Automated Blood Cultures

韩向阳Xiang-Yang Han, MD, PhD

Department of Laboratory MedicineThe University of Texas

M. D. Anderson Cancer Center

Blood Cultures

• Factors affecting bloodstream infections and culture yield

• Parameters• Significance and interpretation• Systems and media• Fastidious organisms• Trends

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.

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.

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.

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 1:5 ratio;• 20 ml blood inoculated into 80 ml broth of ESP 80A

bottle (ratio 1:5).– Pediatric cultures ranging from 1 to 10 ml,

depending on the age.

• 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 1983;5:35-53):• First culture detected 257 (91%);• Two cultures together detected 281 (99%).

Blood Cultures: Parameters-3

• 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.

Blood Cultures: Parameters-4

• 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

Blood Cultures: Parameters-5

• 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

Blood Cultures: Interpretation

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)

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

• 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.

Blood Cultures: CMBCS-2

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

Blood Cultures: CMBCS-3

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

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

Quantitative Blood Cultures: Lysis Centrifugation Method

Blood Cultures: Positive Rates

• Overall positive rate ~10%• HACEK organisms, 0.01% for all

blood cultures or 1% of all blood isolates

Blood Cultures: Common Organisms

• Data from Reimer et al., 19971975-77 1992-93

1 E. coli S. aureus2 S. aureus E. coli3 S. pneumoniae Coag-neg. Staph4 K. pneumoniae K. pneumoniae5 P. aeruginosa Enterococcus spp.6 B. fragilis P. aeruginosa7 Enterococcus spp. S. pneumoniae8 S. pyogenes Viridans streptococci9 C. albicans C. albicans10 P. mirabilis E. cloacae

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

Blood culture for HACEK

• Data from Septi-Chek system (Doern et al., 1996)– Organism Days 1-5 6-7 >=8 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

Blood culture for Brucella

• Bannatyne et al. JCM 1997; 35:2673-4• 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%)

Blood Culture Protocol for Endocarditis?

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

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

Isolation and Pure Culture

Gram Stain

Bacterial Identification: Phenotypic Tests

Antimicrobial Susceptibility Tests

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