This document discusses bloodstream infections (BSIs), including: - BSIs affect 200,000-300,000 people in the US annually with a 20-50% mortality rate. - Timely detection and identification of pathogens is important for optimizing antimicrobial therapy. - Blood cultures are done to detect bacteria or yeasts that have spread from localized infections into the bloodstream. - Key steps for obtaining optimal blood cultures include using aseptic technique, collecting an adequate blood volume (20mL for adults), and obtaining multiple blood culture sets from different sites.

1. Mahen Kothalawala

2. Bloodstream infections (BSIs)
 200,000 to 300,000 cases of BSI (US)
 mortality - 20% to 50%;
 timely detection and identification - most important
functions of the microbiology laboratory.
 It helps to
 establish an infectious etiology and
 provide susceptibility testing for optimization of Antimicrobial
therapy

3. Blood cultures are done to
 Detect the presence of bacteria or yeasts, which may
have spread from a specific site in the body into the
bloodstream.

4. BSI
 Rate of multiplication of microorganisms > the rate of
removal by RES of the body = Blood Stream infection
occur
 Failure of body’s defense systems to clear or failure to
attempts to remove /drain the localized infection –
persistent BSI occur

5. Routes
Organisms in
Circulation
Bactermia
Organisms enter the blood
through the lymphatic system
Focus lung, Meninges, Direct entry to blood stream - in
Joints skin GUT,or Bone infective endocarditis, infected AV fistula, mycotic
aneurisms, suppurative thrombophlebitis and
etc colonized IV catheters

6.  Sudden rush of organisms generally cleared by the
defense system in minutes to hours
 Fixed macrophage in spleen and liver play major role
in clearance- Bacterial virulent factors hinders the
clearance ex capsule etc
 When body response to the infective agents in blood –
Systemic signs of symptoms of illness occur (SIRS) –
the condition is known as sepsis

7. Common sources of BSI
19%
27% Intra Vascular Devises
Genito Urinary Tract
Respiratory Tract
17% Bowel and peritoneum
8% Skin
Billiary Tract
5% 12% Intra Abdominal abcesses
3% 5% Other Known Sites
4% Unknown Sites

8. Clinical Pattern of Blood Stream
Infections
Transient Intermittent Continuous
Manipulation of infected Undrained intra Cardinal feature of endo
sites ex- abdominal abscessese, vascular infections most
abscesses, furuncles, and perinephric abcess, notably acute, sub acute IE
cellulitis Prostatic abscesses et –
Instrumentation of these are common causes
mucosal surfaces colonized of PUO
with regional
flora(Dental, urologic
procedures)
Surgery involving prostate, First few weeks of Typhoid
hysotectomies, suction fever and Brucellosis
curattage, debridement of
infected wounds and burns

9. Breakthrough bacteremia
 Bacteremia which occur while a patient is receiving
systemic therapy with antibacterial agents to which
organism is susceptible
 Can occur early in the infection due to inadequate
antibiotic concentration
 Later due to inadequate drainage of a focus or
impairment host defenses

10. Bacteremia also occur in
 Early in the course of many systemic and localized
infections
 50 to 80% patients with meningitis
 5 to 30% patients with pneumonia
 20 to 70% Septic arthritis
 30 to 50% osteomyelitis
 5 to 90% patients with gonococcal and meningococcal
infections

11. Principles
 Procedure appear very simple
 key elements often are ignored by health-care workers,
thereby yielding false-positive tests (i.e., contaminated
cultures).

12. Key steps
1. best available site for culture,
2. aseptic technique,
3. adequate volume of blood,
4. sufficient number of blood culture sets.
5. Timing of blood culture

13. 1.Selection of the Best Available
Site
 Easily accessible, readily available & minimal
discomfort to the pt
 low contamination rate
 As a rule ,vessels of lower extremes> Upper limb
vessels
 antecubital veins- preferred
 Arterial blood = venous blood
 Avoid blood drawing through lines

14. Blood obtain through intravascular devices
 Often discouraged
 False positive results due to colonizers of the line
(twice than the normal procedure)
 Often carried out to when suspecting Line infections.
 When blood drawn through lines, paired with a
second culture obtained by peripheral venipuncture
aid in interpreting

15. Blood through the CVC
 hematology-oncology patients, blood cultures drawn
through either the central catheter or peripheral vein
show excellent negative predictive value.
 Blood cultures drawn through an indwelling central
venous catheter have low positive predictive value
 Therefore, a positive result from a catheter needs
clinical interpretation and may require confirmation.

