Validation of Molecular and Protein Biomarkers in Sepsis
Validation of Molecular and Protein Biomarkers in Sepsis
Background:

 Sepsis (blood poisoning) is a clinical syndrome characterised by a dysregulated host response
 to infection causing life-threatening organ dysfunction which results in admission to an
 intensive care unit. It typically shows an initial harmful inflammation resulting from the
 immune system's overreaction to a severe infection. It is a major healthcare problem,
 affecting millions of people worldwide. In the UK, it kills over 37,000 people/year, costing
 the NHS £2.5 billion a year, and is increasing in incidence. Despite extensive efforts to
 tackle this burden, at present, however, there are no specific and effective therapies for
 this illness.

 Sepsis is a potentially life-threatening condition caused by a severe infection. When someone
 develops sepsis, inflammation occurs not just at the site of the infection but throughout the
 whole body. This widespread inflammation can be very harmful. It is known that similar
 responses occur in other conditions, not relating to infection.

 The investigators are recruiting patients with severe infections causing organ failure (also
 known as severe sepsis/ septicaemia and septic shock) and also patients where widespread
 inflammation, not related to infection, causes organ failure. In this study the investigators
 hope to find out whether certain groups of genetic and blood based protein markers of sepsis
 can forewarn the clinicians to this condition and also highlight patients who are responding
 well to the treatment.

 Although it is known that the majority of the patients suffering from sepsis will survive
 their ICU stay and leave the hospital alive, there is insufficient data how these patients do
 on a longer term, i.e. after some time at home. To date there is little information on the
 ability of the observed genetic and blood based protein markers to predict the functional
 status of the patients surviving these conditions.
Sepsis, defined as life threatening organ failure resulting from a dysregulated host response
 to infection, remains a leading cause of death in critically ill patients and has been
 included as a health priority in a 2017 WHO resolution. Diagnosis of this disorder is
 challenging because the clinical signs and symptoms of systemic inflammation in sepsis
 overlap with those of non-infectious critical conditions i.e. severe inflammatory response
 syndrome (SIRS) e.g. cardiac arrest and burns. Early and accurate diagnosis of sepsis is
 critical for improving patient outcomes and reducing antibiotic usage. Delays in antibiotic
 administration are associated with worse outcomes; however paradoxically, indiscriminate
 prescription of antibiotics to patients without bacterial infections increases both rates of
 morbidity and antimicrobial resistance. The rate of inappropriate antibiotic prescriptions in
 the hospital setting is estimated at 30 to 50% and would be decreased by access to improved
 diagnostic tests.

 There is currently no gold standard laboratory test that can broadly determine the presence
 and type of infection. Although new polymerase chain reaction (PCR)-based molecular
 diagnostics can profile pathogens directly from blood culture, they suffer from sensitivity
 issues due to dependence on sufficient numbers of pathogens in the blood sample. They are
 also limited to detection of a discrete range of pathogens. As a result, there is a growing
 focus on molecular diagnostics that profile the host immune response. Current sepsis
 groupings are based on clinical criteria such as the presence of shock, infection source, or
 organ failure, but such groupings may not represent the underlying biology driving the host
 response. They have also failed to adequately match patients for novel interventions. If the
 heterogeneity of sepsis truly reflects heterogeneity in the host response, characterisation
 of these underlying host response types will be fundamental to enabling precision sepsis
 therapeutics.

 In a previous multi-centre, clinical-temporal study in three cohorts of patients admitted to
 the intensive clinical care unit (ICU); (i) out of hospital cardiac arrest (n=36 - SIRS
 group)) (ii) pulmonary sepsis (n=84) (iii) abdominal sepsis (n=64) and 30 healthy controls,
 validated potential host immune biomarkers. Using 202 samples from these cohorts, the
 investigators derived a set of gene biomarkers which can identify patients with severe
 inflammation and discriminate sepsis from non-infected inflammation across a broad range of
 clinical conditions. Other biomarkers have been identified for use for other purposes e.g.
 prognosis/severity. Our patent arising from this work has been filed and entered PCT stage.
 From these patented markers a parsimonious set of 17 genes has been further delineated, which
 are under further evaluation. A sub-panel of two gene entities has been identified that can
 accurately detect severe inflammation using receiver operating characteristic/area under the
 curve (ROC) analysis with a value of approximately 0.98. A panel of three/four gene entities
 has been identified for discrimination of SIRS from all sepsis types (ROC 0.89-0.92), all
 depending on sensitivity or specificity range settings.

