microbiology review blood
TRANSCRIPT
Demetrio L. Valle Jr., MD, MSc., FPSP, FASCP, IFCAP
Anatomic and Clinical Pathologist
OUTLINEDIAGNOSTIC BACTERIOLOGY
Blood Cerebrospinal FluidUrineStoolUpper respiratory tractLower respiratory tractPus and exudatesUpdates in Multidrug Resistance Bacteria
BLOOD Expected Organisms
Bacteroides fragilis Brucella Burkholderia pseudomallei Candida albicans and Cryptococcus neoformans Haemophilus influenzae Neisseria meningitidis Non-fermenters other than Pseudomonas aeruginosa Other Enterobacteriaceae Pseudomonas aeruginosa Salmonella typhi and non-typhi Staphylococcus aureus Streptococci (S. pyogenes, S. pneumoniae, viridans
streptococci)
Blood - Media and diagnostic reagentsCOLLECTION MEDIAAEROBIC - Tryptic soy broth (TSB) can be
replaced by any rich broth, e.g. Brain–heart infusion broth
ANAEROBIC- thioglycollate broth or Schaedler broth or Wilkins–Chalgren anaerobe broth
ISOLATION MEDIABlood agar, chocolate agar and MacConkey
agar
Tryptic soy broth (TSB) Thioglycollate broth
Blood - Media and diagnostic reagentsDIAGNOSTIC REAGENTS
Bacitracin disc (Group A β- Streptococci – S. pyogenes –susceptible)
Coagulase plasma (S. aureus) β-Lactamase test reagent (detection of Beta-lactamase enzyme)** Optochin disc (differentiate S. pneumoniae from other α hemolytic
strep) Oxidase reagent Salmonella agglutinating antisera V and XV factors (Identification of Hemophilus influenzae) Haemophilus influenzae type b antiserum Neisseria meningitidis agglutinating serum (polyvalent andspecific groups A, B, C)** penicillin, cephalosporins, cephamycins and carbapenems
Oxidase Test (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) used to detect the presence of oxidase enzymes
produced by a variety of bacteria. oxidase test can be used to differentiate between
genera :Moraxella (+) and Neisseria (+) from Acinetobacter
(-) Aeromonas (+), Plesiomonas shigelloides (+), and
Vibrio (V+) from other Enterobacteriaceae (-) Burkholderia gladioli (-) and B. mallei (V) from B.
cepacia (+) and B. pseudomallei (+) Pseudomonas aeruginosa , Neisseria gonorrhoeae
and Campylobacter jejuni are oxidase-positive pathogens
Oxidase Test V (Heme) & X (NAD) factor test
BACTEREMIAPresence of bacteria in the blood streamCaused by:
Post-operative complicationsIntravascular cathetersLocalized infection
In-patient mortality – 20%Shock and organ failure – 90%
BACTEREMIAEarly detection 77%Differentiation between Gram positive or
Gram negative is one of the most important factors.
WHOLE BLOOD CULTURE
Most sensitive and the gold standard.
It requires incubation, subculturing, biochemical tests.
3-7 days TAT
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METHODS TO IMPROVE BLOOD CULTURE YIELDSKIN PREPARATIONSKIN PREPARATION
Strict aseptic technique (Chandrasekar & Brown, 1994).
Povidone iodine versus 2% tincture of iodine (Little et al., 1999)Tincture of iodine -> significant reduction in
skin flora contamination due to the faster onset of action
0.5% Alcoholic chlorhexidine (Mimoz et al., 1999)Reduced the incidence of blood culture
contamination15 to 30 seconds
METHODS TO IMPROVE BLOOD CULTURE YIELD
TIMING OF BLOOD EXTRACTIONTIMING OF BLOOD EXTRACTIONContinuous Bacteremia Intermittent Bacteremia
Chandrasekar & Brown, 1994 drawing multiple blood culture sets in 24 hour
period have been shown to detect intermittent bacteremia
Li et al., 1994 Similar yields if samples were collected within
2 hours or within 24 hoursMylotte & Tayana, 2000
Patients with antibiotics, samples drawn close to the time antibiotic conc. have reached low levels.
METHODS TO IMPROVE BLOOD CULTURE YIELD
VOLUME OF BLOODVOLUME OF BLOODIs the most important factor ( Shafazand &
Weinacker, 2002)At least 10 mL, provide the highest yield
and lowest number of false-negative blood culture results (Mermel and Maki, 1993).
Extracting more than 30 mL of blood does not improve the sensitivity of blood and contributes to iatrogenic causes of anemia (Weinstein et al., 1983)
METHODS TO IMPROVE BLOOD CULTURE YIELD
ANTIBIOTIC TREATMENTANTIBIOTIC TREATMENTSignificant decrease the yield of blood
cultures (Chandrasekar & Brown, 1994; Leibovici, 1991)
10 mL per 100 mL of culture broth dilutes the concentrations of antibiotics and neutralizing serum bactericidal activity in the culture (Washington & Ilstrup, 1996).
Antibiotic-absorbent resins (BacT/Alert, Biomerioux, France).
Antimicrobial removal device (BACTEC,Beckton Dickinson, MD).
Common causes of bacteremia
Processing of blood culturesIncubation SubculturesFinal processing Antimicrobial susceptibility testing (AST)Detection 0f contaminants
Incubation timeManual
35-37⁰CInspected twice a day for at least 3 days for signs of microbial
growthSedimented red bloodSigns of microbial growth
a floccular deposit on top of the blood layer uniform or subsurface turbidity hemolysis coagulation of the broth a surface pellicle production of gas white grains on the surface or deep in the blood layer.
Perform gram stain
Subculturesfor Gram-negative rods: MacConkey agar,
Kligler iron agar, motilityindole–urease (MIU) medium, Simmons citrate agar;
for small Gram-negative rods: blood agar;for staphylococci: blood agar, mannitol salt
agar;for streptococci: blood agar with optochin,
bacitracin, and tellurite discs, sheep blood agar for the CAMP test, and bile–esculin agar.
SubcultureMicroorganisms may grow without producing
turbidity or visible alteration of the broth. e.g. S. Pneumoniae (autolysis and die rapidly)Subculture in chocolate agar after 18-24 hours
incubation. After 7 days of incubation without growth –
inoculate in thioglycollate broth (incubate for another 3 days)
Antimicrobial susceptibility testingTime is gold in BLOOD CULTURE. Gram stain result – gram positive cocci
(Staph) or gram negative rodsSwab dipped into the turbid broth swab
inoculate in Mueller-Hinton medium (95% of cases correlate with the standardized test)
ContaminantsAseptic skin preparationAseptic procedures during inoculation and
subculturesUsual contaminants
S. epidermidis, P. acnes, diphtheroids, Acinetobacter spp. and Bacillus spp.
True bacteremia if the same organism grows in two bottles of
the same blood specimen;if the same organism grows in cultures from
more than one specimen; if growth is rapid (within 48 hours);if different isolates of one species show the
same biotypes and antimicrobial-susceptibility profiles.