Automated Blood Cultures

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Automated Blood Cultures
??? Xiang-Yang Han, MD, PhD Department of
Laboratory Medicine The University of Texas M.
D. Anderson Cancer Center
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Blood Cultures

• Factors affecting bloodstream infections and
  culture yield
• Parameters
• Significance and interpretation
• Systems and media
• Fastidious organisms
• Trends

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Bloodstream Infection Factors-1

• Bloodstream infection is the most severe form of
  infection and carries a high fatality (20 to
  50).
• Microbial invasion into bloodstream reflects
  failure of initial host defense loss of
  integrity of skin or mucosa, weakened innate or
  acquired immunity, or direct blood inoculation.

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Bloodstream Infection Factors-2

• Microbial factors virulence mechanisms, such as
  toxins, intracellular survival, evasion or
  shielding from host defenses (leukocytes,
  complements, antibodies, etc).
• The presence of antimicrobics in the circulation
  negatively affect culture positivity.

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Blood Cultures Parameters-1

• Timing
• Blood should be drawn before antibiotic therapy,
  if all possible
• The presence of bacteria or fungi in the
  bloodstream is constant in case of endocarditis
• In other cases, microbes in circulation are not
  steady, and the best time to draw blood is during
  the rise of fever.

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Blood Cultures Parameters-2

• Volume
• 20-40 ml for each set of cultures (one aerobic
  bottle and one anaerobic bottle), e.g.,
• 10 ml blood inoculated into 40 ml broth of BACTEC
  bottle to reach 15 ratio
• 20 ml blood inoculated into 80 ml broth of ESP
  80A bottle (ratio 15).
• Pediatric cultures ranging from 1 to 10 ml,
  depending on the age.

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Blood Cultures Parameters-3

• Frequency
• For each episode, 2 to 3 sets of culture should
  be obtained within first 24 hrs
• Data on 282 bacteremic episodes by Weinstein et
  al (RID 1983535-53)
• First culture detected 257 (91)
• Two cultures together detected 281 (99).

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Blood Cultures Parameters-4

• Incubation atomsphere
• The proportion of anaerobic positive cultures is
  decreasing
• Thus, routine anaerobic cultures are not required
  now
• The situation of individual institution and
  patient population needs to be considered, such
  as surgical and OB/GYN patients.

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Blood Cultures Parameters-5

• How long to incubate?
• Many studies have looked into this issue
• Five days are sufficient to detect nearly all
  (99) significant organisms
• Longer incubation mostly picks up contaminants
• A culture turned positive 6-7 days later is
  unlikely to affect patient care

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Blood Cultures Interpretation

• Almost always significant
• Staphylococcus aureus, Escherichia coli and other
  members of Enterobacteriaceae, Pseudomonas
  aeruginosa, Candida spp.
• Common contaminant, but individual judgement
  needed
• Coagulase-neg. staphylococci
• Corneform bacilli
• Alpha-hemolytic streptococci
• Propionibacterium acne

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Blood Cultures Methods

• Manual method (1950s-1970s)
• Incubation for 21 days, visual inspection of
  growth of organisms, and blind subcultures
• Isolator system (lysis centrifugation method)
• Automated method (1980s)
• Automated detection of microbial CO2 production,
  incubation shortened to 7 days, no blind
  subcultures
• Examples BACTEC 460 and 660, later BACTEC NR660.
• Continuously monitoring blood culturing system
  (CMBCS)

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Blood Cultures CMBCS-1

• BioMerieux (Former Organon-Teknika, Durham, NC)
• BacT/Alert series since 1990
• Colorimetric detection for CO2 production
• Every 10 minutes to detect signal and go to
  algorithm for analysis to see if significant
  growth has occurred
• Newer system since 2001 BacT/Alert 3D

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Blood Cultures CMBCS-2

• Becton-Dickinson (Sparks, MD)
• BACTEC series
• Fluorescent detection of CO2 production
• Every 10 minutes to detect signal
• Newer system BACTEC LX 2004 using laser to
  detect CO2 production
• Clinical evaluation in progress.

