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Demetrio L. Valle Jr., MD, MSc., FPSP, FASCP, IFCAP Anatomic and Clinical Pathologist
OUTLINE <ul><li>DIAGNOSTIC BACTERIOLOGY </li></ul><ul><ul><li>Blood  </li></ul></ul><ul><ul><li>Cerebrospinal Fluid </li><...
BLOOD  <ul><li>Expected Organisms </li></ul><ul><ul><li>Bacteroides fragilis </li></ul></ul><ul><ul><li>Brucella </li></ul...
Blood - Media and diagnostic reagents <ul><li>COLLECTION MEDIA </li></ul><ul><li>AEROBIC -  Tryptic soy broth (TSB) can be...
Tryptic soy broth (TSB)  Thioglycollate  broth
 
Blood - Media and diagnostic reagents <ul><li>DIAGNOSTIC REAGENTS </li></ul><ul><ul><li>Bacitracin disc  (Group A  β - Str...
Oxidase Test  ( N,N,N',N'-tetramethyl- p -phenylenediamine dihydrochloride)   <ul><li>used to detect the presence of oxida...
Oxidase Test V (Heme) & X (NAD) factor test
BACTEREMIA <ul><li>Presence of bacteria in the blood stream </li></ul><ul><li>Caused by: </li></ul><ul><ul><li>Post-operat...
BACTEREMIA <ul><li>Early detection    77% </li></ul><ul><li>Differentiation between Gram positive or Gram negative  is on...
WHOLE BLOOD CULTURE <ul><li>Most sensitive and the gold standard. </li></ul><ul><li>It requires incubation, subculturing, ...
METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>SKIN PREPARATION </li></ul><ul><ul><li>Strict aseptic technique (Chandrasek...
METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>TIMING OF BLOOD EXTRACTION </li></ul><ul><ul><li>Continuous Bacteremia  </l...
METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>VOLUME OF BLOOD </li></ul><ul><ul><li>Is the most important factor ( Shafaz...
METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>ANTIBIOTIC TREATMENT </li></ul><ul><ul><ul><li>Significant decrease the yie...
Common causes of bacteremia
Processing of blood cultures <ul><li>Incubation  </li></ul><ul><li>Subcultures </li></ul><ul><li>Final processing  </li></...
Incubation time <ul><li>Manual </li></ul><ul><ul><li>35-37 ⁰C </li></ul></ul><ul><ul><li>Inspected twice a day for at leas...
 
Subcultures <ul><li>for Gram-negative rods: MacConkey agar, Kligler iron agar, motilityindole–urease (MIU) medium, Simmons...
 
Subculture <ul><li>Microorganisms may grow without producing turbidity or visible alteration of the broth.  </li></ul><ul>...
Antimicrobial susceptibility testing <ul><li>Time is gold in BLOOD CULTURE.  </li></ul><ul><li>Gram stain result – gram po...
Contaminants <ul><li>Aseptic skin preparation </li></ul><ul><li>Aseptic procedures during inoculation and subcultures </li...
True bacteremia  <ul><li>if the same organism grows in two bottles of the same blood specimen; </li></ul><ul><li>if the sa...
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Microbiology review blood

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Transcript of "Microbiology review blood"