16. Blood for culture using a existing
line is used in
 especially for patients on hemodialysis or
 for patients with hematologic and other malignancies,
because of convenience and reduction of trauma to the
veins

17. 2. Skin Antisepsis
 avoid false positives – due to skin flora
First Second Remarks
Standard 70% isoproyl alcohol • 1 to 2 % iodine tincture (30S) Contaminatios ans
Practice (30 s) • or iodophore (aquaous iodine Allergic reactions
solution (1.5 to 2 min) more with
iodophhores
Other l 0.2% chlorine peroxide Less contaminations
agents 10% povidone iodine than 1 (two studies)
Other 70% alcohol 2% chlohexidine gluconate Found to be less
agents contamination with
these
0.5% chlorhexidine Safe in neonates < 7d
in 70% alcohol

18. 2002 recommendation
 2% chlorhexidine considered as the agent of choice
 But, not in children < 2yrs

19. Method
 cleansed with 70% isopropyl or ethyl alcohol and
allowed to air dry.
 A second cleansing should be performed using 1% to
2% tincture of iodine or 10% povidone-iodine solution
applied concentrically;
(this should be allowed to air dry before the vein is
punctured.)

20. Contamination of Blood cultures
could be
 Initial Contaminants
Prevent contamination during collection of Blood for culture –
from skin flora – skin flora –CoNS, Diptheroids etc
 Latent Contamination- usually bacterial and is often observed
long after cultures are initiated. Apparently the bacteria are
present endogenously in the initial plant material and are not
obviously pathogenic in situ –from broths or hands of the
operator - they increase in titer and overrun the cultures. Latent
contamination is particularly dangerous because it can easily be
transferred among cultures
 Introduced Contamination – Contamination can also occur as
a result of poor sterile technique or dirty lab conditions.- Fungi
and Bacillus sp

21. 3.Blood Volume
 Direct relationship between the volume and detection
of bacteremia or fungemia
 Blood volume of each culture (Culture set) is the single
most important variable in recovering microorganisms

22.  Several studies using conventional and early
generations auto mated blood culture systems
demonstrated direct relationship between diagnostic
yield and volume of blood
 When volume of blood increased from 2ml to
20ml, the yield increased 30% to 50%
 Cocerill et al showed that for adults optimum volume
is 20ml for a culture

23. Effect of Volume of Blood Cultured on the Diagnostic Yield of Blood Cultures
Mermel, L. A. et. al. Ann Intern Med 1993;119:270-272

24. bacteremia in adults In infants and young Overwhelming infections
children
low order of magnitude magnitude of bacteremia Irrespective of age category
often <1 to 10 CFU/mL). tends to be greater (often bacteriamia would be > 10000
>100 CFU/mL) CFU/ml
Higher volume of blood A comparatively low Generally gives a positive
required from adults, amount of blood is results even with a least
specially in instances where a sufficient sensitive culture systems
prior antibiotics given
Cultures containing lower blood volumes still should be processed for patient care,
with the notation that suboptimal blood volume might give false-negative results.

