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A PROMISING BIOMARKER FOR DETECTION OF SEPSIS
Advanced Clinical Chemistry Course
DR. MAZEN AL-ZAHARNA
Ahmed Adel Abdallah
Islamic University of Gaza
March 2021
OBJECTIVES
• Define sepsis
• Sepsis pathophysiology
• Biomarkers for detection of sepsis
• sCD14 ST (Presepsin)
• Comparison b/w Presepsin, PCT, and CRP.
• Conclusion
• Reference
THE EVOLUTION OF “SEPSIS”
DEFINITION
• The word sepsis is derived from the Greek word for “decomposition” or “decay”.
• In 1991, consensus conference developed initial definitions that sepsis is a systemic
response to infection, manifested by two or more of the SIRS criteria as a result of
infection.
• Severe Sepsis: Sepsis plus sepsis-induced organ dysfunction or tissue hypo perfusion.
• Septic Shock: Sepsis-induced hypotension persisting despite adequate fluid
resuscitation
• In 2001, Definitions of sepsis and septic shock were revised to incorporate the
threshold values for organ damage.
Infection
SIRS
SEPSIS
SEPSIS-1,2 Paradigm
Detection of sepsis and septic shock
At least 2 of 4 criteria:
1. Body temperature: >38°C or <36°C
2. White blood count: >12,000 µL or
<4,000/µL.
OR > 10% immature neutrophils
3. Heart rate: >90bpm
4. Respiratory rate: >20/min
SIRS CRITERIA
SEPSIS-3 DEFINITION
• In 2016, the new definitions of sepsis and septic shock have changed dramatically.
• According to 2016 definition, Sepsis is now defined as life-threatening organ
dysfunction caused by a dysregulated host response to infection.
• Organ dysfunction can be represented by an increase in SOFA (Sequential (sepsis-
related) Organ Function Assessment) score of 2 points or more (associated with
in hospital mortality of 10%)
• Because the new sepsis definition contains “life threatening organ dysfunction,”
the classification “severe sepsis” was felt to be unnecessary
• Clinical criteria for sepsis:
Suspected or documented infection and an acute increase of ⩾2 SOFA points.
• Septic Shock: persisting hypotension requiring vasopressor to maintain MAP 65
mmHg or higher, and Serum lactate level greater than 2 mmol/L (18 mg/dL) despite
adequate volume resuscitation.
• This combination is associated with hospital mortality rates greater than 40%
• Organ dysfunction: an increase in the Sequential [Sepsis-related] Organ Failure
Assessment (SOFA) score of 2 points or more, which is associated with an in-hospital
mortality greater than 10%.
SEPSIS-3 DEF. CONT.
SEPSIS-3 2016 PARADIGM
• Sepsis = Infection + ≥2 pt ↑SOFA score
• Septic Shock = Sepsis + (vasopressor therapy needed to elevate MAP
≥65mm Hg + lactate >2mmol/L despite adequate fluid resuscitation)
Infection
Sepsis:
early signs of
organ failure
Septic Shock:
tissue hypo
perfusion
Detection of sepsis and septic shock
SEQUENTIAL (SEPSIS-RELATED) ORGAN
FAILURE ASSESSMENT (SOFA)
• SOFA uses simple measurements of major organ
function to calculate a severity score.
• The scores are calculated 24 hours after admission to the ICU and every 48 hours.
• The highest scores are most predictive of mortality.
Detection of sepsis and septic shock
SEQUENTIAL (SEPSIS-RELATED) ORGAN
FAILURE ASSESSMENT (SOFA)
The SOFA severity score is based upon the following:
• Respiratory system – the ratio of arterial oxygen tension to fraction of inspired oxygen (PaO2/FiO2)
• Cardiovascular system – the amount of vasoactive medication necessary to prevent hypotension
• Hepatic system – the bilirubin level
• Coagulation system – the platelet concentration
• Neurologic system – the Glasgow coma score
• Renal system – the serum creatinine or urine output
Detection of sepsis and septic shock
Detection of sepsis and septic shock
IDENTIFICATION OF EARLY SEPSIS (QSOFA)
• This score is a modified version of the Sequential (Sepsis-related) Organ Failure Assessment score
(SOFA)
• qSOFA is used to help identifying patients suspected having early sepsis in the ED .
