1. Sepsis Biomarkers Daniel B. Ambrus, MD MSc
Assistant Professor, Department of Hospital Medicine
SLAM Sepsis Symposium, October 31st, 2018

2. Disclosures
I have no actual or potential conflicts of interest with regard to this program/presentation
I am not an expert in sepsis or molecular biology
I did my best to do my homework!

3. Objectives
To establish the added value of biomarkers to clinical assessment of the septic patient
To review the various available biomarkers in sepsis, their performance and their current role
To identify barriers to biomarker implementation and look to the future

4. Sepsis Pathophysiology
Complex, heterogeneous process with multiple contributing factors
Gomes AP, et al. (2016) Pro-Inflammatory Cytokines in Sepsis: Biological Studies and Prospects From In Silico Research. Biol Syst Open Access 5:158

5. What is a Biomarker?
“…a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”
-Group BDW. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clinical Pharmacology and Therapeutics. 2001; 37:2290–2298.

6. Types of Biomarkers
Diagnostic: Establishes a diagnosis that enables a treatment decision rapidly & inexpensively
Monitoring: Measures response to intervention, enabling titration of treatment dose or duration
Surrogate: Not itself a measure of a patient-centered outcome but reliably predicts a clinical outcome
Prognostic: Identifies subgroups who are more likely to benefit from treatment or be harmed
Marshall JC, Reinhart K, International Sepsis F. Biomarkers of sepsis. Crit Care Med. 2009; 37(7): 2290–2298

7. Why Biomarkers? Diagnostic: Need better tools to rule sepsis in or out
SIRS criteria were mainstay since 2001
Most hospitalized patients experience SIRS without sepsis
1 in 8 patients who develop severe sepsis will not have 2/4 SIRS criteria upon first contact
Churpek et al. Am J Respir Crit Care Med 2015 Oct 15; 192(8):

8. Why Biomarkers?
Monitoring: Need to predict favorable response to antibiotics
How do we know which antibiotic to choose?
Up to 40% of patients admitted to intensive care units with a diagnosis of sepsis do not have an infection
2016 SSC guideline requires broad spectrum antibiotic in less than one hour for all patients with suspected sepsis
Stewardship concerns
IDSA did not endorse Surviving Sepsis Campaign guidelines in part over these concerns
IDSA Sepsis Task Force. Clinical Infectious Diseases, Volume 66, Issue 10, 2 May 2018, Pages 1631–1635

9. ROC and AUROC AUROC values: .90-1 = excellent (A) .80-.90 = good (B)
= fair (C)
= poor (D)
= fail (F)

10. Why Biomarkers?
Prognostic: Need to know who is at highest risk for and how to identify them at the first point of contact
Ability to discriminate in- hospital mortality prior to ICU admission by clinical criteria alone:
≥2 qSOFA: AUROC= 0.73; 95% CI, 0.65–0.81
2/4 SIRS: AUROC= 0.57; 95% CI, 0.48–0.66
Finkelsztein, EJ et al. Crit Care 2017; 21, 73

11. What About Lactate?
Diagnostic value: Poor sensitivity & specificity; complex physiology during sepsis is poorly understood
Monitoring value: As part of SEP-1, should be measured and repeated until <2 mmol/L
Prognostic value: Universally accepted as a marker for worse outcomes during sepsis

12. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

13. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$64
Pro-calcitonin
N/A
Up to 5d
Interleukin-8 No
Interleukin-27
Presepsin
Cell Free DNA
Neutrophil CD64
SeptiCyte
Paula Styles

14. We Need Something More
“The task force recognized that sepsis is a syndrome without, at present, a validated criterion standard diagnostic test... The task force determined that there was an important need for features that can be identified and measured in individual patients.”
-Singer M, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016; 315(8):801–810.

