Sepsis Bio markers

1. New Approaches to Sepsis:
Biomarkers and Molecular
Diagnostics.
Dr. Indranil Bhattaharya
MD & WHO Fellow (Pathology)
HOD – Pathology & Laboratory Medicine
Jagjivan Ram Hospital
Western Railway
Mumbai

2. Why give this your attention?
• Microbes – the WMDs in your ICUSepsis is the main killer of General ICU patients
•
Anything that helps you beat it is good news……
We need better diagnostic & prognostic tools

3. Funk and Kumar, Crit Care Clinics 2011; 53-76.
For first 12 hours, 1% mortality
per 5 minute delay

4. Early
Detection is
Paramount
BUT.…

5. Conventional Detection of Sepsis
• 2 main strategies…Detection of bacterial pathogen
– Slow and all too often negative
Detection of host response
– NEWS for fever, tachycardia, tachypnoea
– “Conventional” lab tests (WBC, CRP etc)
– The ICU eyeball test
There might not be an ICU eyeball

7. Enter the goose and the…

8. The Biomarker paradigm…
Sepsis leads to:
– Inflammation
– Coagulation
– Tissue damage and repair
• The sicker you are, the greater the changes…..
•
We can identify biomarkers for these processes
•
We can measure these biomarkers•
We can stratify severity based on biomarker levels
•
We can prognosticate based on biomarker levels

9. Damage Associated Molecular Patterns

10. Pathogen Associated Molecular Patterns

11. Biomarker Candidates
• Multiple, and growing all the time
Some more common in the literature
Linked to the main underlying processes
– Inflammation
– Coagulation
– Tissue damage
– Tissue repair

12. Examples
• Acute phase proteins
–
–
–
–
CRP
Procalcitonin
Pentraxin 3 (PTX3)
Lipopolysaccharide binding protein (LBP)
• Cytokines & chemokines
– IL-1RA, IL-1b, IL-2, IL-6, MCP-1
– TNF-a, TNFR1/2
– HMGBP1
• Cell surface markers
–
–
–
–
Soluble CD14 (presepsin)
Neutrophil CD64 index (CD64in)
mHLA-DR (monocyte HLA-DR levels)
CD-163
• Receptor markers
–
–
–
–
–
VEGF
Soluble VEGF-receptor 1 (sFLT)
Soluble urokinase plasminogen activator (suPAR)
sTREM-1
RAGE (soluble receptor for advanced glycation
end products)
• Coagulation
–- Activated partial thromboplastin time
(aPTT) waveform analysis
– Protein C receptor
– Thrombomodulin
• Endothelial damage
– Heparin binding protein
– E-selectin
– Neopterin
– ICAM-1, VCAM-1
– Angiopoietin-1 and -2
– Syndecan-1 and -2
• Vasodilation
– Copeptin (AVP precursor)
• Cell damage
– MicroRNA
–- Microparticles
• Cell repair
Procollagen III amino propeptide

13. Questions to be answered
• Does the biomarker aid diagnosis?
•Does it provide additional prognostic info?
– For outcome
– For progression/decline
• Better than the ICU eye?
• Better than scoring systems?

14. Sepsis biomarkers are produced
during the host response to infection.
Traditional biomarkers are polypeptides
and/or proteins derived from this response.
Omics-based biomarkers are screening out from
all kinds of molecules of host response while high-
throughout omics technologies are emerging.

15. OMICS
Technologies that measure some characteristic of a large family of cellular
molecules, such as genes, proteins, or small metabolites, have been
named by appending the suffix “-omics,” as in “genomics.”
Omics refers to the collective technologies used to explore the roles,
relationships, and actions of the various types of molecules that make up
the cells of an organism.

16. ROLES OF BIOMARKERS
• Diagnosis:
– Identifying or ruling out sepsis.
– Identifying patients who may benefit from
specific therapies.
• Follow up the response to therapy.
• Prognosis

17. THE PERFECT SEPSIS BIOMARKER
• Should be highly sensitive and specific for sepsis to allow the
differentiation between infectious and non-infectious causes
of inflammation, organ dysfunction and shock.
• Should be present at the onset or even before the appearance
of the clinical signs of sepsis to have prognostic value.
• Should be easy and safe to measure with low cost for the
patients and for the hospital.
• Should be biologically plausible.