  1. 1. Demetrio L. Valle Jr., MD, MSc., FPSP, FASCP, IFCAP Anatomic and Clinical Pathologist
  2. 2. OUTLINE <ul><li>DIAGNOSTIC BACTERIOLOGY </li></ul><ul><ul><li>Blood </li></ul></ul><ul><ul><li>Cerebrospinal Fluid </li></ul></ul><ul><ul><li>Urine </li></ul></ul><ul><ul><li>Stool </li></ul></ul><ul><ul><li>Upper respiratory tract </li></ul></ul><ul><ul><li>Lower respiratory tract </li></ul></ul><ul><ul><li>Pus and exudates </li></ul></ul><ul><ul><li>Updates in Multidrug Resistance Bacteria </li></ul></ul>
  3. 3. BLOOD <ul><li>Expected Organisms </li></ul><ul><ul><li>Bacteroides fragilis </li></ul></ul><ul><ul><li>Brucella </li></ul></ul><ul><ul><li>Burkholderia pseudomallei </li></ul></ul><ul><ul><li>Candida albicans and Cryptococcus neoformans </li></ul></ul><ul><ul><li>Haemophilus influenzae </li></ul></ul><ul><ul><li>Neisseria meningitidis </li></ul></ul><ul><ul><li>Non-fermenters other than Pseudomonas aeruginosa </li></ul></ul><ul><ul><li>Other Enterobacteriaceae </li></ul></ul><ul><ul><li>Pseudomonas aeruginosa </li></ul></ul><ul><ul><li>Salmonella typhi and non-typhi </li></ul></ul><ul><ul><li>Staphylococcus aureus </li></ul></ul><ul><ul><li>Streptococci ( S. pyogenes, S. pneumoniae, viridans streptococci) </li></ul></ul>
  4. 4. Blood - Media and diagnostic reagents <ul><li>COLLECTION MEDIA </li></ul><ul><li>AEROBIC - Tryptic soy broth (TSB) can be replaced by any rich broth, e.g. Brain–heart infusion broth </li></ul><ul><li>ANAEROBIC- thioglycollate broth or Schaedler broth or Wilkins–Chalgren anaerobe broth </li></ul><ul><li>ISOLATION MEDIA </li></ul><ul><li>Blood agar, chocolate agar and MacConkey agar </li></ul>
  5. 5. Tryptic soy broth (TSB) Thioglycollate broth
  6. 7. Blood - Media and diagnostic reagents <ul><li>DIAGNOSTIC REAGENTS </li></ul><ul><ul><li>Bacitracin disc (Group A β - Streptococci – S. pyogenes – susceptible) </li></ul></ul><ul><ul><li>Coagulase plasma ( S. aureus) </li></ul></ul><ul><ul><li>β-Lactamase test reagent (detection of Beta-lactamase enzyme)** </li></ul></ul><ul><ul><li>Optochin disc (differentiate S. pneumoniae from other α hemolytic strep) </li></ul></ul><ul><ul><li>Oxidase reagent </li></ul></ul><ul><ul><li>Salmonella agglutinating antisera </li></ul></ul><ul><ul><li>V and XV factors (Identification of Hemophilus influenzae) </li></ul></ul><ul><ul><li>Haemophilus influenzae type b antiserum </li></ul></ul><ul><ul><li>Neisseria meningitidis agglutinating serum (polyvalent and </li></ul></ul><ul><ul><li>specific groups A, B, C) </li></ul></ul><ul><ul><li>** penicillin, cephalosporins, cephamycins and carbapenems </li></ul></ul>
  7. 8. Oxidase Test ( N,N,N',N'-tetramethyl- p -phenylenediamine dihydrochloride) <ul><li>used to detect the presence of oxidase enzymes produced by a variety of bacteria. </li></ul><ul><li>oxidase test can be used to differentiate between genera : </li></ul><ul><ul><li>Moraxella (+) and Neisseria (+) from Acinetobacter (-) </li></ul></ul><ul><ul><li>Aeromonas (+), Plesiomonas shigelloides (+), and Vibrio (V+) from other Enterobacteriaceae (-) </li></ul></ul><ul><ul><li>Burkholderia gladioli (-) and B. mallei (V) from B. cepacia (+) and B. pseudomallei (+) </li></ul></ul><ul><ul><li>Pseudomonas aeruginosa , Neisseria gonorrhoeae and Campylobacter jejuni are oxidase-positive pathogens </li></ul></ul>
  8. 9. Oxidase Test V (Heme) & X (NAD) factor test
  9. 10. BACTEREMIA <ul><li>Presence of bacteria in the blood stream </li></ul><ul><li>Caused by: </li></ul><ul><ul><li>Post-operative complications </li></ul></ul><ul><ul><li>Intravascular catheters </li></ul></ul><ul><ul><li>Localized infection </li></ul></ul><ul><li>In-patient mortality – 20% </li></ul><ul><li>Shock and organ failure – 90% </li></ul>
  10. 11. BACTEREMIA <ul><li>Early detection  77% </li></ul><ul><li>Differentiation between Gram positive or Gram negative is one of the most important factors. </li></ul>
  11. 12. WHOLE BLOOD CULTURE <ul><li>Most sensitive and the gold standard. </li></ul><ul><li>It requires incubation, subculturing, biochemical tests. </li></ul><ul><li>3-7 days TAT </li></ul>http://www.flickr.com/photos/bjorn_banan/161825278/
  12. 13. METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>SKIN PREPARATION </li></ul><ul><ul><li>Strict aseptic technique (Chandrasekar & Brown, 1994). </li></ul></ul><ul><ul><li>Povidone iodine versus 2% tincture of iodine (Little et al ., 1999) </li></ul></ul><ul><ul><ul><li>Tincture of iodine -> significant reduction in skin flora contamination due to the faster onset of action </li></ul></ul></ul><ul><ul><li>0.5% Alcoholic chlorhexidine (Mimoz et al ., 1999) </li></ul></ul><ul><ul><ul><li>Reduced the incidence of blood culture contamination </li></ul></ul></ul><ul><ul><ul><li>15 to 30 seconds </li></ul></ul></ul>
