BLOOD CULTURE

A KEY INVESTIGATION
FOR DIAGNOSIS
OF BLOODSTREAM
INFECTIONS
 Introduction
“…the laboratory detection of bacteremia and fungemia remains
one of the most important functions of clinical microbiology
Our special laboratories... A positive blood culture establishes or confirms that
there is an infectious etiology of the patient’s illness. Moreover,
thanks go to it provides the etiologic agent and allows antibiotic susceptibility
testing for optimization of therapy.“ (3)
The laboratory detection of bacteremia and fungemia using blood
cultures is one of the most simple and commonly used investigations to
establish the etiology of bloodstream infections.
Rapid, accurate identification of the bacteria or fungi causing
bloodstream infections provides vital clinical information required to
diagnose and treat sepsis.
Sepsis is a complex inflammatory process that is largely under-
recognized as a major cause of morbidity and mortality worldwide.
Dr Susan M. Novak-Weekley There are an estimated 19 million cases worldwide each year,(4) meaning
Ph.D., D(ABMM) that sepsis causes 1 death every 3-4 seconds. (5)
Director of Microbiology, Early diagnosis and appropriate treatment make a critical difference
Kaiser Permanente, when it comes to improving sepsis patient outcomes. Chances of survival
SCPMG Regional Reference Laboratories go down drastically the longer initiation of treatment is delayed. If a
North Hollywood, CA, USA patient receives antimicrobial therapy within the first hour of diagnosis,
chances of survival are close to 80%; this is reduced by 7.6% for every
Wm. Michael Dunne, Jr. hour after. Yet, if a patient initially receives inappropriate antimicrobial
Ph.D. D(ABMM), F(AAM, CCM, IDSA, PIDJ) treatment, they are five times less likely to survive. (6)
Vice President R&D North America, bioMerieux, Inc.
Durham, NC, USA
Adjunct Professor of Pathology & Immunology, This booklet aims to:
l answer key questions commonly asked in relation to
Washington University School of Medicine,
St. Louis, MO, USA blood culture
l provide practical recommendations for routine blood culture
Adjunct Professor of Pediatrics,
Duke University School of Medicine, procedures
l offer an illustrated step-by-step guide to best blood culture
Durham, NC, USA
collection practices.
This booklet is intended to be a useful reference tool for physicians,
for their helpful advice and comprehensive review nurses, phlebotomists, laboratory personnel and all other healthcare
of this booklet. professionals involved in the blood culture process.

1
Definitions
Contents
Bacteremia: the presence of bacteria in the blood. It may be transient,
intermittent or continuous.
Blood culture: blood specimen submitted for culture of microorganisms. l What is a blood culture? 4
It enables the recovery of potential pathogens from patients suspected
of having bacteremia or fungemia. l Why are blood cultures important? 4
Blood culture series: a group of temporally related blood cultures that
are collected to determine whether a patient has bacteremia or fungemia. l When should a blood culture be performed? 5
Blood culture set: the combination of blood culture bottles (one aerobic
and one anaerobic) into which a single blood collection is inoculated.
l What volume of blood should be collected? 6

Bloodstream Infection (BSI): an infection associated with bacteremia l How many blood culture sets should be collected? 8
or fungemia.
Contaminant: a microorganism isolated from a blood culture that was l Which media to use? 10
introduced during specimen collection or processing and is not considered
responsible for BSI (i.e. the isolates were not present in the patient’s l Timing of blood cultures 11
blood when the blood was sampled for culture).
Contamination: presence of microorganisms in the bottle that entered l How to collect blood cultures 12
during sampling but were not actually circulating in the patient’s
bloodstream. l How many days of incubation are recommended? 14
Fungemia: the presence of fungi in the blood.
l Is it a contaminant or a true pathogen? 15
Sepsis: life-threatening organ dysfunction caused by a dysregulated
host response to infection. (1) l Special topic : Infective endocarditis 18
Septicemia: clinical syndrome characterized by fever, chills, malaise,
tachycardia, etc. when circulating bacteria multiply at a rate that exceeds l Processing positive blood cultures 20
removal by phagocytosis. (2)
Septic episode: an episode of sepsis or septic shock for which a blood l Interpretation of results 22
culture or blood culture series is drawn.
l Blood Culture/Sepsis Guidelines 24
Septic shock: a subset of sepsis in which underlying circulatory and
cellular metabolism abnormalities are profound enough to substantially
l References 26
increase mortality. (1)
l Recommendations for blood culture collection 30

Source: Wayne, P.A. Principles and procedures for Blood Cultures; Approved Guideline, CLSI document M47-A.
Clinical and Laboratory Standards Institute (CLSI); 2007 unless otherwise specified.

