaaaaagtt aatttatcaa
ttgattgaat acagttttta ctaattagtt tatcaaacca aatactgtga acgtaccagg
tgtttacaga tttaaatgca tattaccata gaaactatta aagtaactag aactgtcaaa
taacaaaacg gctcatgttt ttaaaatata tgtaactcat tttaaaatat attaaattgt
attccaaacc tgttcttctg tttctgtggc acctaggttt aaaatatgta ttaatgtgta
cccttccatc tgccacggag gatgcaaagg aagagggtct ggaagcccag atcagccgct
tggcagagct gattgggagg ctggagagca aagcactgtg gttcgacctg cagcagcgcc
tgtcagatga agatggcacc aacatgcacc tgcagctggt ccggcaggag atggccgtgt
gccccgagca actgagcgag tttctggact ctctgcgcca gtatctgcgg gggaccactg
gcgtgaggaa ctgcttccac atcactgccg tgaggctctc agatggcttc acctttgtca
tctatgagtt ctgggagaca gaggaggcgt ggaagaggca cctgcagagc cccctgtgta
aggcgttccg gcacgtcaag gtggacacac tgagccagcc tgaggccctc tccaggatct
tggtgccagc tgcttggtgc acggtgggac gggactgaca gcctcccaga ggcccgtgga
ggagcccacc agccccttct tcttgtgaag gaaatcccgt ttttttctag acagacactt
tggtgcagaa gcttcttttc aatccatcct ccacaagaag gtgtttccct gttgttaagt
gaaggaggcc gcccctgccc ccacctgaga aggcagagca gtgtctgtgc tgccaggtcc
tggtgaagcc caaggttgaa gggggcggct tcctggagcc agcacccctg cctcctggtc
ctggcctctc ccctacccct cacatggcca cgcatgaccc acactgacca caccctgccc
agcgatgacc tggatgtctg cattctacca aatgcagaga acagtggaat tatgcaccca
ataacagcag agaggaccag gatgctaaag ataaaagata aaaacaagtt ctactttcct
tcattacaac ccaggaaaga tttaggaaaa gtagtctgtc ctgaattaga ctgtgcaagt
ccaagaaaat atctcttcaa ggatctggat gggagagccc tgggtctgcc tccaccagtt
tctgtatttc ctaaaacaga ggcagaatgg actgcatcct tcttcaacgc aggtacattt
agagaagaac agaaatgtac ataccaattt ctgatgcaag gattcatctg caaacagact
gaccaagtgg tcctaattct tgatagcgct gatgccattt gggcaattca gaagttatat
ccagttgtat ctgtgactag tggttttgtt gatgtcttta gcagtgtaaa tgccaatatt
ccctgctcta cttctgggtc agtgtctact ttctattcta tcttacccat caggcaaatc
accaaagtct gcttcatgga tcaaactcct caagttttgc gcttttttct attggggaac
aaaagtacct ccaagcttct cttggctgta ttctaccatg agctccagag cccccacgtc
ttcttagggg aaagttttat tccacccact ctggttcagt cagcttcctt attgctgaat
gaatctattg gtgccaacta tttcaacatc atggataacc tcttgtatgt tgtcctacaa
ggagaggagc ccattgaaat acgctcaggt gtttccattc acttggccct cactgtgatg
gtttcagtct tagaaaaagg ctgggaaata gtaatactcg aaagactaac taacttctta
cagattggcc aaaaccaaat caggtttatt cacgagatgc ctggccatga agagacctta
aaggccattg ctgacagtag agcaaaaaga aagcgcaatt gccctactgt gacttgcact
agtcattata gaagagttgg tcaacgtagg cctctcatga tggaaatgaa ctcacatagg
gcttcacccc caatgactgt ggaaactatc tcaaaagtga ttgtcattga aattggtgat
tcgccaacag taaggagcac tggaatgatt tcatccttat caagtaacaa attacagaat
ttggctcatc gagtcatcac tgctcaacag actggggtac tagagaatgt tctgaatatg
actatcgggg ccttactagt tactcagtca aagggagtca ttggctatgg aaatacaagc
agttttaaaa ctgggaactt gatatatatt cggccctatg cactttccat cctagtccag
