Transcript LP 2 Engl VI 10.10.2013

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Blood culture
◦ Cultivation of blood sample
◦ Want to isolate and identify the bacteria or fungus
from the blood, in some pathologic conditions.
Bacteriemia – the presence of bacteria in
the blood for a short period of time,
without clinical expression.
Septicaemia – systemic disease associated
with the presence and persistence of the
microorganisms or their toxins in the blood.
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Normally the blood is sterile
In blood culture can grow:
◦ Contaminants bacteria from skin, during sampling
◦ Microorganisms occasionally identified in the blood, in
different situation:
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Skin lesions (catheter, teeth extraction)
Obstruction of billiary or urinary tract)
Profound suppuration
In most of cases, the episode are transitory. In case of patients
with endocard lesions, there is conditions for infectious
endocarditis developing.
◦ Bacteria which are frequent isolated from:
 Infectious endocardytis;
 Enteric fever
 Infectious with Haemophilus influenzae (type b).
 Brucellosis,
 Frequent in acute lobar pneumonia, meningitis,
can develop infections with septic metastasis localization.
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Enterobacteriaceae
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Escherichia coli…………………..............................1751 cases
Klebsiella spp……………………………………..................765
Enterobacter spp………………….............................399
Serratia spp…………………………………………………………..136
Proteus mirabilis……………………………………………………122
Salmonella, all serotypes………………………………..93
Citrobacter spp……………………………………………………….76
complex Enterobacter agglomerans.…………………….44
Morganella morganii…………………………..................26
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Staphylococcus aureus……………………………………….2151
Other microorganisms
Coagulase negative Stafilococci ……………………………1256
Enterococcus spp………………………………………………….794
Streptococcus pneumoniae………………………………...475
Pseudomonas aeruginosa……………………………………451
Streptococi beta-hemolitici…………………………………307
Acinetobacter spp……………………………………………….206
Streptococi viridans……………………………………………154
Stenotrophomonas maltophilia……………………………69
Haemophilus spp………………………………………………….27
Corynebacterium spp…………………………………………..20
sterile transfer kit
culture media
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system of manually manufacture and visual reading;
automatic systems;
systems of centrifugation for mycobacterium
detection and other intracellular bacteria.
piece of oil cloth 40/40 cm
solutions for decontamination:
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liquid soap;
iodine 2%;
ether :
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remove iodine;
dry the skin .
Pathologic product: blood.
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When?
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Onset of disease;
Before antibiotic administration
During shiver
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How much?
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How ?
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Adult: 3 samples x 20 ml, from different veins, at 30‘ interval
Children: 1 sample x 1 – 3 ml.
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Skin washing
Disinfection with iodine
Skin drying with ether
Puncture of the vein with sterile nedle
Introduction of the blood in culture media for blood culture: for every
sample are used 2 flacons: one with aerobic incubation and one with
anaerobic incubation. The bottles are incubated in thermostat at 37 °C and
are kept for 2 weeks.
When can we say that the blood culture of the patient is positive?
- Solid phase: appearance of the colonies;
- Liquid phase:
 turbidity
 destruction / coagulation of erythrocytes
 appearance of gas.
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Next steps:
◦Microscopic examination of the colonies from the solid phase
◦Subculture
◦Biochemical and antigenic identification
◦Antibiogram.
1. Sampling before antimicrobial therapy;
2. Sampling from different veins, for each blood
culture;
3. Avoidance of sampling through i.v. catheter
4. Quick transportation at laboratory, or, if it is not
possible, incubation of bottle at 37C, until the
moment of sending.
5. Isolation of the same bacteria from many bottles.
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Clinical diagnosis
Time of sampling
In case of antimicrobial treatment, the
specification of the antibiotic that was
administered
If the sampling was made during the high
fever.
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The arguments of the microbiolog:
Has clinical significance: isolation of the same bacteria
(species) in 2/3 vials and from different veins.
◦ Contamination: isolation of different bacteria from blood
culture bottles, from the same patient
◦ Quantitative blood culture help to argue the clinical
significance of the conditioned pathogen; it is indicated
when the skin decontamination is difficult.
