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Microbiological Considerations in Diagnosing S. aureus Bacteremia. Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories. Staphylococcus aureus Bacteremia Microbiological Considerations. Overview of Blood Culture Systems Skin Antisepsis

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microbiological considerations in diagnosing s aureus bacteremia

Microbiological Considerations in Diagnosing S. aureus Bacteremia

Patrick R. Murray, Ph.D.

NIH Clinical Center

Chief, Microbiology Laboratories

staphylococcus aureus bacteremia microbiological considerations
Staphylococcus aureus BacteremiaMicrobiological Considerations
  • Overview of Blood Culture Systems
    • Skin Antisepsis
    • Volume of blood and blood-to-broth ratio
    • Number and timing of cultures
    • Methods for detection of microbial growth
  • Interpretation of Culture Results
    • Time to detection of positive cultures
    • Spectrum of organisms recovered in blood cultures
    • Significant vs. contaminated culture
  • Identification of Staphylococci
    • Direct identification vs. use of subculture
    • Coagulase test - slide test, tube test, commercial tests
    • Protein A - commercial tests
    • Genetic probes for S. aureus
    • Fluorescent in situ hybridization (FISH) test
overview of blood culture systems
Overview of Blood Culture Systems
  • Skin antisepsis
    • 70% alcohol followed by 2% tincture of iodine, povidone-iodine, or chlorhexidine
    • <3% considered good practice
  • Volume of blood and blood-to-broth ratio
    • Most septic patients with <1 org/ml of blood; therefore, the volume of blood cultured is related to % positive cultures
    • Volume: 20-30 ml for adults, proportionally less for children
    • Dilution of blood in broth by at least 1:5 ratio (except resin media)
  • Number and timing of cultures
    • Continuous vs. intermittent bacteremia
    • 2-3 cultures over 24-hour period
  • Methods for Detection of Microbial Growth
    • Manual methods - obsolete
    • Lysis-centrifugation system - quantitative culture
    • Automated methods - measure of microbial metabolism, e.g., carbon dioxide production, oxygen consumption
interpretation of culture results
Interpretation of Culture Results
  • Time to detection of positive cultures
    • Most positive cultures detected in first 48 hours of incubation
    • S. aureus detected in <24 hours; other staph >24 hours
    • Culture routinely held 5-7 days
  • Spectrum of organisms recovered in blood cultures
    • 10-15% of cultures typically positive
    • Most common isolates are: CNS, S. aureus, E. coli, Enterococcus, Klebsiella, S. pneumoniae
  • Significant vs. contaminated culture
    • Most isolates of S. aureus, S. pneumoniae, ß-hemolytic streptococci, Enterococcus, Enterobacteriaceae, Ps. aeruginosa, gram-neg. anaerobes, and yeasts are significant
    • Most isolates of CNS, Corynebacterium, Proprionibacterium, and Bacillus are insignificant
    • Significant isolates of CNS are typically associated with a contaminated line or other foreign body.
identification of staphylococci
Identification of Staphylococci
  • Direct identification vs. use of subculture
    • Subculture plate - growth of S. aureus by 4-6 hours
    • Serum separater/clot tube (SST) used to concentrate bacteria
    • Positive broth can be used for the FISH (fluorescent in situ hybridization) test and molecular probes, but a heavier inoculum (e.g., from subculture plate) is needed for the coagulase and protein A tests
identification of staphylococci1
Identification of Staphylococci
  • Coagulase test
    • Measures the ability of Staph to clot plasma; EDTA rabbit plasma is recommended
    • Coagulase can be bound to the surface of the bacteria or freely excreted; bound coagulase (clumping factor) is detected by the “slide” test and commercial latex agglutination tests (rapid), free coagulase is detected by the “tube” test (4-24 hours)
    • All S. aureus isolates are positive by the tube test; 85% are positive by the slide or commercial tests
    • S. schleiferi and S. lugdunensis are positive by the slide test but not the tube test
    • S. intermedius and S. hyicus (animal strains) are positive by the tube test (generally a delayed reaction)
  • Protein A test
    • Can be detected in combination with coagulase by commercial tests
identification of staphylococci2
Identification of Staphylococci
  • Genetic probes for S. aureus
    • The GenProbe AccuProbe system uses a single-stranded DNA probe with a chemiluminescent label that is complementary to rRNA of S. aureus.
    • The test inoculum is prepared from a subculture plate or from a broth culture with a turbidity of a McFarland 1 standard.
    • The total test time for cell lysis, hybridization, and detection takes less than 1 hour.
    • Marlowe et al (JCM 41:1266, 2003) reported the limit of detection with seeded blood cultures was approximately 10,000 CFU/ml with this method. This is at least 10- to 100-fold more sensitive than the limit of detection for the blood culture instrument.
identification of staphylococci3
Identification of Staphylococci
  • Fluorescent in situ hybridization (FISH)
    • Applied Biiosystems (Boston Probes) developed a FISH method using Peptide Nucleic Acid (PNA) probes that target mRNA of specific bacteria (e.g., S. aureus).
    • PNA is a synthetic pseudopeptide that hybridizes to complementary nucleic acid targets. These probes have a higher specificity and more rapid hybridization kinetics compared to traditional DNA probes. Fluorescent labels are attached to the probe to help detect the target organisms.
    • The total test time is approximately 2.5 hours.
    • In three studies using the probes with positive blood culture broths, the sensitivity and specificity was virtually 100%.
      • Oliveira et al. J Clin Microbiol 40: 247-251, 2002
      • Oliveira et al. J Clin Microbiol 41:889-891, 2003
      • Chapin and Musgnug. J Clin Microbiol 41:4324-4327, 2003
identification of staphylococci4
Identification of Staphylococci
  • Fluorescent in situ hybridization (FISH)
    • Applied Biiosystems (Boston Probes) developed a FISH method using Peptide Nucleic Acid (PNA) probes that target mRNA of specific bacteria (e.g., S. aureus).
    • PNA is a synthetic pseudopeptide that hybridizes to complementary nucleic acid targets. These probes have a higher specificity and more rapid hybridization kinetics compared to traditional DNA probes. Fluorescent labels are attached to the probe to help detect the target organisms.
    • The total test time is approximately 2.5 hours.
    • In three studies using the probes with positive blood culture broths, the sensitivity and specificity was virtually 100%.
      • Oliveira et al. J Clin Microbiol 40: 247-251, 2002
      • Oliveira et al. J Clin Microbiol 41:889-891, 2003
      • Chapin and Musgnug. J Clin Microbiol 41:4324-4327, 2003