• A score more than 2 is associated with poor outcomes.
Detection of sepsis and septic shock
Detection of sepsis and septic shock
ETIOLOGY
PATHOGENESIS
• The pathogenesis of the sepsis syndrome or SIRS can
be explained by three mechanisms, all of which involve the release of mediators that result in systemic
inflammatory response.
• Mechanism 1: The Pro-inflammatory Response.
• Mechanism 2: Failure of the Compensatory Anti-inflammatory Response (CARS) to Act
• An imbalance between pro-inflammatory response and anti-inflammatory response is believed to occur
• during infection. This permits the pro-inflammatory mediators to induce an uncontrolled excessive
inflammatory process.
• Mechanism 3: Immunoparalysis
• Mediators of inflammation overwhelm the existing immune system and paralyze it.
PATHOGENESIS, CONT.
PATHOGENESIS, CONT.
• The lysis of Gram-negative bacteria causes them to release lipopolysaccharide.
• The LPS binds to a LPS-binding protein circulating in the blood and this complex, in turn, binds to a
receptor molecule (CD14) found on the surface of body defense cells called macrophages.
• This promote the ability of the toll-like receptor TLR-4 to trigger the macrophage to release cytokines,
including IL-1, IL-6, IL-8, TNF-alpha, and PAF.
• The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate
both the complement pathways and the coagulation pathway.
BIOMARKERS
OF SEPSIS
DIAGNOSIS
• Positive blood cultures remain the gold standard for the diagnosis; however, the false-negative rate is
high and the result is not directly available.
• CRP and PCT have some issues about their diagnostic accuracy, which prevent clinicians from starting
or withholding antimicrobial therapy.
• The results from many published reviews indicate that the sensitivity and specificity for CRP (ranged
from 35 to 100% and from 18 to 84%, respectively) and PCT vary (ranged from 42 to 100% and from 48
to 100%, respectively)
• CRP level increases in 4–6 h and reaches the peak in 48–72 h after the inflammatory onset.
while PCT level increases in 8–24 h and reaches the peak later than 24 h
• Therefore, both PCT and CRP might not be reliable enough as early indicators for sepsis.
• Cluster-of-differentiation 14 (CD14) is a cell surface glycoprotein, exists in two forms,
membrane-bound CD14 (mCD14), and soluble CD14 (sCD14)
• CD14 serves as the receptor expressed on the surface of various kinds of immune
cells, such as monocytes, macrophages and neutrophils. CD14 has high affinity to
bind with Lipopolysaccharide-lipopolysaccharide binding protein (LPS-LBP) complex.
• CD14 has the ability to identify and interact with several ligands of both Gram
positive (e.g., peptoglycan and lipoteichoic acid), Gram negative bacteria (e.g., LPS),
and fungal antigen.
PRESEPSIN (SCD14-ST)
• CD14 activates the intracellular inflammatory response of the Toll-Like receptor 4 (TLR4),
which leads to the triggering the host´s inflammatory cascade against the infectious
pathogenic agent.
• The molecular complex CD14-LPS-LBP is internalized into a phagolysosome.
• CD14-LPS-LBP is exposed to an enzymatic processing that needs cathepsin D.
• sCD14 is cleaved by proteases, releasing a small soluble peptide fragment, called sCD14-
ST or presepsin.
• presepsin is then released in the general circulation by proteolysis and exocytosis.
• The serum level of presepsin (sCD14-ST) elevates in patients with sepsis compared to
healthy controls and patients presenting non-infectious systemic inflammatory response
syndrome (SIRS).
PRESEPSIN (SCD14-ST)
Detection of sepsis and septic shock
• presepsin was first discovered in 2002, as a blood biomarker in patients with sepsis in
Japan .