2 3
What is Providing adequate antibiotic therapy within
the first 24-48 hours leads to: (10-14)
a blood culture? • Decreased infection-related mortality (20-30%)
• Earlier recovery and shorter length of hospital stay
A blood culture is a laboratory test in which blood, taken from the patient, • Less risk of adverse effects
is inoculated into bottles containing culture media to determine whether • Reduced risk of antimicrobial resistance
infection-causing microorganisms (bacteria or fungi) are present in the • Cost reduction (length of stay, therapy, diagnostic testing)
patient’s bloodstream.
Figure 1: Fast effective antimicrobial therapy increases survival
Blood cultures are intended to: chances
• Confirm the presence of microorganisms Adapted from Kumar A, et al. Crit Care Med. 2006;34(6):1589-96. (15)

in the bloodstream
Patient survival rate (%)
• Identify the microbial etiology of 3 MAIN AIMS OF 100 Patients with effective antibiotic therapy
the bloodstream infection
BLOOD CULTURE *: 80

Total patients (%)

• Help determine the source of
• Confirm infectious etiology 60
infection (e.g. endocarditis)
• Identify the etiological agent
• Provide an organism for • Guide antimicrobial
40

susceptibility testing and optimization therapy 20

of antimicrobial therapy
0
0 hours 1 2 3 4 5 6 9 12 24 36

* Adapted from ESCMID (European Society of Clinical Time to antibiotics

Microbiology and Infectious Diseases) guidelines, 2012. (7)

Why are
blood cultures important? When should
a blood culture be performed?
Blood culture is the most widely used diagnostic tool for the detection
of bacteremia and fungemia. It is the most important way to diagnose the Blood cultures should always be requested when a bloodstream infection
etiology of bloodstream infections and sepsis and has major implications or sepsis is suspected.
for the treatment of those patients.
Clinical symptoms in a patient which may lead to
A positive blood culture either establishes or confirms that there is an
infectious etiology for the patient’s illness. (3) A positive blood culture a suspicion of a bloodstream infection are:
also provides the etiologic agent for antimicrobial susceptibility testing, • undetermined fever (≥38°C) or hypothermia (≤36°C)
enabling optimization of antibiotic therapy. (3) Sepsis is one of the most • shock, chills, rigors
significant challenges in critical care, and early diagnosis is one of the • severe local infections (meningitis, endocarditis, pneumonia,
most decisive factors in determining patient outcome. Early identification pyelonephritis, intra-abdominal suppuration…).
of pathogens in the blood can be a crucial step in assuring appropriate • abnormally raised heart rate
therapy, and beginning effective antibiotic therapy as early as possible • low or raised blood pressure
can have a significant impact on the outcome of the disease. (8, 9) • raised respiratory rate

4 5
Blood cultures should be collected: It is also generally recommended that two or three bottle sets (two
• as soon as possible after the onset of clinical symptoms; bottles per set) are used per septic episode, meaning, for adults, 40 to 60 ml
• ideally, prior to the administration of antimicrobial therapy (16). of blood collected from the patient for the 4 to 6 bottles, with 10 ml
per bottle.
If the patient is already on antimicrobial therapy, recovery of micro-
organisms may be increased by collecting the blood sample immediately For each additional milliliter of blood cultured, the yield of microorganisms
before administering the next dose and by inoculating the blood into recovered from adult blood increases in direct proportion up to 30 ml. (19)
bottles containing specialized antimicrobial neutralization media. This correlation is related to the relatively low number of CFU in a milliliter
of adult blood. (3)

* Pediatric
What volume of blood should The optimal volume of blood to be obtained from infants and children
be collected? is less well prescribed, however, available data indicate that the yield of
pathogens also increases in direct proportion to the volume of blood
The optimal recovery of bacteria and fungi from blood depends on cultured. (16, 20) The recommended volume of blood to collect should be
culturing an adequate volume of blood. The collection of a sufficient based on the weight of the patient (see Table 1), and an aerobic
quantity of blood improves the detection of pathogenic bacteria or fungi bottle should be used, unless an anaerobic infection is suspected. (21)
present in low quantities. This is essential when an endovascular infection Specially formulated blood culture bottles are commercially available
(such as endocarditis) is suspected. for use in children <2 years of age. They are specifically designed to
maintain the usual blood-to-broth ratio (1:5 to 1:10) with smaller blood
The volume of blood that is obtained for each blood culture set volumes, and have been shown to improve microbial recovery. (3)
is the most significant variable in recovering microorganisms
from patients with bloodstream infections. (17, 18)
Table 1: Blood volumes suggested for cultures from infants
and children (20)
Blood culture bottles are designed to accommodate the recommended Adapted from Kellogg et al. Frequency of low-level bacteremia in children from birth to fifteen years of
age. J Clin Microbiol. 2000; 38:2181-2185.
blood-to-broth ratio (1:5 to 1:10) with optimal blood volume. Commercial
continuously monitoring blood culture systems may use a smaller
Weight Patient’s Recommended Total %
blood-to-broth ratio (< 1:5) due to the addition of sodium polyanethole- of patient total blood volume of blood volume for of patient’s
sulfonate (SPS) which inactivates inhibitory substances which are present volume for culture (ml) culture total blood
in blood. (3) (ml) (ml) volume
Culture Culture
kg lb
no.1 no.2
* Adults ≤1 ≤2.2 50-99 2 2 4

For an adult, the recommended volume of blood to be obtained per 1.1-2 2.2-4.4 100-200 2 2 4 4
culture is 20 to 30 ml. (3, 16)
2.1-12.7 4.5-27 >200 4 2 6 3
Since each set includes an aerobic and an anaerobic bottle, each
bottle should be inoculated with approximately 10 ml of blood. This 12.8-36.3 28-80 >800 10 10 20 2.5
volume is recommended to optimize pathogen recovery when the bacterial/ >36.3 >80 >2,200 20-30 20-30 40-60 1.8-2.7
fungal burden is less than 1 Colony Forming Unit (CFU) per ml of blood,
which is a common finding.