25. pediatric patients
 Optimum volume of blood culture not defined
certinity
 A linear relationship exists with these patients too.
 Generally pediatric patients have relatively high degree
of bacteremia
 But reported low level of around ≤ 10 CFU/ml
bacteremia among 68% of infants(Up to 2 months)

26. Pediatric patients
Age group Recommended volume
neonates 1 to 2 mL of blood per culture
ages 1 month to 2 years 2 to 3 mLbloo d per culture
older children 3 to 5 mL for blood per culture
for adolescents.[4 10 to 20 mL blood per culture

27.  Kellogg described a total volume of blood for culture
depending on total blood volume ( 4 to 4.5 % of
patients total blood volume)
Weight of the Total blood Recommended V per Total V per % of total
patient V(ml) culture culture blood V
Kg lb Culture I Culture II
≤1 ≤1 50 - 99 2 - 2 4%
1.1-2 2.2-4.4 100-200 2 2 4 4%
2.1-12.7 4.5-27 >200 4 2 6 3%
12.8-36.3 28-80 >800 10 10 20 2.5%
>36.2 >80 >2200 20 20 40 1.8-2.7%

28. Kellogg et al
 recommended blood culture volumes for infants and
children based on weight and estimated total blood
volume of the child.
 The recommendations are based on the premise that
low level bacteremia (1–10 CFU/mL of blood) can
occur in pediatric populations and that its detection
can be optimized by culturing up to 4.5% of a child's
total blood volume.[28]

29. Blood cultures containing volumes of
>30 mL
 May contribute to nosocomial anemia
 Blood may clot in the syringe

30. 4.Number of Blood Culture Sets
 Two or three blood cultures are adequate for detecting
episodes - caused by common microbial pathogens.
 Using conventional, non-automated blood culture
found that total of 20ml per set gives proper result

31. Culture Positive Rate using manual/ or early
generation automated systems
Study Two with patients with
IE Mayo Clinic
91% 99%
99% Study Two Uni of
88% Colarado
80%
65%
1 st Culture 2nd Culture 3rd Culture

32. No of blood culture sets
 are useful to interpreting clinical significance of
positive blood cultures
 One positive test is sufficient for primary pathogens
 Opportunistic pathogens and Regional flora need
more than one culture result for interpretation

33. Based on the data available
 Rarely necessary to collect more than two blood
culture sets per 24 hours.
 It is not appropriate to collect only a single blood
specimen for culture.
 A single blood culture will not have sufficient volume for
optimal detection of bacteremias and fungemias,
 and the significance of a positive result with CONS and
Diptheroids

34. Recommendations for the timing
of blood cultures
Condition or syndrome Recommendation
Suspected acute primary bacterimia or Obtain two or three blood cultures, one right
fungemia – Meningitis, after the other, from different sites
Osteomyelitis,arthritis, or pneumonia
Fever of uncertain origin (ex occult abscess. Obtain two or three blood cultures. One right
Typhoid fever, brucellosis or other after the other, from different sites initially
undiagnosed febrile syndrome If they are negative after 24 to 48 hrs
incubation. Obtain two or more cultures, one
right after other, from different sites
Suspected bactermia or fungema or with Consider alternative blood culture methods
persistantly negative blood cultures designed to entrance recovery of
mycobacteria, fungi, and rare fastidious micro
organisms

35. Common patterns of culture
positivity
Type of bacterimia Example when one blood culture was
positive
Continuous infective endocarditis subsequent cultures positive in
95% to 100% cases
Bacterimia or fungaemia Other causes subsequent cultures positive
not due to endocarditis 75% to 80% cases
Blood culture positivity Chances for subsequent culture
due to contaminants to be positive is 5% highly likely
to be negative

36. Timing of blood cultures
 Chills(and rigors) – occur 1 hr of lag period
 Fever follows chills and rigors
 Therefore, some recommend collection of blood
culture soon before chills/rigors or fever spike

37. Timing of blood cultures
Fever
Bacterimia
Fever spike
Chills / Rigor
Bacteremia –symptoms
appear after ≈ I hr of lag
period

38. Timing of Blood Cultures
 The optimal time for collection - just before the onset of a shaking chill
 not possible to anticipate the precise timing. - common practice to draw
blood cultures when fever is detected
 As a general rule, it is reasonable to obtain two blood culture sets
simultaneously, especially if antibiotic therapy is going to be initiated;
 in less urgent situations - blood cultures may be spaced at intervals. Li et al
found no significant difference in detection of BSI from blood cultures
obtained simultaneously - versus at intervals during a 24-hour period.
 timing of blood cultures should be a clinical decision - acuity of the patient's
illness and whether immediate antimicrobial therapy will be administered.