• Many studies have demonstrated that presepsin (sCD14-ST) can significantly increase
within 2 h and peak at 3 h after the onset of infection.
• Also, studies have shown that presepsin (sCD14-ST) has advantages over PCT, CRP, and IL-
6 in diagnosing the sensitivity and specificity of sepsis and assessing disease severity and
prognosis.
• Presepsin can also be used for monitoring of treatment with antibiotics and can show
effectiveness of antibiotics.
• Presepsin appears to be quite promising and reliable tool for early diagnosis of sepsis
caused by Gram-positive and Gram-negative bacteria or fungi.
PRESEPSIN (SCD14-ST)
Reference interval
COMPARISON BETWEEN PRESEPSIN, PCT, AND CRP
HUMAN PRESEPSIN (SCD14-ST) ELISA KIT
• The kit is based on sandwich enzyme-linked immuno-sorbent assay technology.
• Capture antibody was pre-coated onto 96-well plates.
• The standards, test samples and conjugated antibody added to the wells subsequently
• wash buffer.
• HRP-Streptavidin added and unbound conjugates washed away with wash buffer.
• TMB substrates were used to visualize HRP enzymatic reaction.
• a blue color of product changes into yellow after adding acidic stop solution
• The density of yellow is proportional to the target amount of sample captured in plate
• Read the O.D. absorbance at 450nm, then calculate concentration.
Detection of sepsis and septic shock
PATHFASTTM
• PATHFASTTM Presepsin is a chemiluminescent enzyme immunoassay
(CLEIA) for quantitative measurement of the Presepsin concentration
in whole blood or plasma.
• The result out in less than 17 minutes.
• Take 3
• 1. CULTURES: Take at least one blood cultures before giving antimicrobials. Consider e.g. CSF, urine,
sputum
2. BLOODS: Check point of care lactate, FBC, U&E, LFTS, +/- Coag.
• 3. URINE OUTPUT: Assess urine output and consider urinary catheterization for accurate
measurement in patients with severe sepsis/septic shock.
• Give 3
• 1. OXYGEN: Titrate O2 to saturations of 94 -98%
• 2. FLUIDS: Start IV fluid resuscitation if evidence of hypovolaemia. 500ml bolus of isotonic
crystalloid over 15mins & give up to 30ml/kg, reassessing for signs of hypovolaemia, or
fluid overload.
• 3. ANTIMICROBIALS: Give IV antimicrobials according to local antimicrobial guidelines.
MANAGEMENT
“6 BUNDLE DELIVERED IN 1 HOUR”
REFERENCES
• Memar, M. Y., & Baghi, H. B. (2019). Presepsin: A promising biomarker for the detection of bacterial infections.
Biomedicine & Pharmacotherapy, 111, 649–656. doi:10.1016/j.biopha.2018.12.124
• Fay, K., Sapiano, M. R. P., Gokhale, R., Dantes, R., Thompson, N., Katz, D. E., … Epstein, L. (2020). Assessment of
Health Care Exposures and Outcomes in Adult Patients With Sepsis and Septic Shock. JAMA Network Open,
3(7), e206004. doi:10.1001/jamanetworkopen.2020.6004
• Howell, M. D., & Davis, A. M. (2017). Management of Sepsis and Septic Shock. JAMA, 317(8),
847. doi:10.1001/jama.2017.0131
• Gyawali, B., Ramakrishna, K., & Dhamoon, A. S. (2019). Sepsis: The evolution in definition, pathophysiology,
and management. SAGE Open Medicine, 7, 205031211983504. doi:10.1177/2050312119835043
• Wu, C.-C., Lan, H.-M., Han, S.-T., Chaou, C.-H., Yeh, C.-F., Liu, S.-H., … Chen, K.-F. (2017). Comparison of
diagnostic accuracy in sepsis between presepsin, procalcitonin, and C-reactive protein: a systematic review
and meta-analysis. Annals of Intensive Care, 7(1). doi:10.1186/s13613-017-0316-z
• Taeb, A. M., Hooper, M. H., & Marik, P. E. (2017). Sepsis: Current Definition, Pathophysiology, Diagnosis, and
Management. Nutrition in Clinical Practice, 32(3), 296–308. doi:10.1177/0884533617695243

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Detection of sepsis and septic shock