18. Wang P, Yang Z, He Y, Shu C. Pitfalls in the rapid diagnosis of positive blood culture. Rev. Med. Microbiol. 21(3), 39–43 (2010).

19. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and
associated costs of care. Crit. Care Med. 29(7), 1303–1310 (2001).

20. THE ROC CURVE
• The ROC (Receiver Operating Characteristic) curve
is a widely used tool for comparing diagnostic tests.
• The shape of an ROC curve and the Area Under the
Curve (AUC) helps estimate the discriminative power of a
marker.
• The closer the curve is located to the upper left-
hand corner and the larger the AUC, the better the marker
is at discriminating between septic and non-septic patients.
• A perfect biomarker has an AUC of 1, whereas a non-
discriminating marker has an area of 0.5.

21. Kofoed K, Andersen O, Kronborg G, et al. Use of plasma C-reactive protein, procalcitonin, neutrophils,
macrophage migration inhibitory factor, soluble urokinase-type plasminogen activator receptor, and soluble
triggering receptor expressed on myeloid cells-1 in combination to diagnose infections: a prospective study.
Crit Care 2007; 11: R38.

22. Displayed are ROC curves comparing:
• Soluble urokinase-type plasminogen activator receptor (suPAR),
• Soluble triggering receptor expressed on myeloid cells (sTREM)-1,
• Macrophage migration inhibitory factor (MIF),
• Neutrophil count,
• Procalcitonin (PCT),
• C-reactive protein (CRP),
• Combined three-marker and Six-marker tests for detection of
bacterial versus non-bacterial causes of systemic inflammation.
• The six-marker combination is the best-performing marker
model (ROC-AUC 0.88)

23. C - REACTIVE PROTEIN (CRP)
• Acute phase protein
• Synthesized in liver
• IL-6 (and IL-1 and TNFα) stimulate synthesis
• Binds bacterial polysaccharide/ chromatin
– Activates the classical complement pathway
– Increase the immune inflammatory response in
bacterial infection

24. CLINICAL SIGNIFICANCE (CRP)
Diagnostic use:
• CRP is used mainly as a marker of inflammation.
• Normal concentration in healthy human serum is between 5 and 10
mg/L, increasing with aging
• Higher levels are found in late pregnant women, mild inflammation
and viral infections (10–40 mg/L), active inflammation, bacterial
infection (40–200 mg/L), Severe Bacterial Infections and
Burns (>200 mg/L).
Follow / up treatment:
• Measuring and charting CRP values can prove useful in determining
disease progress or the effectiveness of treatments.

25. Limitations:
• CRP is a non‐specific marker of inflammation, so
does not indicate the organ or organs affected.
• CRP measurements can be used to assess cardio
vasular risk status if they are made in the absence of
acute inflammation.

26. Procalcitonin (PCT), protein that consists of 116 amino acids,
is peptide precursor of calcitonin, hormone synthesized by
the parafollicular C cells of the thyroid and involved in calcium
homeostasis.
Procalcitonin (PCT)
High procalcitonin levels produced during infections are not
followed by a parallel increase in calcitonin or a decrease in
serum calcium levels.

27. Procalcitonin

29. Procalcitonin and prognosis

30. CLINICAL SIGNIFICANCE PROCALCITONIN
Diagnostic:
• To aid in the diagnosis and risk stratification of bacterial sepsis.
• To aid in distinguishing bacterial from viral infections, including
meningitis.
• To monitor therapeutic response to antibacterial therapy and reduce
antibiotic exposure.
• To aid in the diagnosis of systemic secondary infection after surgery
and in severe trauma, burns, and multiorgan failure.

