microbiological considerations in diagnosing s. aureus bacteremia patrick r. murray, ph.d. nih...
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![Page 1: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/1.jpg)
Microbiological Considerations in Diagnosing S. aureus Bacteremia
Patrick R. Murray, Ph.D.
NIH Clinical Center
Chief, Microbiology Laboratories
![Page 2: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/2.jpg)
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
![Page 3: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/3.jpg)
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
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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.
![Page 5: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/5.jpg)
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
![Page 6: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/6.jpg)
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
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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.
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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
![Page 9: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/9.jpg)
![Page 10: Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories](https://reader035.vdocuments.us/reader035/viewer/2022072010/56649dbe5503460f94ab1d32/html5/thumbnails/10.jpg)
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