ccttcagatg gagaagtggg aaatgagctt ccagtgcagc cacaattggt atttttggat
gagcagaatc gaagagtaga gtccctggga cctccttcag agccatggac aatttcagct
tccctggaag gagcatcaga ctcagtgcta aaagggtgca cccaggcaga aactcaagat
ggttatgtta gcttctacaa cttggcagtc ttgatctctg ggtcaaactg gcactttatt
tttactgtca cttctcctcc aggagtcaat tttacagctc gatccaagcc atttgctgtc
ttgcctgtga ctaggaagga gaagtcgacc atcatcctgg ctgcttccct gtcctctgtg
gcctcatggc tggctctgag ctgtctggtg tgctgttggc ttaaaagaag caaaagcaga
aaaacaaaac ctgaagagat tcctgaatcc cagactaata atcaaaatat tcatatccac
atctcatcca aacgccgaga atcacaaggg cccaaaaaag aagacactgt ggtgggagaa
gatatgagaa tgaaggtcat gctgggcaag gtgaaccagt gcccccacca gttgatgaat
ggagtgtcca gaaggaaagt tagccgccac attgtccgag aggaagaggc tgctgtgcct
gctcctggta ctactggcat cacatcccat gggcacatct gtgctccagg tgctcctgct
cagcaggtgt acctgcaaga gactgggaac tggaaggagg gccaagagca gttgctcaga
taccagctgg caggccaaaa tcagctgctg ctgctatgcc cagacttcag acaagagagg
cagcagttgc cagggcaaag tcggctgagt aagcaaagtg gcagcttggg gctttcccaa
gagaagaaag cctcctgcgg ggccactgag gcattctgcc ttcattcagt acacccggaa
actattcagg agcaactgtg atcagggaag ttgggggcat ttggcctgaa aggcagaatg
ttcccagtat ttctggataa taagctgggg aagtgaggac tgtcctgctg ggacaactaa
gaagagagaa tgtggactct gaatcccttt ttcaacttta aaatggaaaa cagtcatata
aatgcttaca gactgaaaaa tgtctcatac atattttgca gctatcatga tcctagttca
atgctaggca aataatggca cttgttaatt atttaccagt ttttaactta agcctgattt
taacaacttg ataatggtgt aatactgact tataccagca ggggttatta ttaagcattc
tctggattga ccacccacaa tgctttgagc ttcttttata gaagggatca tgaaaaggtc
tggtcagcta cagtttaatt ccacactgtt acagaaaagc atttctttat cctgtagtag
catttgaagg gacagttcaa caccctgctc cctgatctgt cagagcttac cttccaacca
caaggcaact tcctggcttt ttacaagtgg attttatttc atggtttaaa ttcaatcatt
attgagaacc tacagtagtg tgagcgtatc aaatttgcac ttcatgagga tttacacatg
aaaattgata ttctcatggg ctttgctgag agtttagtta aggacaaaga tgtaaaaatg
ccaaatttgg gaaaggaaat tttgtatgac cactgttcta tttttcagtg attctctctg
ttagtagctg ggtcacattt acaactggaa aagaactaca ttgggagatc agagaaagcc
aagtggccaa gtcctttatc aaaaagctgc tttttggaga agatgatgca taaagctgtg
atactaagga gaggagagat tatgtcatat gcaactgctc taggctgtgc agggggttta
aatggagatc agaacaaagg ctcacctggt attgatacat ttggtaaggt tgttaaaggc
aatttcacca aattcacaca tttgtacggg gcccatttct tttcaaactg aagaatactt
taaacagcct gactgtgtcc ttggctctgg atccagaagg ccttatgctc caagcatgta
aagaaaagat cgcaggaatg tggagagctt ccaaaaagag attgctctca gattttcctg
aaaatcctga aaaccaggat tttccaatct ctacaatgtt aaggggttgt tgtgtagttg
cattggaaga gagaaaggaa acacactctt tcattgctga attctaaata atgatgccct
gaattagttc tatgagaagt actggcagac agctgttatg tctgcagggc atcaaggggc
aggtgtgtct caccctacta agaattggca gaaatgtttt aacctcttct ttaggcatct
ggtgagtatt gcagatcttc aggctgtccc taaaaggagg taggtagcct tctctgtggt