31. Prognostic:
• To aid the choice and timing of the initiation of antibiotic
treatment (Procalcitonin Algorithm)
• To aid with elucidating prognosis of critically ill patients
with systemic infection
• Predicting the need of antibiotic treatment in sepsis and to
shorten the duration of antibiotics required
• Use as independent predictor of graft failure late after
renal transplantation

32. PROCALCITONIN (PCT)
REFERENCE VALUES (EXCEPT NEWBORN)
SIGNIFICANTLY LOWER IN LEUKOPENIC PATIENTS
• < 0.05ng/ml
• 0.5 - 2ng/ml
• >2ng/ml
• Healthy individuals
Probability of sepsis is low,
local infection possible
• Grey zone, recheck 6-12hrs
later
• Probability of sepsis is high

33. LIMITATIONS
• Procalcitonin, useful in bacterial sepsis, no value in
assessment of fungal or viral infections and shows no
response to local infections with no systemic response.
• Similar to CRP, clinical conditions associated with high
baseline procalcitonin levels include burns, major surgery,
and systemic inflammatory processes.
• Use of procalcitonin, both as an indicator of severe infection
and predictor of antibiotic choices/ duration, has been
center-specific, with insufficient data from
multicenter/multinational studies to support its use as a
routine laboratory marker in clinical practice.

34. PROCALCITONIN VERSUS C- REACTIVE PROTEIN
Use of PCT and CRP have found the diagnostic
performance of CRP and PCT to be rather similar.
In bacteremia, PCT have shown excellent diagnostic
ability; this is in accordance with the suggested
notion that PCT is superior to CRP in diagnosing
systemic infection.
The algorithms based on PCT concentrations as the
main guide can shorten the length of antibiotic
treatment and decrease the use of antibiotics.

35. SOLUBLE CD14 SUBTYPE
(PRESEPSIN)
• In 2004, a new biomarker sCD14-subtypes (presepsin) was found and its
value was shown in the diagnosis and evaluation of sepsis

36. PRESEPSIN
• In 2004, a new biomarker sCD14-subtypes (presepsin)
was found and its value was shown in the diagnosis and
evaluation of sepsis.
• CD14 acts as a co-receptor (along with the Toll-like
receptor TLR 4 and MD-2) for the detection of bacterial
lipopolysaccharide (LPS).
• During inflammation, plasma protease activity
generates soluble CD14 (sCD14) fragments. One of
them, called sCD14 subtype (sCD14-ST), or presepsin.

37. PRESEPSIN VS PCT
In comparison with PCT
Diagnostic accuracy:
• The best diagnostic cutoff for presepsin was 600 pg/ml.
Sensitivity and specificity were 78.95% (95% CI)
• The best diagnostic cutoff in terms of sensitivity and
specificity for PCT was 0.18 ng/ml, corresponding to
89.47% sensitivity

38. PRESEPSIN VS PCT
The ROC curves were significant for both biomarkers, but the AUC
calculated for PCT was wider, demonstrating a better diagnostic
accuracy than Presepsin.

39. PRESEPSIN VS PCT
Prognostic role:
• Analysis of 60-day mortality showed the superiority of
presepsin compared to PCT.
• Presepsin concentrations at the first evaluation in the ED
were higher in non-survivor septic patients than in survivors.
• No significant correlation was found between PCT values
and 60-day mortality.
• These results pointed out the possible prognostic role of
presepsin in predicting in-hospital mortality and promptly
identifying high-risk patients.

40. Pentraxin 3
Pentraxins are liquid-phase PAMP receptors.
Short pentraxins include CRP as is Serum Amyloid P
component (SAP).
Pentraxin-3 involved in:
– Complement activation
– Pathogen opsonisation
– Self versus modified-self versus non-self discrimination

41. Pentraxin-3

42. Is it really that simple?

43. Obviously NOT!

45. Fatty acid b2 oxidation issue in non-survivors v survivors related to carnitine shuttle
(defective fatty acid transfer into mitochondria). Detectable at presentation.

