dr.neha.a.sharma m.d microbiology

1. Laboratory diagnosis of sepsis
Dr.Neha.A.Sharma

2. Definitions
 Sepsis is defined as life-threatening organ dysfunction caused by a
dysregulated host response to infection
 Septic shock is defined as persisting hypotension requiring vasopressors to
maintain MAP ≥65 mm Hg and having a serum lactate level >2 mmol/L
(18mg/dL) despite adequate volume resuscitation
 Organ dysfunction can be identified as an acute change in total SOFA score
≥2 points consequent to the infection
Surviving sepsis campaign 2018 updates

3. SOFA Score
Mendonça, A & Vincent, Jean-Louis & Suter, Peter & Moreno, Rui & Dearden, N & Antonelli, Massimo & Takala, Jukka & Sprung, Charles & Cantraine, F. (2000). Acute renal failure in the ICU:
Risk factors and outcome evaluated by the SOFA score. Intensive care medicine. 26. 915-21. 10.1007/s001340051281.

4. qSOFA
• Systolic blood pressure < 100 mmHg
• Respiratory rate > 20/min
• Glasgow coma scale ≤ 14
• qSOFA ≥2 is indicative of sepsis
Surviving sepsis campaign 2018 updates

5. Epidemiology
• In 2017, an estimated 48·9 million incident cases of sepsis were
recorded worldwide and 11 million sepsis-related deaths were
reported, representing 19·7% of all global deaths
• Study found that the sepsis death rate is 213 per 1,00,000 people
in India, 206 in Pakistan, 183 in Nepal, 136 in Bangladesh, 109 in
Bhutan, 69 in Sri Lanka and 27 in Maldives and 285 in Afghanistan
Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990-2017:
analysis for the Global Burden of Disease Study. Lancet. 2020;395(10219):200-211. doi:10.1016/S0140-
6736(19)32989-7

6. Pathophysiology of sepsis
Bhan C, Dipankar P, Chakraborty P, Sarangi PP. Role of cellular events in the pathophysiology of sepsis. Inflamm Res.
2016;65(11):853-868. doi:10.1007/s00011-016-0970-x

7. • Infection and the subsequent interaction of pathogen motifs
with various receptors present on immune cells activate a
cascade of inflammatory events that aim at removing the
pathogen and restoration of normalcy.
• If the host responses fail to contain the infection, there is a
systemic spread of infection that further leads to progression
of inflammatory changes in a dysregulated manner.
• Thus, excessive inflammatory and anti-inflammatory
responses culminate in irreversible tissue damages, multi-
organ failure, and death
Outline of sepsis development

9. Overview of cellular changes occurring during sepsis
• Entry and recognition of PAMPs by PRRs upregulation of adhesion
molecules, cytokines, and ROS productions.
• These responses, when succeed in clearing the pathogens
upregulation of anti-inflammatory responses and ultimately resolution
and restoration of tissue homeostasis
• Failure of innate & adaptive responses to contain the ongoing infection
locally systemic spread of infection hyperinflamm.immune
response
• To regulate the hyperinflammatory immune cell activities, body’s
immunoregulatory responses are activated at a higher magnitude
ultimately leading to a loss of balance between inflammatory and anti-
inflammatory response and generalized immunosuppressive stage
• Different phenotypic and molecular changes take place in the immune
cells as sepsis progresses from a hyperinflammatory to an
immunosuppressive stage
• Thus, a host response that is designed to protect against pathogen causes
tissue-damaging events leading to multiorgan system failure and death.

10. Lab diagnosis of sepsis
Direct
 Blood culture:
 Gold standard
 Methods :
• Conventional
• Automated
• MALDI-TOF
• Multiplex real
time PCR
Indirect
 Biomarkers :
• Procalcitonin
• CRP
• Endotoxin
Activity Assays
• Cytokines – IL-
10,IL-6 etc
 Invasive fungal
biomarkers :
• Galactomannan
• Beta-D-Glucan
Hematological parameters
• White blood count:
• > 12,000/mm3 or
• < 4,000/mm3 or
• > 10% immature forms
• Neutrophil lymphocyte
ratio > 5
• Platelet count <
80,000/mm3

11. Blood culture
• Gold standard
• Atleast 2 sets - Aerobic and Anaerobic
• Obtained prior to initiation of anti-micobial therapy
• Automated Blood cultures are preferred over conventional
• In presence of intra vascular catheters (> 48 hrs)- one blood
culture set from cath. Alongwith periph.bld cult. To rule out
CRBSI
• Differential time to positivity i.e flashing of blood collected
from cath.2 hrs before periph.bld cult.sample is useful in
diag.of CRBSI
• Disadvtgs.: 30-50% of sepsis patients are culture negative
• Time to diag.sepsis is long

12. MALDI-TOF
• Quick detection of pathogen from blood culture
• MALDI Sepsityper –Bruker Daltronics and VITEK MS blood culture (bioMerieux)
• Advtgs. :
• ID is avail.after culture within 20 min.
• Very low cost per test
• Problems encountered :
• Polymicrobial infections cannot be diagnosed
• Currently overnight incubation of blood culture broth on solid media is required prior
to MALDI-TOF MS analysis
• AST not possible
• Set up cost is very high

13. Multiplex real time PCR
• FilmArray (Biofire Dx/bioMérieux) Blood Culture Identification
Panel
• It tests for 24 bacterial and yeast pathogens plus 3 antibiotic
resistance markers.
• Verigene (Nanosphere) BC-GP and BC-GN assays that use
Gold/Ag nanoparticle probes and micro-array for detection of
bacterial pathogens and several resistance markers