◦ The presence of some bacterial structure involved in the
pathogeny of catheter bactaeremia ( biofilms );
◦ The argumentation of poly-microbial bactaeremia is based
on isolation of at least 2 microorganisms from the same
blood culture, at least two times in 24 hours.
The arguments of the clinician:
◦ The age and the immune status of the patient;
◦ The characteristics of the primary septic focus;
◦ Inflammatory system: leukocyte, CRP, fibrinogen.
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The absence of bacteria from the sample;
Low sensitivity of the method;
Samples collected after treatment with
antibiotics.
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Efficient antibiotic therapy:
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Non – efficient antibiotic therapy:
◦ Decreasing of CRP value
◦ Normalizing of CRP concentration means clinical recovery;
◦ Persistent increasing of CRP at the end of antibiotic
treatment means reappearance of the infectious;
◦ Linear evolution of CRP:
- incorrect antibiotic dosages;
- the resistance of microorganisms to antibiotics;
- forming of a localized suppurate process;
- a non – infectious disease;
- severe prognosis.
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Blood sampling through catheters;
Using just one single bottle instead of three;
Cultivation of some quantity of blood in the same
bottles used for other analysis (CRP, glucose,
calcium).
Woman, 62 old years, is admitted to hospital with
echocardiography diagnosis of sub-acute
endocarditis.
From 3 blood cultures was isolated Enterococcus
spp. Difuzimetric antibiogram has the next results:
Ampicillin – Resistant (R)
Ceftriaxone – Sensitive (S)
Gentamicin (120g) - R
Clindamycin - S
Cotrimoxazol - R
Vancomycin - S
Teicoplanin - S
Linezolid - S
There are 2 types of resistance to beta
lactams:
 Resistance by producing beta lactamase: this
resistance has recently appeared in North
America and in Latin America, but none of this
kind of strains were detected in Europe.
 Resistance by modification of PLP: enterococci
are naturally resistant to cephalosporines,
oxacilins and monobactams.
 (Pyridoxal 5′-phosphate (PLP) is a coenzyme
synthesized by all forms of life).
Phenotype
VanA
VanB
VanD
VanE
MIC vancomycin (ml/l)
60 - > 1024
4 - 1000
64
16
MIC teicoplanin (mg/l)
16 - 512
0.5 - 1
4
0.5
inductible
inductible
constitutive
inductible
Genetic support
Seldom plasmidic
Transposon Tn1546
Seldom chromosomal
chromosomal
Chromosomal
Transferable by
conjugation
+
+
-
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Bacterial species
E. faeecium
E faecalis,
E. avium,
E. gallinarium, E.
flavescens
E. faecium
E. faecalis
E. faecium
E. faecalis
Expression
Interpretative antibiogram
•The resistance to Ampicillin and the high level of
resistance for Gentamicin compromise the synergic
effect of this association;
•This phenotype of resistance is frequent for E.
faecium;
•Sensibility testing for Ceftriaxone is incorrect and
the reported result is false, because is known that
the enterococci are naturally resistant to
cephalosporins;
•The sensitivity for clindamycin and cotrimoxazole is
also false; this can be explained because of the
species identification;
Case 2
Patient, 68 years old, diabetes, AVC, comma, has
received medication through catheter. After 2 weeks present
fever and inflammatory syndrome at the place of catheter
insertion.
Blood culture made through a non catheterized vein
was positive for Klebsiella pneumoniae with the next
sensitivity to antibiotics:
Amoxicillin - R
Gentamicin - R
Amoxicillin +Clavulanic acid - R Amikacin - S
Ticarcillin - R
Ciprofloxacin - R
Piperacilin + Tazobactam - I
Cotrimoxazole - R
Ceftazidim - R (sinergy with clavulanic acid )
Ceftriaxone - S
Imipenem - S
Antibiotic
Wild phenotype
Low level
penicinilase
High level
penicinilase
Aminopenicillins
S
R
R
Aminopenicillins +IBL
S
S
I/R
Carboxipenicillins
S
R
R
Ureidipenicillins
S
I/R
I/R
First generation
cephalosporins
Second generation
cephalosporins
Third generation
cephalosporins
Third generation
cephalosporins + IBL
Cefamicins
S
I
I/R
S
S
S/R
S
S
S
S
S
S
S
S
S
Broad spectrum
cephalosporins
carbapenems
S
S
S
S
S
S
Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, “De l’antibiogramme a
prescription”. Edition bioMerieux, ISBN 973 – 86485-2-1, 2010.