  • 1. A PROMISING BIOMARKER FOR DETECTION OF SEPSIS Advanced Clinical Chemistry Course DR. MAZEN AL-ZAHARNA Ahmed Adel Abdallah Islamic University of Gaza March 2021
  • 2. OBJECTIVES • Define sepsis • Sepsis pathophysiology • Biomarkers for detection of sepsis • sCD14 ST (Presepsin) • Comparison b/w Presepsin, PCT, and CRP. • Conclusion • Reference
  • 3. THE EVOLUTION OF “SEPSIS”
  • 4. DEFINITION • The word sepsis is derived from the Greek word for “decomposition” or “decay”. • In 1991, consensus conference developed initial definitions that sepsis is a systemic response to infection, manifested by two or more of the SIRS criteria as a result of infection. • Severe Sepsis: Sepsis plus sepsis-induced organ dysfunction or tissue hypo perfusion. • Septic Shock: Sepsis-induced hypotension persisting despite adequate fluid resuscitation • In 2001, Definitions of sepsis and septic shock were revised to incorporate the threshold values for organ damage.
  • 7. At least 2 of 4 criteria: 1. Body temperature: >38°C or <36°C 2. White blood count: >12,000 µL or <4,000/µL. OR > 10% immature neutrophils 3. Heart rate: >90bpm 4. Respiratory rate: >20/min SIRS CRITERIA
  • 8. SEPSIS-3 DEFINITION • In 2016, the new definitions of sepsis and septic shock have changed dramatically. • According to 2016 definition, Sepsis is now defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. • Organ dysfunction can be represented by an increase in SOFA (Sequential (sepsis- related) Organ Function Assessment) score of 2 points or more (associated with in hospital mortality of 10%) • Because the new sepsis definition contains “life threatening organ dysfunction,” the classification “severe sepsis” was felt to be unnecessary • Clinical criteria for sepsis: Suspected or documented infection and an acute increase of ⩾2 SOFA points.
  • 9. • Septic Shock: persisting hypotension requiring vasopressor to maintain MAP 65 mmHg or higher, and Serum lactate level greater than 2 mmol/L (18 mg/dL) despite adequate volume resuscitation. • This combination is associated with hospital mortality rates greater than 40% • Organ dysfunction: an increase in the Sequential [Sepsis-related] Organ Failure Assessment (SOFA) score of 2 points or more, which is associated with an in-hospital mortality greater than 10%. SEPSIS-3 DEF. CONT.
  • 10. SEPSIS-3 2016 PARADIGM • Sepsis = Infection + ≥2 pt ↑SOFA score • Septic Shock = Sepsis + (vasopressor therapy needed to elevate MAP ≥65mm Hg + lactate >2mmol/L despite adequate fluid resuscitation) Infection Sepsis: early signs of organ failure Septic Shock: tissue hypo perfusion
  • 12. SEQUENTIAL (SEPSIS-RELATED) ORGAN FAILURE ASSESSMENT (SOFA) • SOFA uses simple measurements of major organ function to calculate a severity score. • The scores are calculated 24 hours after admission to the ICU and every 48 hours. • The highest scores are most predictive of mortality.
  • 14. SEQUENTIAL (SEPSIS-RELATED) ORGAN FAILURE ASSESSMENT (SOFA) The SOFA severity score is based upon the following: • Respiratory system – the ratio of arterial oxygen tension to fraction of inspired oxygen (PaO2/FiO2) • Cardiovascular system – the amount of vasoactive medication necessary to prevent hypotension • Hepatic system – the bilirubin level • Coagulation system – the platelet concentration • Neurologic system – the Glasgow coma score • Renal system – the serum creatinine or urine output
  • 17. IDENTIFICATION OF EARLY SEPSIS (QSOFA) • This score is a modified version of the Sequential (Sepsis-related) Organ Failure Assessment score (SOFA) • qSOFA is used to help identifying patients suspected having early sepsis in the ED . • A score more than 2 is associated with poor outcomes.