14. Biomarkers
WHO defn: Any substance, structure, or process that can be measured in the
body or its products and influence or predict the incidence of outcome or
disease
• Ideal charact.of biomarkers:
• Short half life – rapid incr.with onset of sepsis and rapid decr.
• High sensitivity,high specificity
• Discriminate etiology of sepsis
• Level should not increase with assoc.co-morbidities
• Provide clinicians time to intervene for treatment
• Guide for starting and stopping antibiotics
• Standardized values
• Accurately predict severity of disease
• Help in prognostication
• Test should be easily doable
• Cost effective
• Short turn around time
• Sample requirement small

15. Procalcitonin
• PCT 116 amino acid pro-hormone synth.prim.by C-cells of
thyroid gland
• Seum levels in healthy persons < 0.05 ng/ml
• In systemic inflamm . particul . Bact .infectns it is produced in
a number of other tissues and released into circulation where
its level increases upto 1000 times

16. Limitations of PCT
Falsely raised Falsely low
Neonates < 48 hrs Early course of infection
Major trauma Localized infectns
Surg . Intervention Subacute endocarditis
Severe burns
Treatment with drugs stimul. Release of
pro-inflamm.cytokines
Invasive fung.infections
Ac.plasm.falcip.malaria
Severe cardiogenic shock
Severe liver cirrhosis/viral hepatitis
Small cell lung ca/med.ca thyroid

17. Lactate
• Sepsis may progress rapidly to septic shock that is associated with
decreased delivery of oxygen and nutrients into peripheral tissues.
• Lactate levels serve as an endpoint for resuscitation in patients with
sepsis and septic shock as part of the sepsis bundle
• Limitations :
• Elevated lactate levels can be seen in a wide variety of conditions,
such as cardiac arrest, trauma, seizure or excessive muscle activity
• Elevated levels of lactate are not considered specific for either the
diagnosis of sepsis, or predicting mortality

18. C - Reactive protein
• Synthesized prim.in liver hepatocytes
• Disc in 1930 by Tillet and Francis
• Elevated in inflamm.condns.such as Rhuematoid
arthritis,cardiovasc.dis,infection
• IL-6 is main inducer ,IL-1 enhances effect
• Limitations :
• It is sensitive but not very specific
• Levels rise within 6-24 hrs and remain elevated for 3-5 days

19. Endotoxin activity assays
• Principle : Endotoxin binds to Anti-endotoxin antibodies (IgM)
• It is delivered to neutrophils via complement receptors
• In presence of zymosan and luminol,neutrophils undergo a
respiratory burst accompanied by light emission
• Light produced is quantified by chemiluminometer
• Intensity is proportional to amount of endotoxin
• It is a predictor of mortality in critically ill patients
EAA Level Interpretation
<0.4 Low
0.4-0.6 Intermediate
>0.6 High

20. Cytokines
• Cytokines are regulators produced by the host immune
system in response to an infection or injury which have a role
in the complex pathophysiology of sepsis
• IL-6 levels are increased in patients with infectious
complications
• Studies have shown that IL-6 and IL-10 levels are correlated
with the mortality rate in septic patients
• IL-8 has been used to predict the severity of sepsis in
pediatric patients, although the utility of IL-8 has not been
confirmed in adults
• None of the cytokine markers has been proven to be more
sensitive or specific than PCT or CRP

21. Galactomannan
• Galactomannan is polysaccharide antigen that exists prim.in cell
wall of Aspergillus sp.
• GM is released in blood and other body fluids even in early stages
• Remains for 1-8 weeks
• Detection of GM antigen by ELISA is considered a
diag.microb.criterion for invasive fungal infections by EORTC/MSG

22. Galactomannan
• Galactomannan in serum and/or BAL fluid is a criterion to
classify patiens with Inv.Asp. By EORTC/MSG in 2008
• Serum galac.index ≥ 0.5 in 2 separate serum samples or ≥ 1 in
single serum sample
• BAL fluid galac.index ≥ 1 in 2 aliquots of single BAL fluid
sample
• Bio Rad Platelia Aspergillus EIA is the FDA approved assay
• Limitations :
• False positive with :
• piperacillin/tazobactam
• Fluids containing sod.gluc
• Infectns with histoplasma,paecilomyces,geotrichum

23. (1-3)- Beta- D Glucan
• Polysaccharide cell wall component
• Except Mucor,Rhizopus,Blastomyces,Cryptococcus
• Inhibits leucocyte phagocytosis

26. False positive False negative
Bacteremia ( m.c strep.sp.) Lipemic samples
Hemodialysis cellulose membranes and
filters
Hemolyzed samples
IVIG Zygomycetes,Cryptococcus,Blastomyces
Serum (yellow colour)
Drugs
Crestin,Lentinan,Schizophyllan,Sclerogluc
an

27. Other markers
• TNF alpha
• CD 64
• Soluble form of urokinase –type plasminogen activator
receptor (suPAR)
• Soluble trigging receptor expressed on myeloid cells-1 (Strem-
1)
• D-dimer
• Proadrenomedullin (ProADM)
• Myocardial Biomarkers—troponin, natriuretic peptides and
myoglobin

28. Hour-1 Surviving sepsis campaign Bundle of care
• Measure lactate level.Re-measure within 2-4 hrs if initial
lactate > 2 mmol/L
• Obtain blood cultures prior to administration of antibiotics
• Administer broad- spectrum antibiotics
• Begin rapid administration of 30ml/kg crystalloid for
hypotension or lactate ≥4 mmol/L
• Apply vasopressors if the patient is hypotensive during or
after fluid resuscitation to maintain MAP≥65 mmHg

29. Thank You