Antibiotic
Wild phenotype
(low level
penicillinase)
High level
penicillinase
ESBL
CHN
(high level
cephalosp.)
Aminopenicillins
R
R
R
R
Aminopenicillins +IBL
S
R
R
R
Carboxipenicillins
R
R
R
R
Ureidipenicillins
I
R
R
R
First generation
cephalosporins
Second generation
cephalosporins
Third generation
cephalosporins
Third generation
cephalosporins + IBL
Cefamicins
S
R
R
R
S
I/R
R
R
S
S
R
R
S
S
S
R
S
S
S
R
Broad spectrum
cephalosporins
carbapenems
S
S
R
S
S
S
S
S
(ExtendedSpectrum Betalactamase)
Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, “De l’antibiogramme a prescription”. Edition bioMerieux, ISBN
EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or
epidemiological importance, July 2013
•Hospital bacteria which produce beta-lactamase
with broad spectrum;
•False sensitivity for Ceftriaxone; this phenotype
is known to be resistant for all the
Cephalosporins;
•Associated resistance to antibiotics from other
category;
•The Carbapenems remain active; Ertapenem is
preferred;
•It is compulsory the replacement of the colonized
catheter.
Case 3
Patient C.T., 16 old days, premature born, with i. v. catheter soon
after birth. Since 2 days present fever 38,1°C, pale skin, cold
extremities.
The results of the blood cultures:
 First blood culture: (time 21.30) was positive for Staphylococcus
aureus, after 18 hours of incubation.
 Second blood culture (time 23.00) was negative, after 7 days of
incubation.
Antibiogram:
Sensitive to: meropeneme, norfloxacine, gentamicin, vancomycin,
cotrimoxazol
Resistant to: penicillin, oxacillin, erythromycin, tetracycline
Interpret the results and argue. Indicate you treatment option.
Mechanism
Peniciline G, peniciline
A, carboxipeniciline,
ureidopeniciline
Antibiotic +
beta lactamase
inhibitor
Peniciline M
Cephalosporine
carbapenems
Wild
S
S
S
S
Penicilase
R
S
S
S
Modifying PLP, mecA gene
R
R
R
R
BORSA
R
S/R
R
S
MODRSA
S
S
R
S
BORSA: S. aureus borderline
MODSA: modified S. aureus
•Bacteria is resistant to Oxacillin (Meticillin
resistant), so it will be resistant to all beta lactam
antibiotics, including carbapenems;
•Testing for Vancomycin through difuzimetric
antibiogram it is not indicated, because the
sensitivity to this antibiotic can be tested trough
MIC (minimal inhibitory concentration).
Patient D.C., 28 years, was involved in a traffic
stroke. He received an arthiculary prosthesis at the
knee. 4 days after surgical intervention, the patient
present fever, 39,2°C.
Sampling: 3 blood cultures at each 30 minutes.
Results:
 Blood culture I (time 17.10) and II (time 17.40) was
positive for Staphylococcus epidermidis meticilinresistent
 The third blood culture (time 18.10) was negative
after 7 days of incubation.
Interpret and argue the results. Indicate your
treatment option.
MIC determination:
Prepare inoculum
suspension
Select colonies
Mix well
Standardize
inoculum
suspension
Swab plate
Remove sample
Incubate overnight
Add disks
Transmitted
Light
Reflected
Light
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Microbial Identification - bacteria and yeast
identification (ID)
Antibiotic Susceptibility Testing (AST) and
resistance mechanism detection
Epidemiologic trending and reporting
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