cactgtgttg gatttggaag aaaatataag tagatgttaa ggtcagtgct ctccaggaga
tttcaatcca ggctatctgt gttcagggaa tgatttgcaa gaagttggtt tatagacgtt
tactctggag gcacaagctg gctgccatgc tgagtaagtc ctctttgtag atgatgtatt
ttgcttgggc accaacagaa ttaaaaacga aaaacaaaaa aaaactcgaa tgacttaggc
agaacatgcc ttccctagtt taattagtcc ctgttataac cagttattta tctttttttg
tagattctcg gtcttgaagt catttgcctt tgcaatctct actttagtat actcattgga
tcctcaaaga gccgtaagct ttttggaaaa aaacatttca gaagtgaagg cttttgaggt
ttataaaatg gtagcagtct tagtccttgc cctgcctcag tttacctgca catgctttct
ttcatttcct cggattcttt caccactttt taagtttttt ttccacaggt atttttcctg
agctctatac catgcattta gctgcctagt gagaatatcc agtaggttat cctatagtta
ctatgaagtt aacacagccc aaaaacctac ttctccttgt gtttttttcc aactcaatga
atgacatcac tgcactcaga ccagaaatct agaagtcagt catgactctt cattctattt
cgcccttcac aaagagttag ctgccagctc tgttatttct gccccttctt tacccttctt
ccaattttta ttatcttctt atttatcctc tatgtgtcac aagaccagtc tttctgggag
gcatgtttta tcctatccct ctgcgtagga ataaccataa ttatcttact aattccatcc
cagaaaagcc tgtgatccct gaggccagaa ttgactcttt gggcctaaat agcagctata
aacatcttca gtaacttgcc aggctctagc atgtggcatg gaattttggc atctagctaa
aattcctcaa cccatagtta agctactcct ctttctccta aaaaaaaaaa aaaatgagag
aattagggaa acaccaaaag caaagagagc agctctatgt tcattcatgt ctggagatga
gccaatttga gcaccactgt aggccaactc taaatttatt cagatcgtta acatgggtaa
cccagcaaat atttcccagc agtgacaatc tacacccttt atctaaataa atattgagat
gtgctttttt cctgtgttag ttcaaagaat ctcttcttgc ctcccattgc tgattttctg
tattgatgga ctcatttctc tcaagagatt ggacaagcta aaattctaga gagttcaaaa
gtgtttgaaa gtacatttta agtgttctca ccacaaaaaa tgctagtgtg tgaggtgata
catatgttaa ttagttcaac tgatccattc cacaatgtat acatatttca aactatcgag
ttgtacatga taaatatata caatttttat ttgtcaaata aagaacatct ataaatattt
atagatttca aaattgagaa agaagtatat atggagccac ggttgttttg accatttctc
catgcttttt gtttggggag ggggacattt tttggggggc agggactgtg ttcagggaaa
gtacctgacc aacagaactt caagtctctt ccagagtgcg agactcagct tatctgatca
aggtgaactt gcccttcaat agacttgccc ttgttactgc ccagtgaggc caccaaggac
caagaacagt tgagctctct cagggccttc ccctttcaac aatttagttg aatgactccc
ttccatctat cagtcttact ccaaatatat gaaattacta tacattgaaa tgcctgcaaa
ctatatgcaa aaatgagtat gaggaaatca aatgtaaatc atatgcaaat attattcaac
caagcttcta taaaattgta cgtgtcaata acagtaaaga attttaatta attctgtcat
ttaaaattca tgcattccat attttgcttt cagaacttat agtctatgcc aattttgaaa
cagggccagg gctacccggg gccccctatg cgctatggag agagttcctc ttctccgttc
ccgcggggcc ccccgcgcag tactcggtca ccctccacgg ccaggatgtc ctcatgctcc
ctggtgacct cgttggcttg cagcacgacg ctggccctgg cgccctcctg cactgctcgc
cggctcccgg ccaccctggt ccccaggccc cgtacctctc cgccaacgcc tcgtcatggc
tgccccactt gccagcccag ctggagggca cttgggcctg ccctgcctgt gccctgcggc
tgcttgcagc cacggaacag ctcaccgtgc tgctgggctt gaggcccaac cctggactgc