  • 21. PATHOGENESIS • The pathogenesis of the sepsis syndrome or SIRS can be explained by three mechanisms, all of which involve the release of mediators that result in systemic inflammatory response. • Mechanism 1: The Pro-inflammatory Response. • Mechanism 2: Failure of the Compensatory Anti-inflammatory Response (CARS) to Act • An imbalance between pro-inflammatory response and anti-inflammatory response is believed to occur • during infection. This permits the pro-inflammatory mediators to induce an uncontrolled excessive inflammatory process. • Mechanism 3: Immunoparalysis • Mediators of inflammation overwhelm the existing immune system and paralyze it.
  • 23. PATHOGENESIS, CONT. • The lysis of Gram-negative bacteria causes them to release lipopolysaccharide. • The LPS binds to a LPS-binding protein circulating in the blood and this complex, in turn, binds to a receptor molecule (CD14) found on the surface of body defense cells called macrophages. • This promote the ability of the toll-like receptor TLR-4 to trigger the macrophage to release cytokines, including IL-1, IL-6, IL-8, TNF-alpha, and PAF. • The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway.
  • 25. DIAGNOSIS • Positive blood cultures remain the gold standard for the diagnosis; however, the false-negative rate is high and the result is not directly available. • CRP and PCT have some issues about their diagnostic accuracy, which prevent clinicians from starting or withholding antimicrobial therapy. • The results from many published reviews indicate that the sensitivity and specificity for CRP (ranged from 35 to 100% and from 18 to 84%, respectively) and PCT vary (ranged from 42 to 100% and from 48 to 100%, respectively) • CRP level increases in 4–6 h and reaches the peak in 48–72 h after the inflammatory onset. while PCT level increases in 8–24 h and reaches the peak later than 24 h • Therefore, both PCT and CRP might not be reliable enough as early indicators for sepsis.
  • 26. • Cluster-of-differentiation 14 (CD14) is a cell surface glycoprotein, exists in two forms, membrane-bound CD14 (mCD14), and soluble CD14 (sCD14) • CD14 serves as the receptor expressed on the surface of various kinds of immune cells, such as monocytes, macrophages and neutrophils. CD14 has high affinity to bind with Lipopolysaccharide-lipopolysaccharide binding protein (LPS-LBP) complex. • CD14 has the ability to identify and interact with several ligands of both Gram positive (e.g., peptoglycan and lipoteichoic acid), Gram negative bacteria (e.g., LPS), and fungal antigen. PRESEPSIN (SCD14-ST)
  • 27. • CD14 activates the intracellular inflammatory response of the Toll-Like receptor 4 (TLR4), which leads to the triggering the host´s inflammatory cascade against the infectious pathogenic agent. • The molecular complex CD14-LPS-LBP is internalized into a phagolysosome. • CD14-LPS-LBP is exposed to an enzymatic processing that needs cathepsin D. • sCD14 is cleaved by proteases, releasing a small soluble peptide fragment, called sCD14- ST or presepsin. • presepsin is then released in the general circulation by proteolysis and exocytosis. • The serum level of presepsin (sCD14-ST) elevates in patients with sepsis compared to healthy controls and patients presenting non-infectious systemic inflammatory response syndrome (SIRS). PRESEPSIN (SCD14-ST)
  • 29. • presepsin was first discovered in 2002, as a blood biomarker in patients with sepsis in Japan . • Many studies have demonstrated that presepsin (sCD14-ST) can significantly increase within 2 h and peak at 3 h after the onset of infection. • Also, studies have shown that presepsin (sCD14-ST) has advantages over PCT, CRP, and IL- 6 in diagnosing the sensitivity and specificity of sepsis and assessing disease severity and prognosis. • Presepsin can also be used for monitoring of treatment with antibiotics and can show effectiveness of antibiotics. • Presepsin appears to be quite promising and reliable tool for early diagnosis of sepsis caused by Gram-positive and Gram-negative bacteria or fungi. PRESEPSIN (SCD14-ST)
  • 32. HUMAN PRESEPSIN (SCD14-ST) ELISA KIT • The kit is based on sandwich enzyme-linked immuno-sorbent assay technology. • Capture antibody was pre-coated onto 96-well plates. • The standards, test samples and conjugated antibody added to the wells subsequently • wash buffer. • HRP-Streptavidin added and unbound conjugates washed away with wash buffer. • TMB substrates were used to visualize HRP enzymatic reaction. • a blue color of product changes into yellow after adding acidic stop solution • The density of yellow is proportional to the target amount of sample captured in plate • Read the O.D. absorbance at 450nm, then calculate concentration.
