Microbiology review blood

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  • 1. Demetrio L. Valle Jr., MD, MSc., FPSP, FASCP, IFCAP Anatomic and Clinical Pathologist
  • 2. OUTLINE
    • DIAGNOSTIC BACTERIOLOGY
      • Blood
      • Cerebrospinal Fluid
      • Urine
      • Stool
      • Upper respiratory tract
      • Lower respiratory tract
      • Pus and exudates
      • Updates in Multidrug Resistance Bacteria
  • 3. 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)
  • 4. Blood - Media and diagnostic reagents
    • COLLECTION MEDIA
    • AEROBIC - 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 MEDIA
    • Blood agar, chocolate agar and MacConkey agar
  • 5. Tryptic soy broth (TSB) Thioglycollate broth
  • 6.  
  • 7. Blood - Media and diagnostic reagents
    • DIAGNOSTIC 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 and
      • specific groups A, B, C)
      • ** penicillin, cephalosporins, cephamycins and carbapenems
  • 8. 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
  • 9. Oxidase Test V (Heme) & X (NAD) factor test
  • 10. BACTEREMIA
    • Presence of bacteria in the blood stream
    • Caused by:
      • Post-operative complications
      • Intravascular catheters
      • Localized infection
    • In-patient mortality – 20%
    • Shock and organ failure – 90%
  • 11. BACTEREMIA
    • Early detection  77%
    • Differentiation between Gram positive or Gram negative is one of the most important factors.
  • 12. WHOLE BLOOD CULTURE
    • Most sensitive and the gold standard.
    • It requires incubation, subculturing, biochemical tests.
    • 3-7 days TAT
    http://www.flickr.com/photos/bjorn_banan/161825278/
  • 13. METHODS TO IMPROVE BLOOD CULTURE YIELD
    • SKIN 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 contamination
        • 15 to 30 seconds
  • 14. METHODS TO IMPROVE BLOOD CULTURE YIELD
    • TIMING OF BLOOD EXTRACTION
      • Continuous 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 hours
        • Mylotte & Tayana, 2000
          • Patients with antibiotics, samples drawn close to the time antibiotic conc. have reached low levels.
  • 15. METHODS TO IMPROVE BLOOD CULTURE YIELD
    • VOLUME OF BLOOD
      • Is 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)
  • 16. METHODS TO IMPROVE BLOOD CULTURE YIELD
    • ANTIBIOTIC TREATMENT
        • Significant 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).
  • 17. Common causes of bacteremia
  • 18. Processing of blood cultures
    • Incubation
    • Subcultures
    • Final processing
    • Antimicrobial susceptibility testing (AST)
    • Detection 0f contaminants
  • 19. Incubation time
    • Manual
      • 35-37 ⁰C
      • Inspected twice a day for at least 3 days for signs of microbial growth
      • Sedimented red blood
      • Signs 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
  • 20.  
  • 21. Subcultures
    • for 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.
  • 22.  
  • 23. Subculture
    • Microorganisms 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)
  • 24. Antimicrobial susceptibility testing
    • Time is gold in BLOOD CULTURE.
    • Gram stain result – gram positive cocci (Staph) or gram negative rods
    • Swab  dipped into the turbid broth  swab inoculate in Mueller-Hinton medium (95% of cases correlate with the standardized test)
  • 25. Contaminants
    • Aseptic skin preparation
    • Aseptic procedures during inoculation and subcultures
    • Usual contaminants
      • S. epidermidis, P. acnes, diphtheroids, Acinetobacter spp. and Bacillus spp.
  • 26. 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.