ggctgcctgg gcgctatgag gtccgggcag aggtgggcaa tggcgtgtcc aggcacaacc
tctcctgcag ctttgacgtg gtctccccag tggctgggct gcgggtcatc taccctgccc
cccgcgacgg ccgcctctac gtgcccacca acggctcagc cttggtgctc caggtggact
ctggtgccaa cgccacggcc acggctcgct ggcctggggg cagtgtcagc gcccgctttg
agaatgtctg ccctgccctg gtggccacct tcgtgcccgg ctgcccctgg gagaccaacg
ataccctgtt ctcagtggta gcactgccgt ggctcagtga gggggagcac gtggtggacg
tggtggtgga aaacagcgcc agccgggcca acctcagcct gcgggtgacg gcggaggagc
ccatctgtgg cctccgcgcc acgcccagcc ccgaggcccg tgtactgcag ggagtcctag
tgaggtacag ccccgtggtg gaggccggct cggacatggt cttccggtgg accatcaacg
acaagcagtc cctgaccttc cagaacgtgg tcttcaatgt catttatcag agcgcggcgg
tcttcaagct ctcactgacg gcctccaacc acgtgagcaa cgtcaccgtg aactacaacg
taaccgtgga gcggatgaac aggatgcagg gtctgcaggt ctccacagtg ccggccgtgc
tgtcccccaa tgccacgcta gcactgacgg cgggcgtgct ggtggactcg gccgtggagg
tggccttcct gtggaccttt ggggatgggg agcaggccct ccaccagttc cagcctccgt
acaacgagtc cttcccggtt ccagacccct cggtggccca ggtgctggtg gagcacaatg
tcatgcacac ctacgctgcc ccaggtgagt acctcctgac cgtgctggca tctaatgcct
tcgagaacct gacgcagcag gtgcctgtga gcgtgcgcgc ctccctgccc tccgtggctg
tgggtgtgag tgacggcgtc ctggtggccg gccggcccgt caccttctac ccgcacccgc
tgccctcgcc tgggggtgtt ctttacacgt gggacttcgg ggacggctcc cctgtcctga
cccagagcca gccggctgcc aaccacacct atgcctcgag gggcacctac cacgtgcgcc
tggaggtcaa caacacggtg agcggtgcgg cggcccaggc ggatgtgcgc gtctttgagg
agctccgcgg actcagcgtg gacatgagcc tggccgtgga gcagggcgcc cccgtggtgg
tcagcgccgc ggtgcagacg ggcgacaaca tcacgtggac cttcgacatg ggggacggca
ccgtgctgtc gggcccggag gcaacagtgg agcatgtgta cctgcgggca cagaactgca
cagtgaccgt gggtgcggcc agccccgccg gccacctggc ccggagcctg cacgtgctgg
tcttcgtcct ggaggtgctg cgcgttgaac ccgccgcctg catccccacg cagcctgacg
cgcggctcac ggcctacgtc accgggaacc cggcccacta cctcttcgac tggaccttcg
gggatggctc ctccaacacg accgtgcggg ggtgcccgac ggtgacacac aacttcacgc
ggagcggcac gttccccctg gcgctggtgc tgtccagccg cgtgaacagg gcgcattact
tcaccagcat ctgcgtggag ccagaggtgg gcaacgtcac cctgcagcca gagaggcagt
ttgtgcagct cggggacgag gcctggctgg tggcatgtgc ctggcccccg ttcccctacc
gctacacctg ggactttggc accgaggaag ccgcccccac ccgtgccagg ggccctgagg
tgacgttcat ctaccgagac ccaggctcct atcttgtgac agtcaccgcg tccaacaaca
tctctgctgc caatgactca gccctggtgg aggtgcagga gcccgtgctg gtcaccagca
tcaaggtcaa tggctccctt gggctggagc tgcagcagcc gtacctgttc tctgctgtgg
gccgtgggcg ccccgccagc tacctgtggg atctggggga cggtgggtgg ctcgagggtc
cggaggtcac ccacgcttac aacagcacag gtgacttcac cgttagggtg gccggctgga
atgaggtgag ccgcagcgag gcctggctca atgtgacggt gaagcggcgc gtgcgggggc
tcgtcgtcaa tgcaagccgc acggtggtgc ccctgaatgg gagcgtgagc ttcagcacgt
cgctggaggc cggcagtgat gtgcgctatt cctgggtgct ctgtgaccgc tgcacgccca