6 7
How many blood culture sets
should be collected?
Since bacteria and fungi may not be constantly present in the bloodstream, A contaminant will usually be present in only one bottle of a set of blood
the sensitivity of a single blood culture set is limited. culture bottles, in contrast to a true bloodstream infection, in which
Using continuous-monitoring blood culture systems, a study investigated multiple blood culture bottles/sets will be positive.
the cumulative sensitivity of blood cultures obtained sequentially over
a 24-hour time period. It was observed that the cumulative yield of Therefore, guidelines recommend to collect 2, or preferably 3,
pathogens from three blood culture sets (2 bottles per set), with a blood blood culture sets for each septic episode. (3, 7, 16)
volume of 20 ml in each set (10 ml per bottle), was 73.1% with the first
set, 89.7% with the first two sets and 98.3% with the first three sets. If 2 to 3 sets are taken and cultures are still negative after 24-48 hours
However, to achieve a detection rate of >99% of bloodstream infections, incubation, and the patient is still potentially septic, 2 to 3 additional
as many as four blood culture sets may be needed. (22) cultures may be collected, as indicated in the following diagram. (16)

Figure 2: Cumulative sensitivity of blood culture sets (22) Figure 3: Recommended number of blood culture sets
Adapted from Lee et al. Detection of Bloodstream Infections in Adults: How Many Blood Cultures Are Adapted from Baron, E.J., et al. Cumitech 1C, Blood Cultures IV. Coordinating ed., E.J. Baron. ASM
Needed? J Clin Microbiol. 2007; 45:3546-3548 Press, Washington, D.C. 2005

Detection sensitivity
100%
98.3% Collect 2 to 3 sets If culture is negative
of bottles after 24-48 h incubation
89.7% (aerobic + anaerobic) for and patient is still
90%
each septic episode potentially septic without
an identified source
80%
73.1%
70%

20 ml 40 ml 60 ml Collect 2 to 3
If culture is negative
additional sets of bottles
after 24 h incubation
(aerobic + anaerobic)

A single blood culture bottle or set should never be drawn from

adult patients, since this practice will result in an inadequate volume
of blood cultured and a substantial number of bacteremias
may be missed. (3, 22) Repeat protocol Prolong Investigate
if necessary incubation non-microbial
etiology

8 9
Which media to use?
Microorganisms causing bloodstream infections are highly varied Which bottle should be inoculated first?
(aerobes, anaerobes, fungi, fastidious microorganisms…) and, in addition
If using a winged blood collection set, then the aerobic bottle
to nutrient elements, may require specific growth factors and/or a special
should be filled first to prevent transfer of air in the device into the
atmosphere.
anaerobic bottle.
In cases where the patient is receiving antimicrobial therapy, specialized
If using a needle and syringe, inoculate the anaerobic bottle first to
media with antibiotic neutralization capabilities should be used. Antibiotic
avoid entry of air.
neutralization media have been shown to increase recovery and provide
faster time to detection versus standard media. (23-26) If the amount of blood drawn is less than the recommended volume*,
then approximately 10 ml of blood should be inoculated into the aerobic
bottle first, since most cases of bacteremia are caused by aerobic and
facultative bacteria. In addition, pathogenic yeasts and strict aerobes
l It is recommended that each adult routine blood culture set
(e.g. Pseudomonas) are recovered almost exclusively from aerobic bottles.
include paired aerobic and anaerobic blood culture bottles.
Any remaining blood should then be inoculated into the anaerobic
l T he blood drawn should be divided equally between the bottle. (8)
aerobic and anaerobic bottles. * For recommended volumes, see page 6 “What volume of blood should be collected?

l If an anaerobic bottle is not used, it should always be replaced

by an additional aerobic bottle to ensure that a sufficient
volume of blood is cultured. (27)
Timing of blood cultures
Studies have shown that the time interval between collecting two blood
A blood culture medium must be: culture samples is not considered to be a critical factor as the diagnostic
l sensitive enough to recover: yield remains the same. (7)
• a broad range of clinically relevant microorganisms, even the most Guidelines recommend that the first two/three sets (2 bottles/set)
fastidious (Neisseria, Haemophilus…) of blood culture be obtained either over a brief time period
• m icroorganisms releasing small amounts of CO 2 (Brucella, (e.g. within 1 hour) or as a single sample taken at one time. (3, 7, 16)
Acinetobacter…) The possible impact that the blood culture collection method used
l versatile: able to provide a result for all types of sample collection (e.g. single or multiple venipunctures, winged collection set or needle
(adults, infants, patients receiving antibiotic therapy, sterile body fluids…) and syringe) may have on contamination rates should be considered. (7)
Drawing blood at spaced intervals, such as 1 to 2 hours apart, is only
recommended to monitor continuous bacteremia/fungemia in patients
with suspected infective endocarditis or other endovascular (i.e. catheter-
related) infections. (16)

