heathcare is very important

1. CLINICAL MICROBIOLOGY
Sepsis
Clinical case and diagnostics
UNIVERSITY OF EAST SARAJEVO
Faculty of Medicine in Foča

2. Sepsis
 Systemic inflammatory reaction in response to invasion by microorganisms.

3.  Gram-bacteria (60%)
 Gram+ cocci (20%)
 Fungi (2-5%)
 Mycobacteria
 Viruses
 Protozoa
 The penetration of microorganisms into the bloodstream is not decisive for the onset of
sepsis. Systemic spread of micro-organism products also causes sepsis.
Etiology

4. Gram-bacteria

5.  Risk factors for Gram-bacteremia:
 Diabetes mellitus
 Lymphoproliferative diseases
 Liver cirrhosis
 Burns
 Invasive procedures
 Artificial valves...
 Drugs that cause neutropenia
 Risk factors for Gram+ bacteremia:
 Venous catheters
 Burns
 Intravenous drug addiction
 Artificial valves...
Etiology

6.  A local infection in the urogenital tract, biliary tract, lungs, skin or digestive system can
spread to the bloodstream.
Pathogenesis

7. Pathogenesis
 A local infection in the urogenital tract, biliary tract, lungs, skin or digestive system can
spread to the bloodstream.

8.  Microorganisms can be
introduced directly into the
bloodstream.
 In a small number of cases,
there are no visible sites of
primary infection.
 The penetration of microorganisms into the bloodstream is not
decisive for the onset of sepsis. Systemic spread of micro-
organism products also causes sepsis.
Pathogenesis

9.  The host's immune system recognizes certain molecules of microorganisms as foreign.
 Lipopolysaccharide (LPS) is a product of Gram- bacteria and a very powerful stimulator
of the immune response.
 Peptidoglycan and lipoteichoic acid of Gram+ bacteria, certain polysaccharides and
exotoxins cause a similar effect to LPS. The immune response to these molecules is less
well studied.
 LPS is a potent activator of macrophages. It binds to CD14 and TLR4 receptors on
macrophages and dendritic cells.
 Systemic changes in patients with disseminated bacterial infection represent a reaction to
cytokines whose production is initiated and stimulated by LPS.
Pathogenesis

10.  Neutrophilia. Bone marrow response to circulating cytokines, primarily G-CSF.
Increased production and release of neutrophils that replace those consumed in the
inflammatory reaction.
 Elevated temperature. LPS (exogenous pyrogen) stimulates leukocytes to produce
the cytokines IL-1 and TNF-α (endogenous pyrogens) which enhance
cyclooxygenase-2 enzyme activity and increase the synthesis of prostaglandins from
aradidonic acid in vascular and perivascular cells of the hypothalamus, which raises
the temperature.
 Acute phase proteins. Plasma proteins synthesized primarily in the liver. In
response to LPS, their plasma concentration increases.
 C-reactive protein
 Fibrinogen
 Serum amyloid A
 The synthesis of these molecules is regulated by IL-6, IL-1 and TNF-α
 Fibrinogen - erythrocyte sedimentation
A mild form of sepsis

11.  Disseminated intravascular coagulation. Increased expression of pro-coagulant
proteins (tissue factor) and decreased anti-coagulant activity of endothelial cells
(consequence of TNF-α). Inflammation and intravascular thrombosis develop in
many organs.
 Tissue damage. Activation of neutrophils before exiting the vascular bed damages
endothelial cells and reduces blood flow. The lungs and liver are particularly
sensitive. Neutrophilic damage to the lung endothelium allows fluid to escape from
the blood vessels into the air space of the lungs - ARDS (Adult Respiratory Distress
Syndrome). Liver damage leads to reduced gluconeogenesis and a drop in
glycemia.
A severe form of sepsis

12. Disseminated intravascular coagulation

13. ARDS

14. Presentation of a patient with sepsis

15.  Mister White, a 35-year-old journalist, was healthy all his life. A few days after a
surgical intervention in the oral cavity, he noticed frequent fatigue, sleepiness,
muscle pain and loss of appetite. He did not consult a doctor. The next day, he
developed a high fever, rapid breathing and a disturbance of consciousness.
 Sepsis is easily suspected in a patient with a local infection who suddenly develops a
fever.
 Hyperventilation is often a useful clue to diagnosis even though its cause is not clear
enough.Disorientation and other signs of encephalopathy can also be early signs of
sepsis.
Presentation of a patient with sepsis