tccctggggg tcctaccatc tcttacacct tccgctccgt gggcaccttc aatatcatcg
tcacggctga gaacgaggtg ggctccgccc aggacagcat cttcgtctat gtcctgcagc
tcatagaggg gctgcaggtg gtgggcggtg gccgctactt ccccaccaac cacacggtac
agctgcaggc cgtggttagg gatggcacca acgtctccta cagctggact gcctggaggg
acaggggccc ggccctggcc ggcagcggca aaggcttctc gctcaccgtg ctcgaggccg
gcacctacca tgtgcagctg cgggccacca acatgctggg cagcgcctgg gccgactgca
ccatggactt cgtggagcct gtggggtggc tgatggtggc cgcctccccg aacccagctg
ccgtcaacac aagcgtcacc ctcagtgccg agctggctgg tggcagtggt gtcgtataca
cttggtcctt ggaggagggg ctgagctggg agacctccga gccatttacc acccatagct
tccccacacc cggcctgcac ttggtcacca tgacggcagg gaacccgctg ggctcagcca
acgccaccgt ggaagtggat gtgcaggtgc ctgtgagtgg cctcagcatc agggccagcg
agcccggagg cagcttcgtg gcggccgggt cctctgtgcc cttttggggg cagctggcca
cgggcaccaa tgtgagctgg tgctgggctg tgcccggcgg cagcagcaag cgtggccctc
atgtcaccat ggtcttcccg gatgctggca ccttctccat ccggctcaat gcctccaacg
cagtcagctg ggtctcagcc acgtacaacc tcacggcgga ggagcccatc gtgggcctgg
tgctgtgggc cagcagcaag gtggtggcgc ccgggcagct ggtccatttt cagatcctgc
tggctgccgg ctcagctgtc accttccgcc tgcaggtcgg cggggccaac cccgaggtgc
tccccgggcc ccgtttctcc cacagcttcc cccgcgtcgg agaccacgtg gtgagcgtgc
ggggcaaaaa ccacgtgagc tgggcccagg cgcaggtgcg catcgtggtg ctggaggccg
tgagtgggct gcaggtgccc aactgctgcg agcctggcat cgccacgggc actgagagga
acttcacagc ccgcgtgcag cgcggctctc gggtcgccta cgcctggtac ttctcgctgc
agaaggtcca gggcgactcg ctggtcatcc tgtcgggccg cgacgtcacc tacacgcccg
tggccgcggg gctgttggag atccaggtgc gcgccttcaa cgccctgggc agtgagaacc
gcacgctggt gctggaggtt caggacgccg tccagtatgt ggccctgcag agcggcccct
gcttcaccaa ccgctcggcg cagtttgagg ccgccaccag ccccagcccc cggcgtgtgg
cctaccactg ggactttggg gatgggtcgc cagggcagga cacagatgag cccagggccg
agcactccta cctgaggcct ggggactacc gcgtgcaggt gaacgcctcc aacctggtga
gcttcttcgt ggcgcaggcc acggtgaccg tccaggtgct ggcctgccgg gagccggagg
tggacgtggt cctgcccctg caggtgctga tgcggcgatc acagcgcaac tacttggagg
cccacgttga cctgcgcgac tgcgtcacct accagactga gtaccgctgg gaggtgtatc
gcaccgccag ctgccagcgg ccggggcgcc cagcgcgtgt ggccctgccc ggcgtggacg
tgagccggcc tcggctggtg ctgccgcggc tggcgctgcc tgtggggcac tactgctttg
tgtttgtcgt gtcatttggg gacacgccac tgacacagag catccaggcc aatgtgacgg
tggcccccga gcgcctggtg cccatcattg agggtggctc ataccgcgtg tggtcagaca
cacgggacct ggtgctggat gggagcgagt cctacgaccc caacctggag gacggcgacc
agacgccgct cagtttccac tgggcctgtg tggcttcgac acagagggag gctggcgggt
gtgcgctgaa ctttgggccc cgcgggagca gcacggtcac cattccacgg gagcggctgg
cggctggcgt ggagtacacc ttcagcctga ccgtgtggaa ggccggccgc aaggaggagg
ccaccaacca gacggtgctg atccggagtg gccgggtgcc cattgtgtcc ttggagtgtg
tgtcctgcaa ggcacaggcc gtgtacgaag tgagccgcag ctcctacgtg tacttggagg
gccgctgcct caattgcagc agcggctcca agcgagggcg gtgggctgca cgtacgttca
