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Blood culture

Definitions: Definitions:
Blood culture Cultivation of blood sample Want to isolate and identify the bacteria or fungus from the blood, in some pathologic conditions. Bacteriemia Bacteriemia the presence of bacteria in the blood for a short period of time, without clinical expression. Septicaemia Septicaemia systemic disease associated with the presence and persistence of the microorganisms or their toxins in the blood.

Physiopathology
Normally the blood is sterile In blood culture can grow:
Contaminants bacteria from skin, during sampling Microorganisms Microorganisms occasionally identified in the blood, in different situation:
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, rucellosis, Frequent Frequent in acute lobar pneumonia, meningitis, can develop infections with septic metastasis localization.

Microorganisms izolated Microorganisms izolated from patients with bacteremia (Diekema et al, 1999)
Enterobacteriaceae
Escherichia coli..............................1751 cases coli..............................1751 cases Klebsiella spp..................765 Enterobacter spp.............................399 Serratia spp..136 Proteus mirabilis122 mirabilis122 Salmonella, Salmonella, all serotypes..93 serotypes..93 Citrobacter spp.76 complex Enterobacter agglomerans..44 agglomerans..44 Morganella morganii..................26 morganii..................26

Other microorganisms microorganisms


Staphylococcus aureus.2151 aureus.2151 Coagulase negative Stafilococci 1256 Stafilococ Enterococcus spp.794 Streptococcus pneumoniae...475 pneumoniae...475 Pseudomonas aeruginosa451 aeruginosa451 Streptococi beta-hemolitici307 betaAcinetobacter spp.206 Streptococi viridans154 Stenotrophomonas maltophilia69 maltophilia69 Haemophilus spp.27 Corynebacterium spp..20

Procedure necessary: ecessary:


sterile transfer kit culture media
system of manually manufacture and visual reading; system automatic systems; systems of centrifugation for mycobacterium detection and other intracellular bacteria.

piece of oil cloth 40/40 cm solutions for decontamination:


liquid soap; iodine 2%; ether :
remove iodine; dry the skin .

Diphasic culture media

Procedure
Pathologic product: blood. When?
At the beginning of the disease; Before antibiotic administration During the high fever

How much?
Adult: 3 samples x 20 ml, from different veins Children: 1 sample x 1 3 ml.

How ?
Skin washing Disinfection with iodine 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:
became turbid destruction / coagulation of erythrocytes appearance of gas.

Next:
Microscopic examination of the colonies from the solid phase Subculture Biochemical and antigenic identification Antibiogram. Antibiogram.

Quality criteria for blood culture:


Sampling before antimicrobial therapy; Sampling from different veins, for each blood culture; Avoidance of sampling through i.v. catheter i.v. Quick transportation at laboratory, or, if it is not possible, incubation of bottle at 37C, until the 37 moment of sending. Isolation of the same bacteria from many bottles.

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3. 4.

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Analysis requesting specifications:

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.

Results interpretation
Microbiologist reasons: reasons: Has clinical significance: isolation of the same bacteria (species) in significance: more bottles and from different veins. Contamination: 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 polyof at least 2 microorganisms from the same blood culture, at least two times in 24 hours. Physicians reasons: The age and the immune status of the patient; The characteristics of the primary septic focus; Inflammatory system: leukocyte, CRP, fibrinogen

The analysis of the bacterial growing absence

The absence of bacteria from the sample; Low sensitivity of the method; Samples collected after treatment with antibiotics.

The efficiency of antimicrobial therapy


Efficient antibiotic therapy: Decreasing of CRP value Normalizing Normalizing of CRP concentration means clinical recovery; Non efficient antibiotic therapy: 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.

Wrong techniques:

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).

Case 1 Case
Woman, 62 old years, is admitted to hospital with echocardiography diagnosis of sub-acute endocarditis. subFrom 3 blood cultures was isolated Enterococcus spp. Difuzimetric antibiogram has this results: Ampicil Ampicillin Resistant (R) Ceftriaxone Ceftriaxone Sensitive (S) Gentamicin (120g) - R (120 Clindamy Clindamycin - S Cotrimoxazol - R Vancomy Vancomycin - S Teicoplanin - S Linezolid - S

Antibiotics: mechanisms of actions

Interpretative antibiogram antibiogram

The resistance to Ampicillin and the high level of resistance


for Gentamicin compromise the synergic effect of this compromise association;

This phenotype of resistance is frequent for E. faecium;; faecium Sensibility testing for Ceftriaxone is wrong / not possible, and
the transmitted 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, 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

Interpretative antibiogram antibiogram


Hospital bacteria who produce beta-lactamase with broad beta
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 imposed the replacement of the colonized catheter.

Caz 3
Patient C.T., 16 old days, premature born, with i. v. catheter soon after birth. Pat T., days, Since 2 days present fever 38,1C, pale skin, cold extremities. ,1

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: Antibiogram: Sensitive to: meropeneme, norfloxacine, gentamicin, vancomycin, cotrimoxazol to: meropeneme norfloxacine vancomy Resistant to: penicillin, oxacillin, erythromycin, tetracycline to: penicill oxacill ery romy tetracycline Interpret the results and argue. Indicate you treatment option.

Interpretative antibiogram

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).

Case 4 Case
Patient D.C., 28 years, was involved in a traffic stroke. He Pat years, received an articulary prosthesis at the knee. 4 days after surgical intervention, the patient present fever, 39,2C. 39,2 Sampling: 3 blood cultures at each 30 minutes.

Results: Blood culture I (time 17.10) and II (time 17.40) was (time (time positive for Staphylococcus epidermidis meticilin-resistent meticilin-res 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

Measure Zones
Transmitted Light

Reflected Light