16.  Mister White then reports to the doctor. During the examination, the doctor
confirmed a high temperature, rapid breathing and a disturbance of consciousness,
but he also discovered a rapid heartbeat and a drop in blood pressure. He also
noticed skin lesions.
 Hypotension and disseminated intravascular coagulation create ischemic necrosis
of peripheral tissues. Bacterial toxins can spread hematogenously and cause diffuse
skin changes. Skin lesions can sometimes point to a specific trigger.
Presentation of a patient with sepsis

17.  The doctor asks the patient if he has had nausea, vomiting or other gastrointestinal
complaints.
 Gastrointestinal manifestations (nausea, vomiting, diarrhea, ileus, ulcer, cholestatic
jaundice) may precede other signs of sepsis. In most cases, it is a hepatocellular
disorder. Prolonged or severe hypotension can cause acute liver damage.
Presentation of a patient with sepsis

18.  Laboratory findings: thrombocytopenia, increased lactates, hypoglycemia,
increased S-reactive protein, fibrinogen, complement components, accelerated
sedimentation.
 Serum lactates are elevated primarily due to incomplete hepatic clearance.
Impaired gluconeogenesis leads to hypoglycemia. Cytokines IL-6, IL-1 and TNF-α
induce the synthesis of acute phase proteins in the liver: C-reactive protein,
fibrinogen, complement components. Thrombocytopenia suggests DIK.
Presentation of a patient with sepsis

19.  Blood was taken for microorganism isolation and final diagnosis. A Gram-causing
agent of the infection was discovered, which spread into the bloodstream.
 Sepsis can quickly kill the patient. Successful treatment requires prompt treatment
of local infection, ensuring hemodynamics of the cardiovascular system and
treatment of other sepsis symptoms.
Presentation of a patient with sepsis

20. Laboratory diagnosis
of sepsis
 Microbiological confirmation of clinically diagnosed
sepsis is established by isolating microorganisms from
blood.
 Blood for blood culture is taken by venipuncture and
inoculated into a medium for isolating microorganisms
next to the patient's bed.
 It is a critical point for possible sample contamination.
 The percentage of contamination ranges from 0.6% to 6%,
on average 2 - 3%.

21. Taking a sample for
blood culture
 Everything must be done with gloves.
 It is necessary to disinfect the place of venipuncture, usually first with
alcohol and then with iodine disinfectant, wait for the optimal time
required for the effective effect of the disinfectant (at least 1 minute).
 After disinfection, the vein should not be palpated.
 After venipuncture, the iodine disinfectant should be removed with a
swab soaked in alcohol.
 The practice of "two needles" (one needle is used for venipuncture, and
the other is used to puncture the vial and inoculate the substrate) was
abandoned in the 1980s due to the risk of HIV infection.
 The use of systems specially adapted for blood collection for
hemoculture, with a protected attachment for puncturing the bottle, is
the best protection against contamination and the safest for healthcare
workers.

22. Taking a sample for blood
culture

23.  During sepsis, relatively low numbers of microorganisms are often present in the
blood - typically <30/mL, so it is recommended to:
 younger than 10 years: 1 mL of blood for each year of life
 older than 10 years: 20-30 mL of blood
 For blood culture, it is optimal to take three blood samples from the patient within
24 hours. Most often, blood is taken at intervals of half an hour to one hour.
 Blood should be taken at the first symptoms or signs of a rise in temperature, and
you should not wait for a rise to 38.5°C.
 Blood is sown in a ratio of 1:9 (blood: medium).
 Substrates adapted to the isolation of aerobic bacteria, anaerobic bacteria,
Mycobacteria, and fungi (molds/yeasts) can be used.
 Usually, the sample is seeded in one set of vials each - for aerobic and anaerobic
bacteria.
Taking a sample for blood culture

24. Taking a
sample for
blood
culture

25. Taking a sample for
blood culture
 The main ingredient of all bases is nutritionally rich broth.
 The pads may contain "resins" that inactivate antibiotics, if the patient has
already received treatment.
 Blood culture media can be adapted for conventional diagnostics (manual
processing) or for culturing in automatic systems.
 Cloudiness, appearance of hemolysis, appearance of gas, appearance of
colonies in blood culture are observed.
 Blood cultures are incubated for up to 7 days.
 Under special circumstances, e.g. suspicion of brucellosis, up to 21 days or even
longer.
 The problem of interpreting the findings: what is a pathogen and what is a
contaminant (presence of signs and symptoms of the disease, biochemical
markers, history of the disease, isolation of microorganisms from two or more
vials).