gcaacaagac gctggtgctg gatgagacca ccacatccac gggcagtgca ggcatgcgac
tggtgctgcg gcggggcgtg ctgcgggacg gcgagggata caccttcacg ctcacggtgc
tgggccgctc tggcgaggag gagggctgcg cctccatccg cctgtccccc aaccgcccgc
cgctgggggg ctcttgccgc ctcttcccac tgggcgctgt gcacgccctc accaccaagg
tgcacttcga atgcacgggc tggcatgacg cggaggatgc tggcgccccg ctggtgtacg
ccctgctgct gcggcgctgt cgccagggcc actgcgagga gttctgtgtc tacaagggca
gcctctccag ctacggagcc gtgctgcccc cgggtttcag gccacacttc gaggtgggcc
tggccgtggt ggtgcaggac cagctgggag ccgctgtggt cgccctcaac aggtctttgg
ccatcaccct cccagagccc aacggcagcg caacggggct cacagtctgg ctgcacgggc
tcaccgctag tgtgctccca gggctgctgc ggcaggccga tccccagcac gtcatcgagt
actcgttggc cctggtcacc gtgctgaacg agtacgagcg ggccctggac gtggcggcag
agcccaagca cgagcggcag caccgagccc agatacgcaa gaacatcacg gagactctgg
tgtccctgag ggtccacact gtggatgaca tccagcagat cgctgctgcg ctggcccagt
gcatggggcc cagcagggag ctcgtatgcc gctcgtgcct gaagcagacg ctgcacaagc
tggaggccat gatgctcatc ctgcaggcag agaccaccgc gggcaccgtg acgcccaccg
ccatcggaga cagcatcctc aacatcacag gagacctcat ccacctggcc agctcggacg
tgcgggcacc acagccctca gagctgggag ccgagtcacc atctcggatg gtggcgtccc
aggcctacaa cctgacctct gccctcatgc gcatcctcat gcgctcccgc gtgctcaacg
aggagcccct gacgctggcg ggcgaggaga tcgtggccca gggcaagcgc tcggacccgc
ggagcctgct gtgctatggc ggcgccccag ggcctggctg ccacttctcc atccccgagg
ctttcagcgg ggccctggcc aacctcagtg acgtggtgca gctcatcttt ctggtggact
ccaatccctt tccctttggc tatatcagca actacaccgt ctccaccaag gtggcctcga
tggcattcca gacacaggcc ggcgcccaga tccccatcga gcggctggcc tcagagcgcg
ccatcaccgt gaaggtgccc aacaactcgg actgggctgc ccggggccac cgcagctccg
ccaactccgc caactccgtt gtggtccagc cccaggcctc cgtcggtgct gtggtcaccc
tggacagcag caaccctgcg gccgggctgc atctgcagct caactatacg ctgctggacg
gccactacct gtctgaggaa cctgagccct acctggcagt ctacctacac tcggagcccc
ggcccaatga gcacaactgc tcggctagca ggaggatccg cccagagtca ctccagggtg
ctgaccaccg gccctacacc ttcttcattt ccccggggag cagagaccca gcggggagtt
accatctgaa cctctccagc cacttccgct ggtcggcgct gcaggtgtcc gtgggcctgt
acacgtccct gtgccagtac ttcagcgagg aggacatggt gtggcggaca gaggggctgc
tgcccctgga ggagacctcg ccccgccagg ccgtctgcct cacccgccac ctcaccgcct
tcggcgccag cctcttcgtg cccccaagcc atgtccgctt tgtgtttcct gagccgacag
cggatgtaaa ctacatcgtc atgctgacat gtgctgtgtg cctggtgacc tacatggtca
tggccgccat cctgcacaag ctggaccagt tggatgccag ccggggccgc gccatccctt
tctgtgggca gcggggccgc ttcaagtacg agatcctcgt caagacaggc tggggccggg
gctcaggtac cacggcccac gtgggcatca tgctgtatgg ggtggacagc cggagcggcc
accggcacct ggacggcgac agagccttcc accgcaacag cctggacatc ttccggatcg
ccaccccgca cagcctgggt agcgtgtgga agatccgagt gtggcacgac aacaaagggc
tcagccctgc ctggttcctg cagcacgtca tcgtcaggga cctgcagacg gcacgcagcg
ccttcttcct ggtcaatgac tggctttcgg tggagacgga ggccaacggg ggcctggtgg
