2. Bloodstream infections (BSIs) 200,000 to 300,000 cases of BSI
(US) mortality - 20% to 50%; timely detection and identification -
most importantfunctions 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
ofremoval by RES of the body = Blood Stream infectionoccur Failure
of bodys defense systems to clear or failure toattempts to remove
/drain the localized infection persistent BSI occur 5.
RoutesOrganisms in Circulation BactermiaOrganisms enter the
bloodthrough the lymphatic systemFocus lung, Meninges,Direct entry
to blood stream - in Joints skin GUT,or Boneinfective endocarditis,
infected AV fistula, mycoticaneurisms, suppurative thrombophlebitis
andetc 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 peritoneum8% Skin
Billiary Tract 5%12% Intra Abdominal abcesses 3% 5% Other Known
Sites4% Unknown Sites 8. Clinical Pattern of Blood
StreamInfectionsTransient IntermittentContinuousManipulation of
infected Undrained intraCardinal feature of endosites ex-abdominal
abscessese,vascular infections mostabscesses, furuncles,
andperinephric abcess,notably acute, sub acute IEcellulitis
Prostatic abscesses et Instrumentation of these are common
causesmucosal surfaces colonized of PUOwith regionalflora(Dental,
urologicprocedures)Surgery involving prostate, First few weeks of
Typhoidhysotectomies, suctionfever and Brucellosiscurattage,
debridement ofinfected 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 meningococcalinfections 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 steps1. 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 AvailableSite Easily accessible, readily available &
minimaldiscomfort to the pt low contamination rate As a rule
,vessels of lower extremes> Upper limbvessels 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 asecond culture obtained by peripheral venipunctureaid 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 existingline 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 floraFirstSecondRemarksStandard 70% isoproyl
alcohol 1 to 2 % iodine tincture (30S) Contaminatios ansPractice
(30 s) or iodophore (aquaous iodine Allergic reactionssolution (1.5
to 2 min)more with iodophhoresOtherl 0.2% chlorine peroxideLess
contaminationsagents 10% povidone iodine than 1 (two
studies)Other70% alcohol 2% chlohexidine gluconate Found to be
lessagents contamination with these 0.5% chlorhexidineSafe 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 andallowed to air dry. A second cleansing should be
performed using 1% to2% tincture of iodine or 10% povidone-iodine
solutionapplied concentrically;(this should be allowed to air dry
before the vein ispunctured.) 20. Contamination of Blood
culturescould be Initial ContaminantsPrevent contamination during
collection of Blood for culture from skin flora skin flora CoNS,
Diptheroids etc Latent Contamination- usually bacterial and is
often observedlong after cultures are initiated. Apparently the
bacteria arepresent endogenously in the initial plant material and
are notobviously pathogenic in situ from broths or hands of
theoperator - they increase in titer and overrun the cultures.
Latentcontamination is particularly dangerous because it can easily
betransferred among cultures Introduced Contamination Contamination
can also occur asa result of poor sterile technique or dirty lab
conditions.- Fungiand 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 adultsIn infants and young Overwhelming
infectionschildrenlow order of magnitudemagnitude of
bacteremiaIrrespective of age categoryoften 10000>100 CFU/mL)
CFU/mlHigher volume of blood A comparatively low Generally gives a
positiverequired from adults,amount of blood isresults even with a
leastspecially in instances where a sufficientsensitive culture
systemsprior antibiotics givenCultures 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
definedcertinity A linear relationship exists with these patients
too. Generally pediatric patients have relatively high degreeof
bacteremia But reported low level of around 10 CFU/mlbacteremia
among 68% of infants(Up to 2 months) 26. Pediatric patientsAge
group Recommended volumeneonates1 to 2 mL of blood per cultureages
1 month to 2 years 2 to 3 mLbloo d per cultureolder children3 to 5
mL for blood per culturefor adolescents.[410 to 20 mL blood per
culture 27. Kellogg described a total volume of blood for
culturedepending on total blood volume ( 4 to 4.5 % ofpatients
total blood volume) Weight of the Total blood Recommended V
perTotal V per % of total patientV(ml) culturecultureblood V
KglbCulture I Culture II 1150 - 992-2 4%1.1-22.2-4.4 100-200224
4%2.1-12.7 4.5-27 >200426 3%12.8-36.3 28-80 >800101020 2.5%
>36.2>80>2200202040 1.8-2.7% 28. Kellogg et al recommended
blood culture volumes for infants andchildren based on weight and
estimated total bloodvolume of the child. The recommendations are
based on the premise thatlow level bacteremia (110 CFU/mL of blood)
canoccur in pediatric populations and that its detectioncan be
optimized by culturing up to 4.5% of a childstotal 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 systemsStudy 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
ofpositive blood cultures One positive test is sufficient for
primary pathogens Opportunistic pathogens and Regional flora