10 11
 10 Key Steps to Good Sample Collection:
For an illustrated step-by-step, see page 30.
Two to three additional blood culture sets can be performed if the 1 Prior to use, examine the bottles for evidence of damage,
first 2-3 blood cultures are negative after 24-48 hours incubation in cases deterioration or contamination. Do not use a bottle containing
of severe infection or in order to increase detection sensitivity (in cases of media which exhibits turbidity or excess gas pressure, as these
pyelonephritis for example). This also depends on the microorganisms are signs of possible contamination.
involved: while sensitivity is relatively good for organisms like Escherichia 2 Check the expiry date printed on each bottle. Discard bottles
coli or Staphylococcus aureus, it is lower for Pseudomonas aeruginosa, that have expired.
streptococci or fungi. (28) 3 Strictly follow the collection protocol in use in the healthcare setting,
including standard precautions for handling blood at the bedside.
4 Blood culture bottles should be clearly and correctly labelled,
including patient identification, date and collection time, puncture
How to collect blood cultures 5
site (venipuncture or intravascular device).
Each blood culture set should include an aerobic and an anaerobic
Sample collection is a crucial step in the blood culture process. Standard bottle.
precautions must be taken, and strict aseptic conditions observed 6 Blood for culture should be drawn from veins, not arteries. (30)
throughout the procedure. Compliance with blood culture collection 7 It is recommended to avoid drawing blood from a venous or
recommendations can significantly improve the quality and clinical arterial catheter, since these devices are often associated with
value of blood culture investigations and reduce the incidence of higher contamination rates. (31)
sample contamination and “false-positive” readings. 8 Carefully disinfect the skin prior to collection of the sample using
an appropriate disinfectant, such as chlorhexidine in 70% isopropyl
alcohol or tincture of iodine in swab or applicator form. (3)
A properly collected sample, that is free of contaminants, is key 9 Transport the inoculated bottles and the completed blood
to providing accurate and reliable blood culture results. culture request to the clinical microbiology laboratory as quickly
as possible, preferably within 2 hours per CLSI. (3) Any delay in
testing the inoculated bottles may potentially lead to an
It is recommended that blood cultures should be collected only by increased risk of false negative results. If delays are expected, it
members of staff (medical, nursing, phlebotomist or technician) who is important to refer to the manufacturer’s Instructions for Use
have been fully trained and whose competence in blood culture collection (IFU) for guidance. As an example for guidance regarding delays,
has been assessed. (29) the ESCMID guidelines recommend that blood culture bottles
for testing in continuous monitoring systems should be stored
temporarily at room temperature, whereas bottles for manual
testing should be incubated as soon as possible. (32) Again, refer
to the manufacturer’s IFU for guidance.
The use of vacuum tube transport systems can facilitate the
rapid transmission of bottles to the microbiology laboratory.
However these systems should be used with caution if using
glass bottles. (33)
10 All blood cultures should be documented in the patient’s notes,
including date, time, collection site and indications.

12 13
How many days of incubation Is it a contaminant or a true
are recommended? pathogen?
The current recommendation, and standard incubation period, Contamination of blood cultures during the collection process can
for routine blood cultures performed by continuous-monitoring produce a significant level of false-positive results, which can have a
blood systems is five days. (34) negative impact on patient outcome.
A false positive is defined as growth of bacteria in the blood culture
However, published data suggest that three days may be adequate
bottle that were not present in the patient’s bloodstream, and were
to recover up to 95 to 98% of clinically significant microorganisms.
most likely introduced during sample collection.
A study by Bourbeau, et al. (JCM, 2005) showed the number of significant
microorganisms isolated per day for 35,500 consecutive blood cultures Contamination can come from a number of sources: the patient’s skin,
collected over 30 months, of which 2,609 were clinically significant the equipment used to take the sample, the hands of the person taking
isolates and 1,097 were contaminants. (35) the blood sample, or the environment.

Figure 4: Clinically significant isolates per day (35) Collecting a contaminant-free blood sample is critical to providing
Adapted from Bourbeau PP et al. Routine incubation of BacT/ALERT® FA and FN blood culture bottles
for more than 3 days may not be necessary. J Clin Microbiol. 2005;43:2506-2509
a blood culture result that has clinical value.
80%
69%
60%
Certain microorganisms such as coagulase-negative staphylococci,
viridans-group streptococci, Bacillus spp, Propionibacterium spp.,
40% diphtheroids, Micrococcus spp. rarely cause severe bacterial infections
26%
20% or bloodstream infections. These are common skin contaminants,
3% 1% 1% and although they are capable of causing serious infection in the appropriate
0%
Day 1 Day 2 Day 3 Day 4 Day 5 setting, their detection in a single blood culture set can reasonably be
identified as a possible contaminant without clinical significance. However,
These results demonstrate that 98% of clinically significant isolates were it is important to consider that coagulase-negative staphylococci are the
recovered within the first 3 days of incubation and 95% within 2 days of primary cause of both catheter- and prosthetic device-associated infections
incubation. and may be clinically significant in up to 20% of cases. (37)
The most difficult interpretation problem for the physician is whether
Incubation of Fastidious Microorganisms the organism recovered from a blood culture is a true pathogen causing
Another study by Cockerill, et al. (CID, 2004) demonstrated that, when using bloodstream infection, or a contaminant. If it is a contaminant, the
a continuous-monitoring blood culture system, 99.5% of non-endocarditis patient may be treated unnecessarily with antibiotics, leading to additional
bloodstream infections and 100% of endocarditis episodes were detected patient risks. Interpretation of true pathogen versus contaminant should
within 5 days of incubation. (19) This data suggests that extended incubation be based on whether the blood has been collected with a venipuncture
periods previously recommended for detection of the fastidious micro- or an intra-vascular device, and multiplicity of isolation of the same species.
organisms* that sometimes cause endocarditis, are no longer necessary This illustrates the crucial nature of having collection site information
when using continuous-monitoring blood culture systems. (16) included with the blood culture request sent to the laboratory.
* including Brucella, Capnocytophaga and Campylobacter spp., and the HACEK group (Haemophilus (except
H. influenzae) species, Aggregatibacter (previously Actinobacillus) species, Cardiobacterium hominis, Eikenella
corrodens and Kingella species) (36)