  13. 14. METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>TIMING OF BLOOD EXTRACTION </li></ul><ul><ul><li>Continuous Bacteremia </li></ul></ul><ul><ul><li>Intermittent Bacteremia </li></ul></ul><ul><ul><ul><li>Chandrasekar & Brown, 1994 </li></ul></ul></ul><ul><ul><ul><ul><li>drawing multiple blood culture sets in 24 hour period have been shown to detect intermittent bacteremia </li></ul></ul></ul></ul><ul><ul><ul><li>Li et al ., 1994 </li></ul></ul></ul><ul><ul><ul><ul><li>Similar yields if samples were collected within 2 hours or within 24 hours </li></ul></ul></ul></ul><ul><ul><ul><li>Mylotte & Tayana, 2000 </li></ul></ul></ul><ul><ul><ul><ul><li>Patients with antibiotics, samples drawn close to the time antibiotic conc. have reached low levels. </li></ul></ul></ul></ul>
  14. 15. METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>VOLUME OF BLOOD </li></ul><ul><ul><li>Is the most important factor ( Shafazand & Weinacker, 2002) </li></ul></ul><ul><ul><li>At least 10 mL, provide the highest yield and lowest number of false-negative blood culture results (Mermel and Maki, 1993). </li></ul></ul><ul><ul><li>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) </li></ul></ul>
  15. 16. METHODS TO IMPROVE BLOOD CULTURE YIELD <ul><li>ANTIBIOTIC TREATMENT </li></ul><ul><ul><ul><li>Significant decrease the yield of blood cultures (Chandrasekar & Brown, 1994; Leibovici, 1991) </li></ul></ul></ul><ul><ul><ul><li>10 mL per 100 mL of culture broth dilutes the concentrations of antibiotics and neutralizing serum bactericidal activity in the culture (Washington & Ilstrup, 1996). </li></ul></ul></ul><ul><ul><ul><li>Antibiotic-absorbent resins (BacT/Alert, Biomerioux, France). </li></ul></ul></ul><ul><ul><ul><li>Antimicrobial removal device (BACTEC,Beckton Dickinson, MD). </li></ul></ul></ul>
  16. 17. Common causes of bacteremia
  17. 18. Processing of blood cultures <ul><li>Incubation </li></ul><ul><li>Subcultures </li></ul><ul><li>Final processing </li></ul><ul><li>Antimicrobial susceptibility testing (AST) </li></ul><ul><li>Detection 0f contaminants </li></ul>
  18. 19. Incubation time <ul><li>Manual </li></ul><ul><ul><li>35-37 ⁰C </li></ul></ul><ul><ul><li>Inspected twice a day for at least 3 days for signs of microbial growth </li></ul></ul><ul><ul><li>Sedimented red blood </li></ul></ul><ul><ul><li>Signs of microbial growth </li></ul></ul><ul><ul><ul><li>a floccular deposit on top of the blood layer </li></ul></ul></ul><ul><ul><ul><li>uniform or subsurface turbidity </li></ul></ul></ul><ul><ul><ul><li>hemolysis </li></ul></ul></ul><ul><ul><ul><li>coagulation of the broth </li></ul></ul></ul><ul><ul><ul><li>a surface pellicle </li></ul></ul></ul><ul><ul><ul><li>production of gas </li></ul></ul></ul><ul><ul><ul><li>white grains on the surface or deep in the blood layer. </li></ul></ul></ul><ul><ul><li>Perform gram stain </li></ul></ul>
  19. 21. Subcultures <ul><li>for Gram-negative rods: MacConkey agar, Kligler iron agar, motilityindole–urease (MIU) medium, Simmons citrate agar; </li></ul><ul><li>for small Gram-negative rods: blood agar; </li></ul><ul><li>for staphylococci: blood agar, mannitol salt agar; </li></ul><ul><li>for streptococci: blood agar with optochin, bacitracin, and tellurite discs, sheep blood agar for the CAMP test, and bile–esculin agar. </li></ul>
  20. 23. Subculture <ul><li>Microorganisms may grow without producing turbidity or visible alteration of the broth. </li></ul><ul><ul><li>e.g. S. Pneumoniae (autolysis and die rapidly) </li></ul></ul><ul><ul><li>Subculture in chocolate agar after 18-24 hours incubation. </li></ul></ul><ul><ul><li>After 7 days of incubation without growth – inoculate in thioglycollate broth (incubate for another 3 days) </li></ul></ul>
  21. 24. Antimicrobial susceptibility testing <ul><li>Time is gold in BLOOD CULTURE. </li></ul><ul><li>Gram stain result – gram positive cocci (Staph) or gram negative rods </li></ul><ul><li>Swab  dipped into the turbid broth  swab inoculate in Mueller-Hinton medium (95% of cases correlate with the standardized test) </li></ul>
  22. 25. Contaminants <ul><li>Aseptic skin preparation </li></ul><ul><li>Aseptic procedures during inoculation and subcultures </li></ul><ul><li>Usual contaminants </li></ul><ul><ul><li>S. epidermidis, P. acnes, diphtheroids, Acinetobacter spp. and Bacillus spp. </li></ul></ul>
  23. 26. True bacteremia <ul><li>if the same organism grows in two bottles of the same blood specimen; </li></ul><ul><li>if the same organism grows in cultures from more than one specimen; </li></ul><ul><li>if growth is rapid (within 48 hours); </li></ul><ul><li>if different isolates of one species show the same biotypes and antimicrobial-susceptibility profiles. </li></ul>
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