39. Variables which govern the culture results
1. Culture Medium
2. Ratio of Blood to Broth
3. Inactivation of Antimicrobial Agents
4. Duration of Incubation of Blood Cultures

40. 1. Culture Medium
 No medium optimally supports the growth of all
potential bloodstream pathogens.
 Even same basal medium (e.g., soybean casein
digested broth) - not perform equally
 Manufacturers supplement the basal media with
proprietary supplements

41. 2.Ratio of Blood to Broth
 Inhibitory substances in blood impairs bacterial
growth – False negatives
 Different methods are used to nullify the effects

42. Factors in blood which inhibit Mechanisms available to Systems
microbial growth counteract the mechanisms
Complement, lysozyme, and Dilution with culture broth below Manual/
phagocytic leukocytes the critical threshhold or action automated
Antimicrobial agents –Inhibition of Dilution with broth and addition of Automated
susceptible agents resins (adsorption), Activated
charcoal or enzymes- β lactamases
**optimal dilution of blood in broth is 5- to 10-fold (i.e., a blood :
broth ratio of 1:5 to 1:10).

43. 3.Inactivation of Antimicrobial
Agents
System Method available Advantage
BACTEC, BD Biosciences, media containing resins that Media containing
Sparks, Md bind antimicrobial agents, resins/charcoal detect more
BacT/Alert, Organon adding activated charcoal to episodes of bacteremia than
Teknika Corp., Durham, some of its medium others media which doesn’t
N.C.) formulations. containing additives
specially gram positives*
this is a one reason for
shifting of major pathogens
of bacteremia from BNB to
GPC
** Resin containing media are very costly than ordinary media

44. 4. Duration of Incubation of Blood
Cultures
 Variable
 As most clinical microbiology laboratories use
Continuous Monitoring systems – for majority of
pathogens 5 d of incubation is enough
 Some recommend – 4 d
 Longer incubation - for fastidious pathogens
Bartonella, Legionella, Brucella, and certain fungi.

45.  prolonged incubation for culture neg IE (e.g., the
HACEK group of fastidious gram-negative bacteria) is
controversial
 Blood cultures for the detection of mycobacteria
should be incubated for four weeks.(HIV/AIDS and
Immunocompromised

46. Available culture systems
Three types
1. Manual (conventional)
2. Lysis-centrifugation
3. Automated continuous-monitoring

47. Manual system
 Transfer of organisms from culture bottle to media
 Blind subculture
 Subculture when changes occur in medium – turbidity,
froth, deposits, pellicles and hemolysis
 High rates of false positivty and false negativity
 Labour intensive

48. Lysis Centrifugation
 The Isolator blood culture system (Wampole
Laboratories, Cranbury, N.J
 Principle of lysis-centrifugation- Blood is inoculated to culture
tubes that contain a lysis solution. After lysis of the blood cells and
tube is centrifuged
 Is unique as the only non-broth-based blood culture system
 After removing the supernatant , pellet is inoculated directly to
agar media that support the growth of potential blood pathogens
including bacteria, fungi, and mycobacteria.

49.  Advantages
 Best system available to detect dimorphic fungi and
Bartonella species[7] ;
 Disadvantages
 labor-intensive, more processing needed
 More manipulations may place technologists at an
increased risk of - blood-borne pathogens & category III and IV organisms
 Reduced detection of anaerobes, Haemophilus
species, and pneumococci – due to delayed processing

50. Automated systems
Early systems New system
The BACTEC radiometric system – Continuous-monitoring blood culture
Replaced in most countries due to safety systems (CMBCS).
labor reduction and more rapid detection
of microbial growth
1. BacT/Alert (Organon Teknika Corp.,
Durham, N.C.),
2. BACTEC 9000 (BD Biosciences, Sparks,
Md.),
3. and ESP (Trek Diagnostic Systems,
Westlake, Ohio). ms