14 15
 Figure 5: Example of a laboratory-based algorithm to
determine blood culture contamination (42)
In contrast to patients with infective endocarditis or other true positive Adapted from Richter et al. Minimizing the workup of blood culture contaminants: implementation and
evaluation of a laboratory-based algorithm. J Clin Microbiol. 2002;40:2437-2444.
bloodstream infections, patients whose blood cultures grow contaminants
usually have only a single blood culture that is positive. This information
is of great practical value for physicians, and underlines the importance of
taking two to three blood culture sets from different anatomical sites. (16) Potential contaminant*
isolated from blood
culture
Contamination rates can be most effectively reduced by strict
compliance with hand hygiene rules and best practices for
blood collection, particularly during the stages of skin antisepsis,
venipuncture and sample transfer to blood culture bottles.
Additional draws +/- Evaluation by
48 hours? NO qualified personnel
However, even when the best blood collection protocols are used, it
may not be possible to reduce the contamination rate below 2%. (38) YES
The American Society for Microbiology and CLSI recommend targeting
contamination rates not exceeding 3% of the total of collected sets. (3, 16)
Probable
Positive with same contaminant;
organism? NO AST** not performed
Impact of contamination rates unless requested
YES
A contaminated blood culture can result in unnecessary antibiotic therapy,
increased length of hospitalization and higher costs.
It has been found that each false positive result can lead to:
Viridans group Evaluation by
l Increased length of stay - on average 1 day. (39) streptococci? NO qualified personnel
l 39% increase in intravenous antibiotic charges. (39)
l $5,000 to $8,720 additional charges. (40, 41) YES
l 20% increase in laboratory charges. (39)

l 3 days longer on antibiotics. (39)

Pathogen;
set up AST**

** Microorganisms such as coagulase-negative staphylococci, Streptococcus viridans, Bacillus spp,

Propionibacterium spp., diphtheroids, Micrococcus spp.
** AST: Antimicrobial Susceptibility Testing

16 17
Special topic:
Infective Endocarditis
Blood culture is essential in the diagnosis of infective endocarditis How many cultures?
(infection of the heart valves). In this elusive disease, blood cultures In order to distinguish between contamination and true bacteremia, a
may need to be taken repeatedly during febrile episodes, when bacteria total of three to five blood culture sets should be sufficient.
are shed from the heart valves into the bloodstream. For patients with
l Initially, two to three blood culture sets should be obtained from patients
infective endocarditis, positive blood cultures will be obtained in over
with suspected infective endocarditis. If the first 2-3 sets are negative
90% of cases, if optimal culture conditions are respected. (43)
after 24-48 hours, collect two to three more sets of cultures. (3)
Often patients with suspected infective endocarditis have been put on
Acute Infective Endocarditis antibiotics prior to blood collection. This is the most common reason for
“culture-negative” infective endocarditis. It is therefore important
This is a fulminant illness progressing rapidly over days to weeks, which
to use a blood culture medium that has antimicrobial neutralization
may be caused by highly virulent pathogens, such as Staphylococcus aureus.
capacity in order to sustain microbial growth in the presence of antibiotics
When suspected, the severity of this disease requires blood cultures to
(see page 10 “Which media to use?”). (48, 49)
be drawn immediately to avoid unnecessary delays in treatment.
However, “culture-negative” endocarditis may also be due to fastidious
l  ultiple blood culture sets should be drawn during a 30-minute
M
microorganisms, such as Aspergillus spp., Brucella spp., Coxiella burnetii,
period prior to administration of empiric antimicrobial therapy. (44)
Chlamydia spp. and HACEK* microorganisms.
Subacute Infective Endocarditis l Since current continuous-monitoring blood culture systems can recover

If sub-acute infection is suspected, there is usually not an urgent need all HACEK and other fastidious organisms within a 5-day period,
to initiate empiric therapy. It is more important to attempt to establish extending incubation beyond this period is no longer considered to
the microbiological diagnosis. be necessary. However, if all blood culture bottles are negative after
5 days, and infectious endocarditis is still suspected, all bottles should
l  ultiple blood culture sets should be obtained prior to initiation of
M
be subcultured to chocolate agar. (50)
antimicrobial therapy, with sets spaced 30 minutes to one hour apart.
This may help document a continuous bacteremia, and could be of
additional clinical value. (3)

Fungal Infective Endocarditis

Once a rare occurrence, the incidence of fungal endocarditis is increasing
considerably. (45) Candida species are the most common fungal pathogens
involved in infective endocarditis. (46) If optimum collection conditions
are observed, the yield for positive blood cultures in fungal endocarditis
for Candida spp. is 83 to 95 %. (47)

