Detection of sepsis and septic shock

Definition of sepsis and septic shock.
The new definition of sepsis 2016 conference.
SIRS, SOFA, QSOFA
Most common pathogen causing sepsis.
Pathogenesis and pathophysiology of sepsis
Biomarkers for detection of sepsis and septic shock
Preseason, sCD14 Subtype marker
Comparison of Procalcitonin and CRP with presepsin.
Mechanism of presepsin detection.
Management of sepsis.

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 .

5. SEPSIS-1,2 Paradigm

6. 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

7. 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.

8. 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.

9. 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)

10. 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.

11. 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

12. 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.

13. Etiology

14. 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.

15. PATHOGENESIS, cont.

16. 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.  

17. Biomarkers of sepsis

18. 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.

19. 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 ( S CD14-ST )

20. 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 ( S CD14-ST )

21. 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 ( S CD14-ST )

22. Reference interval

23. Comparison between presepsin, pct, and crp

24. 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.

25. PATHFAST TM PATHFAST TM  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.

26. 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”

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