51. CMBCS
 Has modular incubation and agitation units,
 a central computer,
 monitoring of each bottle in the system for microbial growth at
10- to 24-minute intervals,
 culture bottles that accept up to 10 mL of blood per bottle.
 Results of individual bottle readings from the incubator modules
are transmitted to the system's computer for data storage and
analysis, and growth curves are calculated according to
sophisticated algorithms.
 Testing and data accumulation occur around the clock.
 these systems have been able to detect evidence of microbial
growth 1 to 1.5 days sooner
 result in fewer instrument false-positive signals than the earlier
systems.

52. Specific Pathogen groups
 Some microorganisms are isolated infrequently-
Nutritionally fastidious
 These organism groups may not be detected using
routine blood culture methods and systems.
1. Anerobes
2. Nutritionally Variant Streptococci
3. Fungi
4. Bartonells sp
5. Mycobacteria

53. Anaerobic Bacteria
 decrease in the incidence of anaerobic bacteremia for
only about 3% of all BSIs,
 Currently, no consensus about the routine use of
anaerobic bottles
 some authorities recommend their selective use only -
in patients at risk for anaerobic infections.

54. Fungi
 Increasing Trend as the etiologic agents of BSIs.
 Incubation temperature varies
 filamentous fungi grow better at 27° to 30° C,
 while yeasts may show better growth at 37° C.
 The duration of incubation
 most yeasts grow in two to three days,
 the dimorphic fungi may take as long as three to six weeks.
 Variety of media,
 tryptic soy,
 columbia,
 throglycollate and
 brain heart infusion (BHI) broths all have been successful for growing yeasts
and molds from blood.
 Biphasic media (e.g., Castañeda bottles) traditionally have been considered best
for growth of fungi from blood.
 Lysis Cenrifugation is better for intracellular fungi

55. Bartonella Species
 Bartonella henselae and Bartonella quintana are
isolated best by using the Isolator system
 anecdotal reports of isolation using the BACTEC blood
culture system as well.

56. Mycobacteria
 Blood cultures for mycobacteria are usually restricted
to immunocompromised patients, particularly those
with AIDS
 Either the Isolator system, or the BACTEC radiometric
system using 13A blood culture medium can be used
for mycobacterial blood cultures.
 From the Isolator tube the sediment is inoculated onto
Middlebrook 7H11 media.

57. Nutritionally Variant Streptococci
(NVS)
 Similar to viridans group
 Require pyridoxal for growth
 Separate species – Abiotrophia
 considered whenever broth-based cultures appear positive
with gram-positive cocci, but usual subcultures are negative.
1. a subculture to blood agar supplemented with 0.001% pyridoxal o
2. demonstration of satelliting on a blood agar plate, disc 0.001%
pyridoxal or along a staphylococcal streak,

58. Interpretation of positive blood
culture
1. Organisms – considered as true pathogens
2. Organisms of questionable significance

59. Organisms – considered as true pathogens
 Common blood isolates that always or nearly always
(>90%) represent true infection include
 Staphylococcus aureus,
 Streptococcus pneumoniae,
 Escherichia coli
 and other members of the Enterobacteriaceae family,
 Pseudomonas aeruginosa,
 and Candida albicans.

60. Other microorganisms
 Organisms rarely (<5%) represent true bacteremia
 Corynebacterium species,
 Propionibacterium acnes,
 More problematic are group
 viridans streptococci,
 enterococci, and
 coagulase-negative staphylococci (CoNS),
***represent true bacteremia in 38%, 78%, and 15% of
cases

61. CoNS
 Common blood culture contaminants, and important
pathogens in
 patients with intravascular devices
 and implanted prosthetic materials.
 Some authorities suggest that the number of positive
blood culture bottles - predicts the clinical significance of CoNS
 Systematic evaluations find this criterion unreliable

62.  RAPID METHODS FOR IDENTIFICATION
OF ORGANISMS IN BLOOD CULTURES
 Future ?