* HACEK = Haemophilus (except H. influenzae) species, Aggregatibacter (previously Actinobacillus) species,

Cardiobacterium hominis, Eikenella corrodens and Kingella species. (36)

18 19
Processing positive blood
cultures
Today, continuously-monitored blood culture systems provide the Recent technological advances such as MALDI-TOF (Matrix-Assisted
optimum solution for blood sample processing. Generally accepted Laser Desorption Ionization Time of Flight) provide the ability to rapidly
incubation periods can vary from 5-7 days, with 5 days being most deliver definitive organism identification. Molecular diagnostics can
popular. (27) The study discussed in Figure 4 shows that 98% of all positive identify the most common pathogens in positive blood cultures as
specimens were detected within the first 3 days (see page 14). (35) well as specific antibiotic resistance genes associated with bloodstream
infections. Rapid identification allows physicians to prescribe more
targeted and effective antimicrobial therapy earlier to positively
Patients who progress to septic shock have a 7.6% increase in
influence outcomes. (54-56)
mortality every hour while not on appropriate therapy. (15)
Additionally, antibiotic susceptibility testing techniques should be
Following an instrument-flagged positive event, the bottle is removed performed on positive blood cultures to provide the clinician with
from the system and a Gram stain and subculture is performed. a complete result. Appropriate use of antibiotics is crucial in cases
of bloodstream infections and sepsis. Accurately determining the
l If the sample is Gram stain positive, the morphology of the organism antimicrobial resistance profile of the causative pathogen in order to
should be reported immediately to the physician. Subcultures or select the most effective antibiotic therapy can have a significant impact
rapid techniques (e.g. molecular diagnostics) should be initiated on patient outcomes.
immediately in order to provide further organism identification and
antibiotic susceptibility testing should be performed as soon as possible. When processed correctly, blood cultures provide clinically
l If a sample is Gram stain negative, no report is made to the clinician
relevant information that can help improve patient outcomes,
unless there is growth on subculture. decrease length of hospital stay and reduce use of antibiotics.

A positive blood culture is a critical result and must be reported as soon

as available, due to the immediate impact on patient care decisions.
When reports are delivered rapidly, studies have shown broadly
improved outcomes and efficiencies in patient management. (51, 52)
A study by Barenfanger, et al. (Am J Clin Pathol, 2008) validated that
Gram stains of positive blood cultures are a very important factor
influencing appropriate therapy and patient outcomes. The study
documented a statistically significant increase in the mortality rate for
patients who had blood cultures processed after a delay (i.e. Gram stain
performed ≥1 hour after being detected as positive; P = 0.0389). The
timely removal and reporting of Gram stain results have a positive
impact on patient care and this study supports the need for 24/7 coverage
of blood culture instruments. (53)

20 21
Interpretation of results
The microbiology laboratory can provide useful information to clinicians Laboratories should consult with their medical director to create an
to help them determine whether a blood culture sample is a true positive algorithm which helps determine whether or not an isolated organism
or a false positive (contaminant). For example, the identity of the micro- is a contaminant vs. an infective agent.
organism isolated can help determine if the culture is contaminated, and
Models, such as the algorithm below, can give guidance only on the
the number of cultures positive with the same organism can help pre-
interpretation of blood culture results. (42, 57, 58) These guidelines
dict true infections. (57) Time to positivity is also a factor used to deter-
should be used in conjunction with clinical guidelines, e.g. patient’s full
mine potential contamination as contaminants usually have a delayed
blood count, presence of catheters, radiological findings, etc.
(longer) time-to-detection due to a lower overall bio-load.

Figure 6: Example of interpretation algorithm for blood

culture results
1 2
More than one positive bottle Only one positive bottle

monomicrobial polymicrobial culture if pathogenic organism: if normal skin flora: if viridans streptococci
culture (from the appropriate Listeria, S. aureus, Propionibacterium, or coagulase-negative
+ clinical setting Brucella, Haemophilus, corynebacterium, staphylococci and
clinical symptoms (e.g. transplants, Enterobacteriaceae, … Bacillus, consistent with clinical
(e.g. endocarditis, intraabdominal infection, coagulase-negative setting (e.g. indwelling
meningitis, pneumonia…) immunocompromized staphylococci catheter, prosthetic
patient…) heart valve, immuno-
compromized patient)

probable probable
probable probable
bloodstream infection bloodstream bloodstream
bloodstream infection contamination
infection infection

Negative blood cultures

Repeat blood samples
but clinical symptoms

Consider non-infectious etiology

Investigate viral etiology or

non-culturable microorganism

22 23
Blood Culture/Sepsis COUNTRY
REGION
GUIDELINES

Guidelines France l R EMIC 2015. Automatisation des cultures microbiennes :

quel cahier des charges ? Chapitre 11
http://www.sfm-microbiologie.org/

International Guidelines Germany l R einhart K et al., Prevention, diagnosis, therapy and