15. Procalcitonin: Vital Statistics
Quantitative
?Diagnostic, Monitoring, Prognostic
Normally made in small amounts by thyroid, infection causes it to be expressed by other tissues
Levels rise within 4-6 hours of infection and fall similarly quickly upon resolution of infection
Peaks after 12-48h

16. Procalcitonin: Diagnostic Value
May add diagnostic value to clinical assessment, not endorsed in guidelines
Meta-analysis by Tang et al., Lancet 2007:
Ability to discriminate sepsis from non-inflammatory SIRS: AUC =0.78
Authors concluded this was poor performance
Meta-analysis by Wacker et al., Lancet 2013:
Ability to discriminate sepsis from non-inflammatory SIRS: AUC =0.85
Authors concluded this was good performance
Data does not support starting antibiotics based on PCT levels
Randomized trial by Jensen et al., Crit Care Med 2011:
Procalcitonin guided antibiotic escalation led to worse outcomes

17. Procalcitonin: Monitoring Value
Algorithms in clinical trials have been shown to reduce antibiotic duration and even mortality
2016 SSC: “Procalcitonin levels can support decision to shorten the duration of antibiotics in patients with known sepsis, or stop antibiotics in patients with limited evidence for sepsis.”
IDSA: “Our interpretation of the literature is that …procalcitonin guidance for duration of antibiotic therapy is feasible and safe in critically ill patients with infections.”
Areas of uncertainty:
Possible “prompting effect” in clinical trials
Heterogeneity between studies and among algorithms used for de-escalation
No guidance on how to operationalize in day-to-day practice

18. Procalcitonin: Prognostic Value
Consistent association between higher levels of PCT and worse outcomes including end-organ damage, mortality, etc.
Boussky, et al.: 2005 prospective cohort of critically ill patients with pneumonia
Those who died in the ICU had mean initial PCT of 5.6 vs in those who survived
Wanner, et al.: 2000 serial assessment of PCT levels in trauma patients
Patients eventually diagnosed with severe MODS had initial mean PCT 5.7 vs. 1.1 for patients with uneventful recovery

19. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

20. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Interleukin-27
Presepsin
Cell Free DNA
Neutrophil CD64
SeptiCyte
Paula Styles

21. Interleukin-8 (IL-8)
What it is: Chemokine produced by macrophages to mobilize neutrophils
Expression is upregulated among patients with septic shock within 24 hours (best validated in pediatric)
Implication as prognostic marker
Pediatric: high IL-8 associated with 10 fold mortality risk
Adult: high IL-8 AUROC for 28-day mortality was 0.74

22. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

23. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Research Only
1-4d ?
Interleukin-27
Presepsin
Cell Free DNA
Neutrophil CD64
SeptiCyte
Paula Styles

24. Interleukin-27 (IL-27): Vital Statistics
Cytokine dimer made of IL-27.p28 gene + Epstein-Barr virus induced gene (EBI-3) subunits
Quantitative
Pro and anti-inflammatory properties
Expression due to inflammation is robust during early childhood, declining into adulthood
Diagnostic implications

25. Interleukin-27 (IL-27): Diagnostic in Pediatric
Developed as a diagnostic marker using gene mapping of sepsis in children
Good correlation with sepsis in pediatric population (AUROC in various studies/populations)
Poorer performance in adults (AUROC ~0.68)
Possibly due to declining expression of EBI-3 in adulthood
Improved performance in non-pulmonary origin sepsis

26. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

27. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Research Only
1-4d ?
Interleukin-27
Presepsin
Cell Free DNA
Neutrophil CD64
SeptiCyte
Paula Styles

28. Presepsin (sCD14-ST): Vital Statistics
What it is: Circulating fragment of macrophage surface receptor CD-14, cleaved by proteases in response to inflammation
Pharmacodynamics: Lab models demonstrate detection within 2h of insult, peak levels within 3h
Results within 1-4 hours depending on assay used
Lab notes: Quantitative
Clinical value: Diagnostic, monitoring and prognostic implications