  • 34. PATHFASTTM • PATHFASTTM Presepsin is a chemiluminescent enzyme immunoassay (CLEIA) for quantitative measurement of the Presepsin concentration in whole blood or plasma. • The result out in less than 17 minutes.
  • 35. • Take 3 • 1. CULTURES: Take at least one blood cultures before giving antimicrobials. Consider e.g. CSF, urine, sputum 2. BLOODS: Check point of care lactate, FBC, U&E, LFTS, +/- Coag. • 3. URINE OUTPUT: Assess urine output and consider urinary catheterization for accurate measurement in patients with severe sepsis/septic shock. • Give 3 • 1. OXYGEN: Titrate O2 to saturations of 94 -98% • 2. FLUIDS: Start IV fluid resuscitation if evidence of hypovolaemia. 500ml bolus of isotonic crystalloid over 15mins & give up to 30ml/kg, reassessing for signs of hypovolaemia, or fluid overload. • 3. ANTIMICROBIALS: Give IV antimicrobials according to local antimicrobial guidelines. MANAGEMENT “6 BUNDLE DELIVERED IN 1 HOUR”
  • 36. REFERENCES • Memar, M. Y., & Baghi, H. B. (2019). Presepsin: A promising biomarker for the detection of bacterial infections. Biomedicine & Pharmacotherapy, 111, 649–656. doi:10.1016/j.biopha.2018.12.124 • Fay, K., Sapiano, M. R. P., Gokhale, R., Dantes, R., Thompson, N., Katz, D. E., … Epstein, L. (2020). Assessment of Health Care Exposures and Outcomes in Adult Patients With Sepsis and Septic Shock. JAMA Network Open, 3(7), e206004. doi:10.1001/jamanetworkopen.2020.6004 • Howell, M. D., & Davis, A. M. (2017). Management of Sepsis and Septic Shock. JAMA, 317(8), 847. doi:10.1001/jama.2017.0131 • Gyawali, B., Ramakrishna, K., & Dhamoon, A. S. (2019). Sepsis: The evolution in definition, pathophysiology, and management. SAGE Open Medicine, 7, 205031211983504. doi:10.1177/2050312119835043 • Wu, C.-C., Lan, H.-M., Han, S.-T., Chaou, C.-H., Yeh, C.-F., Liu, S.-H., … Chen, K.-F. (2017). Comparison of diagnostic accuracy in sepsis between presepsin, procalcitonin, and C-reactive protein: a systematic review and meta-analysis. Annals of Intensive Care, 7(1). doi:10.1186/s13613-017-0316-z • Taeb, A. M., Hooper, M. H., & Marik, P. E. (2017). Sepsis: Current Definition, Pathophysiology, Diagnosis, and Management. Nutrition in Clinical Practice, 32(3), 296–308. doi:10.1177/0884533617695243

Notes de l'éditeur

  1. https://www.atsu.edu/faculty/chamberlain/Website/lectures/lecture/sepsis.htm https://www.youtube.com/watch?v=Ig4dQgcpQDM&ab_channel=CentralBloodBank