follow-up care of sepsis: 1st revision of S-2k guidelines of
WHO guidelines on drawing blood: best practices in Phlebotomy. the German Sepsis Society (Deutsche Sepsis-
World Health Organization 2010. Gesellschaft e.V. (DSG)) and the German Interdisciplinary
http://whqlibdoc.who.int/publications/ 2010/9789241599221_eng.pdf Association of Intensive Care and Emergency Medicine
(DIVI). German Medical Science, 2010, Vol. 8: 1-86
Surviving sepsis campaign: international guidelines for management http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2899863/pdf/
of severe sepsis and septic shock: 2012. GMS-08-14.pdf
Dellinger RP., et al. Crit Care Med. 2013;41:580-637.
South l  uideline for the optimal use of blood cultures. SAMJ
G
http://www.survivingsepsis.org/guidelines/Pages/default.aspx
Africa 2010; Vol. 100, No. 12: 839-843 SAMJ
The Third International Consensus Definitions for Sepsis and http://www.fidssa.co.za/Guidelines/Guideline_for_the_optimal_
use_of_blood_cultures.pdf
Septic Shock (Sepsis-3).
Singer M., et al. JAMA. 2016;315(8):801-810. UK l K Standards for Microbiology Investigations.
U
http://jama.jamanetwork.com/article.aspx?articleid=2492881 Investigation of Blood Cultures (for Organisms other
than Mycobacterium species). Bacteriology | B 37 |
Issue no: 8 | Issue date: 04.11.14 | Page: 1 of 51. Issued
by the Standards Unit, Health Protection Agency, PHE.
National Guidelines https://www.gov.uk/government/uploads/system/uploads/
attachment_data/file/372070/B_37i8.pdf
COUNTRY l T aking blood cultures - a summary of best practice:
GUIDELINES Saving lives reducing infection, delivering clean and
REGION
safe care. London: Department of Health; 2007.
Australia l A ustralia Clinical Excellence Commission Sepsis Kills http://webarchive.nationalarchives.gov.uk/20120118164404/
Program: Adult Blood Culture Sampling Guide v2 2012 hcai.dh.gov.uk/files/2011/03/Document_Blood_culture_
SHPN (CEC) 120077 FINAL_100826.pdf
http://www.cec.health.nsw.gov.au/__data/assets/pdf_
file/0005/259412/adult-blood-culture-sampling-guideline.pdf USA l A merican Society for Microbiology: Cumitech 1C, 2005
(EJ Baron et al.) ASM Press
Brazil l Elmor de Araujo MR, Hemocultura: recomendações l Clinical and Laboratory Standards Institute (CLSI®),
de coleta, processamento e interpretação dos resultados, document M47-A, Vol 27, 2007 (ML Wilson et al.)
J Infect Control 2012; 1: 08-19 l Emergency Nurses Association (ENA). Clinical Practice
http://www.iqg.com.br/pbsp/img_up/01355393320.pdf Guideline: Prevention of Blood Culture Contamination
https://www.ena.org/practice-research/research/CPG/Documents/
Europe l European Society for Clinical Microbiology and Infectious BCCCPG.pdf
Diseases, European Manual for Clinical Microbiology, l E. Septimus. CDC Clinician Guide for Collecting Cultures. 2015
1st Edition, 2012. http://www.cdc.gov/getsmart/healthcare/implementation/ clinician
https://www.escmid.org/escmid_library/manual_of_microbiology/ guide.html

24 25
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1999;115(2):462-74 BacT/ALERT FA Plus and FN Plus Blood Culture Media with BacT/ALERT FA and
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28 29
 Recommendations 3. Prepare venipuncture site 6. Other blood tests
for blood culture collection If skin is visibly soiled, clean with soap and water. Apply a disposable If blood is being collected for other tests, an insert placed into the adapter
tourniquet and palpate for a vein. Apply clean examination gloves cap may be required. The insert is used to guide blood collection tubes
A SUMMARY OF GOOD PRACTICE (sterile gloves are not necessary). onto the needle.
A) Using winged blood collection set Cleanse the skin using an appropriate disinfectant, such as chlorhexidine
in 70% isopropyl alcohol or tincture of iodine in swab or applicator
If other blood tests are requested, always collect the blood culture first.
(preferred method of collection)59, 60, 61
form. The venipuncture site is not fully clean until the disinfectant
has fully evaporated.
1. Prepare blood collection kit
Confirm the patient’s identity and gather all required materials before
beginning the collection process.
Do not use blood culture bottles beyond their expiration date, or 7. Finish the procedure
bottles which show signs of damage, deterioration or contamination. Discard the winged collection set into a sharps container and cover the
It is recommended to mark the blood culture bottle about 10 ml above puncture site with an appropriate dressing. Remove gloves and wash
the media level to indicate the correct filling level. 4. Venipuncture
hands before recording the procedure, including indication for culture,
Attach a winged blood collection set to a collection adapter cap. time, site of venipuncture, and any complications.
To prevent contaminating the puncture site, do not re-palpate the Ensure additional labels are placed in the space provided on the
prepared vein before inserting the needle. Insert the needle into the bottle label and do not cover the bottle barcodes, and that the tear-off
prepared vein. barcode labels are not removed. If additional labels contain a barcode,
they should be positioned in the same manner as the bottle barcode.
Inoculated bottles should be transported to the laboratory for testing as
quickly as possible, preferably within 2 hours per CLSI.(3) If delays are
expected, it is important to refer to the manufacturer’s Instructions for
Use for guidance.
5. Culture bottle inoculation
2. Prepare bottles for inoculation
Place the adapter cap over the aerobic bottle and press straight down
Wash hands with soap and water then dry, or apply an alcohol hand rub to pierce the septum. Hold the bottle upright, below the level of the draw
or another recognized effective hand rub solution. site, and use the graduation lines to accurately gauge sample volume *.
Remove the plastic “flip-cap” from the blood culture bottles and disinfect Add 10 ml of blood per adult bottle and up to 4 ml per pediatric bottle.
the septum using an appropriate and recognized effective disinfectant, such Once the aerobic bottle has been inoculated, remove the adapter cap and
as chlorhexidine in 70% isopropyl alcohol, 70% isopropyl alcohol, or repeat the procedure for the anaerobic bottle.
tincture of iodine in swab or applicator form. Use a fresh swab/applicator The use of blood collection sets without blood collection adapters is
for each bottle. Allow bottle tops to dry in order to fully disinfect. not recommended. * Avoid holding the blood culture bottle in a horizontal or upside down position or drawing blood
with a needle connected directly to the adaptor cap, as fill level cannot be monitored during
collection and there is a possible risk of media reflux into the bloodstream.
These recommendations illustrate the best practices for blood culture collection based on
the World Health Organization recommendations (WHO guidelines on drawing blood: best
practices in phlebotomy. 2010. ISBN 978 92 4 159922 1). Best practices may vary between
healthcare facilities; refer to guidelines applicable in your facility.