agaaggaggt gctggccgcg agcgacgcag cccttttgcg cttccggcgc ctgctggtgg
ctgagctgca gcgtggcttc tttgacaagc acatctggct ctccatatgg gaccggccgc
ctcgtagccg tttcactcgc atccagaggg ccacctgctg cgttctcctc atctgcctct
tcctgggcgc caacgccgtg tggtacgggg ctgttggcga ctctgcctac agcacggggc
atgtgtccag gctgagcccg ctgagcgtcg acacagtcgc tgttggcctg gtgtccagcg
tggttgtcta tcccgtctac ctggccatcc tttttctctt ccggatgtcc cggagcaagg
tggctgggag cccgagcccc acacctgccg ggcagcaggt gctggacatc gacagctgcc
tggactcgtc cgtgctggac agctccttcc tcacgttctc aggcctccac gctgaggcct
ttgttggaca gatgaagagt gacttgtttc tggatgattc taagagtctg gtgtgctggc
cctccggcga gggaacgctc agttggccgg acctgctcag tgacccgtcc attgtgggta
gcaatctgcg gcagctggca cggggccagg cgggccatgg gctgggccca gaggaggacg
gcttctccct ggccagcccc tactcgcctg ccaaatcctt ctcagcatca gatgaagacc
tgatccagca ggtccttgcc gagggggtca gcagcccagc ccctacccaa gacacccaca
tggaaacgga cctgctcagc agcctgtcca gcactcctgg ggagaagaca gagacgctgg
cgctgcagag gctgggggag ctggggccac ccagcccagg cctgaactgg gaacagcccc
aggcagcgag gctgtccagg acaggactgg tggagggtct gcggaagcgc ctgctgccgg
cctggtgtgc ctccctggcc cacgggctca gcctgctcct ggtggctgtg gctgtggctg
tctcagggtg ggtgggtgcg agcttccccc cgggcgtgag tgttgcgtgg ctcctgtcca
gcagcgccag cttcctggcc tcattcctcg gctgggagcc actgaaggtc ttgctggaag
ccctgtactt ctcactggtg gccaagcggc tgcacccgga tgaagatgac accctggtag
agagcccggc tgtgacgcct gtgagcgcac gtgtgccccg cgtacggcca ccccacggct
ttgcactctt cctggccaag gaagaagccc gcaaggtcaa gaggctacat ggcatgctgc
ggagcctcct ggtgtacatg ctttttctgc tggtgaccct gctggccagc tatggggatg
cctcatgcca tgggcacgcc taccgtctgc aaagcgccat caagcaggag ctgcacagcc
gggccttcct ggccatcacg cggtctgagg agctctggcc atggatggcc cacgtgctgc
tgccctacgt ccacgggaac cagtccagcc cagagctggg gcccccacgg ctgcggcagg
tgcggctgca ggaagcactc tacccagacc ctcccggccc cagggtccac acgtgctcgg
ccgcaggagg cttcagcacc agcgattacg acgttggctg ggagagtcct cacaatggct
cggggacgtg ggcctattca gcgccggatc tgctgggggc atggtcctgg ggctcctgtg
ccgtgtatga cagcgggggc tacgtgcagg agctgggcct gagcctggag gagagccgcg
accggctgcg cttcctgcag ctgcacaact ggctggacaa caggagccgc gctgtgttcc
tggagctcac gcgctacagc ccggccgtgg ggctgcacgc cgccgtcacg ctgcgcctcg
agttcccggc ggccggccgc gccctggccg ccctcagcgt ccgccccttt gcgctgcgcc
gcctcagcgc gggcctctcg ctgcctctgc tcacctcggt gtgcctgctg ctgttcgccg
tgcacttcgc cgtggccgag gcccgtactt ggcacaggga agggcgctgg cgcgtgctgc
ggctcggagc ctgggcgcgg tggctgctgg tggcgctgac ggcggccacg gcactggtac
gcctcgccca gctgggtgcc gctgaccgcc agtggacccg tttcgtgcgc ggccgcccgc
gccgcttcac tagcttcgac caggtggcgc agctgagctc cgcagcccgt ggcctggcgg
cctcgctgct cttcctgctt ttggtcaagg ctgcccagca gctacgcttc gtgcgccagt
ggtccgtctt tggcaagaca ttatgccgag ctctgccaga gctcctgggg gtcaccttgg