needmore than one culture result for interpretation 33. Based on
the data available Rarely necessary to collect more than two
bloodculture sets per 24 hours. It is not appropriate to collect
only a single bloodspecimen for culture. A single blood culture
will not have sufficient volume foroptimal detection of bacteremias
and fungemias, and the significance of a positive result with CONS
andDiptheroids 34. Recommendations for the timing of blood
culturesCondition or syndrome RecommendationSuspected acute primary
bacterimia or Obtain two or three blood cultures, one rightfungemia
Meningitis,after the other, from different
sitesOsteomyelitis,arthritis, or pneumoniaFever of uncertain origin
(ex occult abscess. Obtain two or three blood cultures. One
rightTyphoid fever, brucellosis or other after the other, from
different sites initiallyundiagnosed febrile syndromeIf they are
negative after 24 to 48 hrsincubation. Obtain two or more cultures,
oneright after other, from different sitesSuspected bactermia or
fungema or withConsider alternative blood culture
methodspersistantly negative blood culturesdesigned to entrance
recovery ofmycobacteria, fungi, and rare fastidious microorganisms
35. Common patterns of culture positivity Type of
bacterimiaExamplewhen one blood culture waspositive
Continuousinfective endocarditis subsequent cultures positive in95%
to 100% casesBacterimia or fungaemiaOther causessubsequent cultures
positivenot due to endocarditis 75% to 80% casesBlood culture
positivityChances for subsequent culture due to contaminantsto 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
bloodculture soon before chills/rigors or fever spike 37. Timing of
blood culturesFever BacterimiaFever spike Chills / RigorBacteremia
symptomsappear after I hr of lagperiod 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 drawblood cultures when fever is detected As a
general rule, it is reasonable to obtain two blood culture
setssimultaneously, especially if antibiotic therapy is going to be
initiated; in less urgent situations - blood cultures may be spaced
at intervals. Li et alfound no significant difference in detection
of BSI from blood culturesobtained simultaneously - versus at
intervals during a 24-hour period. timing of blood cultures should
be a clinical decision - acuity of the patientsillness and whether
immediate antimicrobial therapy will be administered. 39. Variables
which govern the culture results1. Culture Medium2. Ratio of Blood
to Broth3. Inactivation of Antimicrobial Agents4. 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
toSystemsmicrobial growthcounteract the mechanismsComplement,
lysozyme, and Dilution with culture broth below Manual/phagocytic
leukocytes the critical threshhold or action automatedAntimicrobial
agents Inhibition of Dilution with broth and addition of
Automatedsusceptible agentsresins (adsorption), Activatedcharcoal
or enzymes- 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 AgentsSystemMethod available
AdvantageBACTEC, BD Biosciences, media containing resins that Media
containing Sparks, Md bind antimicrobial agents, resins/charcoal
detect more BacT/Alert, Organonadding activated charcoal to
episodes of bacteremia thanTeknika Corp., Durham,some of its medium
others media which doesnt 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 systemsThree types1. Manual (conventional)2.
Lysis-centrifugation3. 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 (WampoleLaboratories, Cranbury, N.J
Principle of lysis-centrifugation- Blood is inoculated to
culturetubes that contain a lysis solution. After lysis of the
blood cells andtube is centrifuged Is unique as the only
non-broth-based blood culture system After removing the supernatant
, pellet is inoculated directly toagar media that support the
growth of potential blood pathogensincluding 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 anincreased risk of - blood-borne pathogens
& category III and IV organisms Reduced detection of anaerobes,
Haemophilusspecies, and pneumococci due to delayed processing 50.
Automated systemsEarly systems New systemThe BACTEC radiometric
system Continuous-monitoring blood cultureReplaced 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 at10-
to 24-minute intervals,culture bottles that accept up to 10 mL of
blood per bottle. Results of individual bottle readings from the
incubator modulesare transmitted to the systems computer for data
storage andanalysis, and growth curves are calculated according
tosophisticated algorithms. Testing and data accumulation occur
around the clock. these systems have been able to detect evidence
of microbialgrowth 1 to 1.5 days sooner result in fewer instrument
false-positive signals than the earliersystems. 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.Anerobes2.Nutritionally Variant
Streptococci3.Fungi4.Bartonells sp5.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., Castaeda 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 o2. demonstration of satelliting on a blood agar plate,
disc 0.001% pyridoxal or along a staphylococcal streak, 58.
Interpretation of positive bloodculture1. Organisms considered as
true pathogens2. 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 (