 Better diagnostics for sepsis-driven inflammation are needed in both inpatient and outpatient
 settings. In low-acuity outpatient settings, contributing circa 80% of total UK antibiotic
 use, a simple diagnostic to discriminate a septic inflammatory process from an innocuous,
 self-limiting condition, would assist in appropriate antimicrobial use, appropriate triage,
 avoiding further investigations, and appropriate escalation / admissions. In higher-acuity
 settings, causes of non-infectious inflammation are important to exclude; a decision model
 for antibiotic prescription should include a non-infected, non-healthy cause. A reliable
 diagnostic, such as ours, needs to distinguish all three presentations: non-infected
 inflammation, sepsis, and relative health. It will represent a major step-change in provision
 of diagnostic/stratification capability, vastly improve decision and patient management
 pathways and potentially reducing antibiotic overuse in the acute medical and critical-care
 environment.

 These biomarkers once validated in an independent cohort via qPCR for mRNA and their
 commensurate proteins, together with an accompanied easy-to-use, clinically oriented scoring
 system will represent a complete data package which can be rolled out internally, subject to
 the appropriate further accreditation and/or leveraged for development of point-of-care
 devices by commercial partners. This latter option could prove useful for dissemination of
 the test to other patent-appropriate global territories.

 There is an urgent need to validate these findings in several ways; Using an independent
 patient cohort, where laboratory scientists are blinded to the clinical phenotypes of the
 recruited patients and clinicians are not aware of the gene expression data.

 Using a bioinformatics approach to validate the results in already published datasets

 This project will use different approaches to validate these novel SIRS or sepsis-associated
 biomarkers identified by Artificial Neural Network (ANN) and parametric data mining of
 previously published datasets and further validated previously from a previous well
 characterised clinical cohort;

 1. Public Health England laboratories will assess biomarker mRNA expression using a qPCR
 approach, with RNA purified from patient and control whole blood

 2. Public Health England and Cardiff laboratories will assess protein biomarkers using
 ELISA assays

 3. Proteomic analysis of blood by external collaborators

 Further data analysis will be conducted using ROC curve analysis and arithmetic algorithms
 and/or other statistical/bioinformatics methods. Assessment of specificity and sensitivity
 and positive and negative predictive values using well established methods will also be
 conducted to evaluate the performance of the biomarker panels in discriminating patient
 control and disease groups.
Inclusion Criteria:

 -  Inclusion Criteria for community-acquired Sepsis and Septic Shock

 1. Age => 18

 2. Admission to hospital within 72 hours of symptoms onset and development of
 clinical signs of sepsis

 3. Diagnosis of sepsis

 -  SEPSIS is defined as a (1) DEFINED FOCUS OF INFECTION AND (2) SOFA>2, with
 at least ONE organ specific SOFA subscore =2.

 1. (1) 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, etc).

 2. (2) The SOFA score criteria are described in the Appendix [7]

 -  SEPTIC SHOCK is defined as SEPSIS plus the presence of hypotension requiring
 the use of vasopressors to maintain mean arterial pressure of 65 mmHg or
 greater and having a serum lactate level greater than 2 mmol/L persisting
 after adequate fluid resuscitation [8]

 Inclusion Criteria for Critically Ill patients without infection

 1. Patients admitted to the ICU following out-of hospital cardiac arrest OR Patients
 admitted to the ICU following major trauma (ISS>12) OR Patients admitted to the ICU
 following pancreatitis

 2. Have multiorgan failure as defined by SOFA>2, with at least ONE organ specific SOFA
 subscore =2.

 3. Patients must not be receiving antibiotics for treatment of known or suspected
 infection

 4. patient already has or will require arterial cannulation as part of standard treatment

 Exclusion Criteria:

 -  1. Patients who are on immune-modulating therapy (e.g., methotrexate, prednisolone
 >5mg/day, or other immunosuppressants) for any length of time within 6 months of index
 admission 2. Patients with acquired cellular immune deficiency (E.g. active HIV
 infection or AIDS); 3. Patients with concurrent blood-borne viral infections (E.g.
 Hepatitis B or C) 4. Patients with any haematological malignancy in their past medical
 history; 5. Patients who are on chronic haemodialysis; 6. Solid organ transplant
 recipients; 7. Patients who have biopsy, image or endoscopy proven liver cirrhosis; 8.
 Patients who are not expected to survive beyond 90 days due to the advancement of
 their underlying disease.