gcctggtggt gctcggggta gcctacgccc agctggccat cctgctcgtg tcttcctgtg
tggactccct ctggagcgtg gcccaggccc tgttggtgct gtgccctggg actgggctct
ctaccctgtg tcctgccgag tcctggcacc tgtcacccct gctgtgtgtg gggctctggg
cactgcggct gtggggcgcc ctacggctgg gggctgttat tctccgctgg cgctaccacg
ccttgcgtgg agagctgtac cggccggcct gggagcccca ggactacgag atggtggagt
tgttcctgcg caggctgcgc ctctggatgg gcctcagcaa ggtcaaggag ttccgccaca
aagtccgctt tgaagggatg gagccgctgc cctctcgctc ctccaggggc tccaaggtat
ccccggatgt gcccccaccc agcgctggct ccgatgcctc gcacccctcc acctcctcca
gccagctgga tgggctgagc gtgagcctgg gccggctggg gacaaggtgt gagcctgagc
cctcccgcct ccaagccgtg ttcgaggccc tgctcaccca gtttgaccga ctcaaccagg
ccacagagga cgtctaccag ctggagcagc agctgcacag cctgcaaggc cgcaggagca
gccgggcgcc cgccggatct tcccgtggcc catccccggg cctgcggcca gcactgccca
gccgccttgc ccgggccagt cggggtgtgg acctggccac tggccccagc aggacacccc
ttcgggccaa gaacaaggtc caccccagca gcacttagtc ctccttcctg gcgggggtgg
gccgtggagt cggagtggac accgctcagt attactttct gccgctgtca aggccgaggg
ccaggcagaa tggctgcacg taggttcccc agagagcagg caggggcatc tgtctgtctg
tgggcttcag cactttaaag aggctgtgtg gccaaccagg acccagggtc ccctccccag
ctcccttggg aaggacacag cagtattgga cggtttctag cctctgagat gctaatttat
ttccccgagt cctcaggtac agcgggctgt gcccggcccc accccctggg cagatgtccc
ccactgctaa ggctgctggc ttcagggagg gttagcctgc accgccgcca ccctgcccct
aagttattac ctctccagtt cctaccgtac tccctgcacc gtctcactgt gtgtctcgtg
tcagtaattt atatggtgtt aaaatgtgta tatttttgta tgtcactatt ttcactaggg
ctgaggggcc tgcgcccaga gctggcctcc cccaacacct gctgcgcttg gtaggtgtgg
tggcgttatg gcagcccggc tgctgcttgg atgcgagctt ggccttgggc cggtgctggg
ggcacagctg tctgccaggc actctcatca ccccagaggc cttgtcatcc tcccttgccc
caggccaggt agcaagagag cagcgcccag gcctgctggc atcaggtctg ggcaagtagc
aggactaggc atgtcagagg accccagggt ggttagagga aaagactcct cctgggggct
ggctcccagg gtggaggaag gtgactgtgt gtgtgtgtgt gtgcgcgcgc gcacgcgcga
gtgtgctgta tggcccaggc agcctcaagg ccctcggagc tggctgtgcc tgcttctgtg
taccacttct gtgggcatgg ccgcttctag agcctcgaca
cccccccaac ccccgcacca
Set out below are the nucleotide sequences of the biomarkers described herein. Exemplary target regions within the biomarker sequences are underlined, and exemplary probe sequences are underlined and shown in bold text.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1616557.3 | Sep 2016 | GB | national |
This application is a continuation of U.S. patent application Ser. No. 16/335,830, filed Mar. 22, 2019, which is the National Stage of International Application No. PCT/GB2017/052945, filed Sep. 29, 2017, which claims priority to Great Britain Application No. 1616557.3, filed Sep. 29, 2016, the disclosures of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16335830 | Mar 2019 | US |
| Child | 18316443 | US |