46. Microparticles?
• Small vesicles shed from membranes of
apoptotic and stress-activated cells
– Endothelial cells, RBCs, monocytes, platelets

47. FUTURE SEPSIS BIOMARKERS
Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1)
TREM-1 is expressed on macrophages and neutrophils,
identified as an amplifier of immune response and enhances
leukocyte activation in the presence of microbial products.
A solubleTREM-1 variant (sTREM-1) detected in several
body fluids
Recent studies found elevated sTREM-1 plasma levels in
patients with non-infectious conditions, such as
inflammatory bowel disease and chronic obstructive
pulmonary disease, and in patients undergoing cardiac
surgery

48. TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS-1
(TREM-1)
• A recently published meta-analysis of the diagnostic
value of sTREM-1 concluded that sTREM-1 represents
a reliable biological marker of bacterial infection
• Because of deficient studies and the heterogeneity
in study design and setting and limited size of the
studies published make it impossible to draw firm
conclusions on the diagnostic accuracy of sTREM-1.

49. Cytokines

50. CYTOKINES
Measurements of single
cytokines will not have great
impact on the future diagnosis
of sepsis.
Monitoring of a panel of
cytokines and receptors will be
important to determine the
level of dysregulation of the
innate immune system and to
guide future sepsis treatments.

51. FUNGAL BIOMARKERS

52. Fungal Biomarkers: GALACTOMANNAN (GM)
ELISA for the detection of Galactomannan (GM), an
Aspergillus sp. cell wall component
GM assay specific for Aspergillus spp.
Cross-reactivity with other fungal species such as Blastomyces
dermatitidis, Penicillium spp., and Fusarium spp.
Different studies reported highly variable sensitivities.
In a meta-analysis of 27 studies, the overall sensitivity was
reported to be 71%, and the specificity was reported to be
89%.
Early Diagnosis of Sepsis Using Serum Biomarkers;Terence Chan; Frank Gu Expert Rev Mol Diagn. 2011;11(5):487-496. © 2011 Expert Reviews Ltd.

53. Fungal Biomarkers: (133)-D-Glucan
Beta - Glucan (BG) component of the fungal cell wall
- present in a wider variety of fungal species,
including Candida spp.
Different studies reported variable
sensitivities - with a sensitivity of 69.9%
being observed for a large multicenter study
using just one sample per patient.
Early Diagnosis of Sepsis Using Serum Biomarkers;Terence Chan; Frank Gu Expert Rev Mol Diagn. 2011;11(5):487-496. © 2011 Expert Reviews Ltd.

54. BIOMARKER COMBINATIONS
• No single marker have high accuracy for fast and
accurate guidance of treatment of sepsis patients.
• Therefore, search for a single “magic bullet” marker might
ultimately be fruitless, but a combination of markers could
improve diagnosis, prognosis and treatment efficacy and
survival. Instead of a single marker, a combination of
markers may be right approach to crack the “sepsis code”.
• A combination of the three best-performing markers (CRP,
PCT and Neutrophil count) and all six markers were found to be
more accurate in detecting inflammatory response caused by
bacterial infection than individual markers alone.

55. BIOMARKER COMBINATIONS
In the future, multi-marker panels will
probably add to the diagnostic
accuracy and risk assessment in sepsis.

56. CONCLUSIONS
• Accurate and timely diagnosis of Sepsis helps
to enable rapid treatment, improve outcomes
and reduce unnecessary antibiotic therapy.
• Current evidence suggests that CRP will remain
an important marker of inflammation and infection.
• PCT will enhance the clinicians’ ability to diagnose
infection in critically ill patients and guides early
stopping of antibiotic therapy.

57. CONCLUSIONS
• Sepsis biomarker that has attracted a lot of attention
during the last years are sTREM-1 and Presepsin.
On the one hand there are studies showing that sTREM-1 &
Presepsin are ideal sepsis biomarker, and on the other hand
there are studies showing that they are as accurate as a toss
of a coin.
• Given the complexity and variability of sepsis it is
understandable that no single biomarker possesses all of the
“Perfect Biomarker” qualities.
Combining information from several sepsis markers is simple
and may facilitate diagnosis and risk assessment in septic
patients.

58. THANK YOU