29. Presepsin (sCD14-ST)
Diagnostic value: Excellent ability to discriminate between SIRS and Sepsis (AUROC )
Performance remained high 2-3 days after sepsis onset
Monitoring & Prognostic value: Increasing levels are highly correlated with illness severity (APACHE-III and SOFA scores)

30. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

31. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Research Only ?
Interleukin-27
1-4d
$10
Presepsin
1-4h $6
Cell Free DNA
Neutrophil CD64
SeptiCyte
Paula Styles

32. Cell Free DNA (cfDNA)
What it is: Product of cell apoptosis. Normally found in low levels but high cell turnover in sepsis
Pharmacodynamics: Detectable within 6 hours, peaks after 24 hours
Lab notes: Quantitative, usually requires time consuming spectrophotometer or PCR, but new point of care assay show promise
Potential uses: Mainly prognostic implications

33. cfDNA: Clinical Value
Diagnostic value: Two small studies concluded AUROC to discriminate sepsis vs. inflammation
Prognostic value: Significantly higher levels in non- survivors, AUROC for mortality 0.81

34. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

35. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Research Only ?
Interleukin-27
1-4d
$10
Presepsin
1-4h $6
Cell Free DNA
Variable
Neutrophil CD64
SeptiCyte
Paula Styles

36. Neutrophil CD64 (nCD64): Vital Statistics
What it is: Monocyte surface receptor, marked upregulation after exposure to pathogen
Pharmacodynamics: Levels rise within 2 hours, remain elevated for a few days after exposure
Lab notes: Quantitative, requires flow cytometry, ~10$ per test
Potential Uses: Diagnostic, Monitoring and Prognostic implications

37. nCD64: Clinical Applications
Diagnostic value: Among adults, ability to identify bacterial infection/sepsis is excellent (AUROC ~0.95)
nCD64 index proposed in one study
Index >1.19 had ST 94%, SP 88% for infection
Index < 1.19 associated with 100% NPV for positive blood cultures
Monitoring value: Persistent elevation of nCD64 in pts who receive ineffective antibiotic therapy
Prognostic value: Expression associated with clinical markers of severity and worse in-hospital mortality

38. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

39. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Research Only ?
Interleukin-27
1-4d
$10
Presepsin
1-4h $6
Cell Free DNA
Variable
Neutrophil CD64
SeptiCyte
Paula Styles

40. SeptiCyte: Vital Statistics
Tests whole blood expression of four genes: CEACAM4, LAMP1,PLAC8, PLA2G7
Quantitative, reported in bands 1-4
Approved by FDA April 6th, 2017
Diagnostic implications
AUROC for SIRS vs Sepsis

41. Name ? Diagnostic Marker? Monitoring Marker? Surrogate Marker?
Jacobs et al. Expert Rev Anti Infect Ther October ; 14(10): 929–941
Name
Diagnostic Marker?
Monitoring Marker?
Surrogate Marker?
Prognostic Marker?
Lactic Acid ✖ ✔
Pro-calcitonin
Interleukin-8
Interleukin-27
Presepsin
Cell Free DNA ?
Neutrophil CD64
SeptiCyte

42. Name Available at UMass FDA Approval Turnaround Time Cost Per Item
Lactic Acid
Yes 1h
$14-47
Pro-calcitonin
12h-5d
$25
Interleukin-8 No
Research Only ?
Interleukin-27
1-4d
$10
Presepsin
1-4h $6
Cell Free DNA
Variable
Neutrophil CD64
SeptiCyte
<24h
Paula Styles

43. What the Future Holds SeptiCyte NCD64 IL-8 Presepsin IL-27 Lactic Acid
Procalcitonin
NCD64
IL-8
Presepsin
IL-27
SeptiCyte
Lactic Acid
Cell Free DNA

45. Thank You Michelle Kelly Sepsis Committee
Paula Stiles and laboratory staff