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

2. Blood Cultures Factors affecting bloodstream infections and culture yield Parameters Significance and interpretation Systems and media Fastidious organisms Trends

3. 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.

4. 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.

5. 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.

6. 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.

7. 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

8. 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

9. 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

10. 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

11. 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)

12. 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

14. 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

16. 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

17. 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

18. Blood Cultures: CMBCS Summary SystemManufacturerCO 2 detection method Detection Interval BacT/AlertBioMerieuxColorimetric10 minutes BACTEC series Becton- Dickenson Fluorescence10 minutes VersaTrekTrek Diagnostics Manometric12 minutes

19. Quantitative Blood Cultures: Lysis Centrifugation Method

20. Blood Cultures: Positive Rates Overall positive rate ~10% HACEK organisms, 0.01% for all blood cultures or 1% of all blood isolates

21. Blood Cultures: Common Organisms Data from Reimer et al., 1997 1975-771992-93 1E. coliS. aureus 2S. aureusE. coli 3S. pneumoniaeCoag-neg. Staph 4K. pneumoniaeK. pneumoniae 5P. aeruginosaEnterococcus spp. 6B. fragilisP. aeruginosa 7Enterococcus spp.S. pneumoniae 8S. pyogenesViridans streptococci 9C. albicansC. albicans 10P. mirabilisE. cloacae

22. Blood Cultures: Time of Detection Data from BACTEC 9240 (Pat Murray, 1997) –Streptococcus10.3 hr –Enterobacteriaceae14.0 hr –Enterococcus15.1 hr –Staphylococcus aureus17.8 hr –Pseudomonas18.5 hr –Coag-neg. Staph22.9 hr –Yeast65.1 hr

23. Blood culture for HACEK Data from Septi-Chek system (Doern et al., 1996) –Organism Days 1-56-7>=8Mean –Haemophilus aphrophilus1941 3.3 –A. actinomycetemcomitans91- 3.7 –Cardiobacterium hominis16-- 2.9 –Eikenella corrodens101- 3.4 –Kingella spp.32- 3.8 –Brucella spp.812 5.6 –Francisella tularensis5-- 3.6 –Nutritionally v. strep121123 4.4 –Total 191216

24. Blood culture for Brucella Bannatyne et al. JCM 1997; 35:2673-4 BACTEC 9240 System –DaysNo. isolated –1-348 (49.5%) –4-542 43.4%) –6-74 (4.1%) –8-93 (3.1%) –100 –Total97 (100%)

25. Blood Culture Protocol for Endocarditis? Not necessary (Baron EJ et al., CID 2005;

26. 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

27. Isolation and Pure Culture

28. Gram Stain

29. Bacterial Identification: Phenotypic Tests

30. Antimicrobial Susceptibility Tests

31. 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