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Blood Cultures CMBCS-3

• Trek (Former Difco, then ESP, Accumed, Cleveland,
  OH)
• ESP series
• Manometric detection of CO2 production
• Every 12 minutes detection
• Newer system VersaTrek, 2004

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Blood Cultures CMBCS-4

• Comparison of 3 systems
• They are comparable overall
• BacT/Alert FAN bottle (containing
  antimicrobic-removing substance) performs
  slightly better than standard bottles and other
  systems

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Blood Cultures CMBCS Summary
System Manufacturer CO2 detection method Detection Interval
BacT/Alert BioMerieux Colorimetric 10 minutes
BACTEC series Becton-Dickenson Fluorescence 10 minutes
VersaTrek Trek Diagnostics Manometric 12 minutes
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Quantitative Blood Cultures Lysis
Centrifugation Method
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Blood Cultures Positive Rates

• Overall positive rate 10
• HACEK organisms, 0.01 for all blood cultures or
  1 of all blood isolates

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Blood Cultures Common Organisms

• Data from Reimer et al., 1997
• 1975-77 1992-93
• E. coli S. aureus
• S. aureus E. coli
• S. pneumoniae Coag-neg. Staph
• K. pneumoniae K. pneumoniae
• P. aeruginosa Enterococcus spp.
• B. fragilis P. aeruginosa
• Enterococcus spp. S. pneumoniae
• S. pyogenes Viridans streptococci
• C. albicans C. albicans
• P. mirabilis E. cloacae

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Blood Cultures Time of Detection

• Data from BACTEC 9240 (Pat Murray, 1997)
• Streptococcus 10.3 hr
• Enterobacteriaceae 14.0 hr
• Enterococcus 15.1 hr
• Staphylococcus aureus 17.8 hr
• Pseudomonas 18.5 hr
• Coag-neg. Staph 22.9 hr
• Yeast 65.1 hr

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Blood culture for HACEK

• Data from Septi-Chek system (Doern et al., 1996)
• Organism Days 1-5 6-7 gt8 Mean
• Haemophilus aphrophilus 19 4 1 3.3
• A. actinomycetemcomitans 9 1 - 3.7
• Cardiobacterium hominis 16 - - 2.9
• Eikenella corrodens 10 1 - 3.4
• Kingella spp. 3 2 - 3.8
• Brucella spp. 8 1 2 5.6
• Francisella tularensis 5 - - 3.6
• Nutritionally v. strep 121 12 3 4.4
• Total 191 21 6

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Blood culture for Brucella

• Bannatyne et al. JCM 1997 352673-4
• BACTEC 9240 System
• Days No. isolated
• 1-3 48 (49.5)
• 4-5 42 43.4)
• 6-7 4 (4.1)
• 8-9 3 (3.1)
• 10 0
• Total 97 (100)

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Blood Culture Protocol for Endocarditis?

• Not necessary (Baron EJ et al., CID 2005

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Blood Culture Media

• Aerobic media
• Standard aerobic (SA) bottles for BACTEC,
  BacT/Alert, and Trek
• Anaerobic media
• Standard anaerobic (SN) bottles for BACTEC,
  BacT/Alert, and Trek
• Mycobacterial media based on Middlebrook 7H9
  broth
• Additives to remove antimicrobics
• Resins in BACTEC bottles
• FAN in BacT/Alert bottles

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Isolation and Pure Culture
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Gram Stain
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Bacterial Identification Phenotypic Tests
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Antimicrobial Susceptibility Tests
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Bacterial Identification Genotypic Tests

• PCR sequencing of the 16S rRNA gene
• DNA extraction from colony or positive liquid
• PCR amplification
• Sequencing
• Matching with database
• Correlating with culture features
• Report final identification

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