30
 Recommendations 2. Prepare bottles for inoculation 5. Culture bottle inoculation
for blood culture collection Wash hands with soap and water then dry, or apply an alcohol hand rub Collect the sample. Transfer the blood into the culture bottles, starting
or another recognized effective hand rub solution. with the anaerobic bottle. Hold the bottle upright and use the graduation
A SUMMARY OF GOOD PRACTICE Remove the plastic “flip-cap” from the blood culture bottles and disinfect lines to accurately gauge sample volume. Add 10 ml of blood per adult
B) Using needle and syringe the septum using an appropriate and recognized effective disinfectant,
such as chlorhexidine in 70% isopropyl alcohol, 70% isopropyl alcohol,
bottle and up to 4 ml per pediatric bottle. Once the anaerobic bottle has
been inoculated, repeat the procedure for the aerobic bottle.
or tincture of iodine in swab or applicator form. Use a fresh swab/
applicator for each bottle. Allow bottle tops to dry in order to fully
disinfect.
Conventional needles and syringes should be replaced
wherever possible with winged blood collection sets, which
are safer.(59, 60, 61) 6. Finish the procedure
They should only be used if prevention measures to Accidental Discard the needle and syringe into a sharps container and cover the
Blood Exposure are strictly applied*. Needles must not be puncture site with an appropriate dressing. Remove gloves and wash
3. Prepare venipuncture site
recapped, purposely bent or broken by hand, removed from hands before recording the procedure, including indication for culture,
disposable syringes or otherwise manipulated by hand. If skin is visibly soiled, clean with soap and water. Apply a disposable time, site of venipuncture, and any complications.
tourniquet and palpate for a vein. Apply clean examination gloves Ensure additional labels are placed in the space provided on the
(sterile gloves are not necessary). bottle label and do not cover the bottle barcodes, and that the tear-off
Cleanse the skin using an appropriate disinfectant, such as chlorhexidine barcode labels are not removed. If additional labels contain a barcode,
in 70% isopropyl alcohol or tincture of iodine in swab or applicator they should be positioned in the same manner as the bottle barcode.
1. Prepare blood collection kit form. The venipuncture site is not fully clean until the disinfectant Inoculated bottles should be transported to the laboratory for testing as
has fully evaporated. quickly as possible, preferably within 2 hours per CLSI. (3) If delays are
Confirm the patient’s identity and gather all required materials before expected, it is important to refer to the manufacturer’s Instructions for
beginning the collection process. Use for guidance.
Do not use blood culture bottles beyond their expiration date, or
bottles which show signs of damage, deterioration or contamination.
It is recommended to mark the blood culture bottle about 10 ml above
the media level to indicate the correct filling level.
4. Venipuncture
Attach the needle to a syringe.
To prevent contaminating the puncture site, do not re-palpate the
prepared vein before inserting the needle. Insert the needle into the
prepared vein. * Refer to recognized guidelines such as those issued by the WHO or CDC:
http://www.who.int/injection_safety/phleb_final_screen_ready.pdf
http://www.cdc.gov/niosh/docs/2000-108/pdfs/2000-108.pdf
These recommendations illustrate the best practices for blood culture collection based on
the World Health Organization recommendations (WHO guidelines on drawing blood: best
practices in phlebotomy. 2010. ISBN 978 92 4 159922 1). Best practices may vary between
healthcare facilities; refer to guidelines applicable in your facility.
05-16 / 9304764 010/GB/C / This document is not legally binding. bioMérieux reserves the right to modify specifications without notice / BIOMERIEUX, the blue logo, BacT/ALERT are used, pending and /or registered trademarks belonging to bioMérieux S.A. or one of its subsidiaries, or one of its companies /
CLSI is a trademark belonging to Clinical Laboratory and Standards Institute, Inc. / Any other trademark is the property of its respective owners / Photos: bioMérieux, Callimedia, Fotolia, GettyImages / bioMérieux S.A. 673 620 399 RCS Lyon / Printed in France / théra RCS Lyon B 398 160 242

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