Laboratory diagnosis of Blood Stream Infections (BSIs)

Laboratory Diagnosis of Blood Stream
Infections
Dr. Suprakash Das
Assist. Prof.

Introduction True/Pseudo?
 The presence of viable bacteria in the blood, as determined by their growth
in a blood culture, is known as bacteremia.
 It is a laboratory finding that may or may not indicate infection.
 Blood cultures may become positive because of contamination during
phlebotomy, leading to false-positive results, a phenomenon termed
pseudobacteremia.
 Such contamination is most often caused by skin commensals, such as
coagulase-negative staphylococci (CoNS).
 Pseudobacteremia is not an indication of infection and does not require
therapy.
 Although bacteremia may be a transient, self limiting phenomenon without
clinical consequences, it frequently reflects the presence of serious
infection.

Introduction True/Pseudo?
 Growth of CoNS or other skin microbiota from a blood culture does not
always represent pseudobacteremia however; it may indicate true
bacteremia, depending on the clinical situation
 Life-threatening infections caused by bacteremia are a concern in patients
who are immunocompromised through drug or chemotherapeutic
intervention or as the result of pre-existing disease and subsequent
immunosuppression.
Bacteremia is often associated with
 Hospitalization,
 Insertion of foreign bodies, such as catheters, into blood vessels,
 Other types of procedures.

Introduction  True/Pseudo?
 Even when growth of an organism from a blood culture reflects a true-
positive result, bacteremia may not be associated with any physical signs
or symptoms of severe infection, a condition known as Occult
(unsuspected) bacteremia.
 Occult bacteremia frequently occurs in children less than 2 years of age
and is most often caused by Streptococcus pneumoniae.
 Because of the lack of clinical evidence for serious infection in such
patients, the diagnosis of bacteremia may be overlooked, with potentially
catastrophic consequences if treatment is delayed.
 Usually, however, bacteremia that reflects true infection results in systemic
physiologic responses that indicate the presence of a serious infection.

Introduction Newer Definitions
 In the past, the term Septicemia was used to indicate bacteremia plus a clinical
presentation of physical signs and symptoms of bacterial invasion and toxin
production.
 The term septicemia is still used clinically and in the collection of epidemiologic
data on causes of death.
 Because of its imprecision in defining a disease state, however, it is not suitable
for categorizing all patients who have bacteremia-related infections or for
designing clinical trials.
 To facilitate the study of the pathogenesis and treatment of the consequences of
severe infections, including those associated with bacteremia,
 An international consensus conference of the American College of Chest
Physicians and the Society of Critical Care Medicine devised standardized
definitions of the response to infection.

Classification of Bacteremia
Site of Origin
 Primary bacteremia occurs when the bacteria are present in an endovascular source such
as an infected cardiac valve or an infected intravenous (IV) catheter, whereas
 Secondary bacteremia occurs when the bacteria come from an infected extravascular
source, such as lungs in patients with pneumonia.
 A case in which the source of bacteremia remains undefined is termed bacteremia of
unknown origin.
 Classification in this manner has important clinical consequences because it determines
the appropriate therapy and prognosis.
 For example, a secondary bacteremia from an infected focus, such as an abscess, may
require surgical therapy to remove the abscess or source of infection, in addition to
antimicrobials to eliminate the infection.
 Bacteremia of unknown origin generally has a poorer prognosis than primary or
secondary bacteremia.

Causative Agent
 Categorized by the general class of microorganism or specific pathogen that has
invaded the bloodstream.
Gram-positive bacteremia is caused by such organisms as
S. pneumoniae,
Staphylococcus aureus, or
Enterococcus faecium, whereas
Gram-negative bacteremia is caused by such organisms as
Escherichia coli or
Pseudomonas aeruginosa.
Anaerobic bacteremia is caused by such organisms as Bacteroides fragilis, whereas
Polymicrobial bacteremia is caused by a mixture of organisms.

 General classification of bacteremia in this fashion can provide initial clues to the underlying
source of a bacteremia and guide therapy, even before organisms have been identified.
 For example, CoNS bacteremia in a hospitalized patient is frequently caused by infection of an
indwelling vascular device, whereas
 Polymicrobial bacteremia with a mixture of enterococci and gram-negative organisms is
frequently caused by invasion of the bloodstream by gastrointestinal (GI) microbiota from
bowel perforation.
Place of Acquisition
Community-acquired bacteremia, as the term suggests, occurs in individuals living in the
general community, whereas
Nosocomial bacteremia (“nosocomial” meaning hospital acquired) occurs in patients who are
hospitalized or living in a nursing home or other health care facility.
 Nosocomial bacteremia is conventionally defined as any bacteremia occurring more than 72
hours after hospital admission.

 Certain bacteremias are more often community acquired. For example,
more than 90% of cases of S. pneumoniae bacteremia are acquired in the
community.
 Others, such as those caused by P. aeruginosa or Enterococcus spp., are
more likely to be nosocomial.
 The place of acquisition may thus be extremely significant in guiding initial
therapy.
 For example, nosocomial bacteremia is more likely to be caused by drug
resistant organisms that express β-lactamases or other resistance factors that
inactivate first-line antimicrobial agents,
 Although this distinction is currently blurred in the case of hospital-acquired
and community-acquired methicillin-resistant Staphylococcus aureus
(MRSA).

Duration
 Bacteremia may also be classified by the duration of a bacteremic episode.
 Bacteremic episodes may be transient, intermittent, or continuous.
 The frequency, time, and number of blood cultures to be collected may depend on
the type of bacteremic episode that the patient is experiencing.
Transient bacteremia usually occurs after a procedural manipulation of a
specific body site colonized by indigenous microbiota, causing the organisms to
enter blood.
 Such sites include the mouth and the GI and urogenital tracts.
 Transient bacteremia may appear for a brief period following a dental,
colonoscopic, or cystoscopic procedure.
 The organisms involved are normally rapidly cleared by the host immune defense,
so their presence is rarely symptomatic.

Intermittent bacteremia can occur because of the presence of abscesses
somewhere in the body or as a clinical manifestation of certain types of infections,
such as meningococcemia, gonococcemia, or pneumonia.
 In intermittent bacteremia, organisms are periodically released from the primary
site of infection into blood.
Continuous bacteremia occurs when the organisms are coming from an
intravascular source and are consistently present in the bloodstream.
 Infective endocarditis is the most common clinical manifestation associated
with continuous bacteremia, although other endovascular sources, such as
infected intravascular catheters or septic thrombi, can also result in continuous
bacteremia.
 Microbial biofilms on foreign body implants and in tissue can contribute to
continuous bacteremia by the periodic release of planktonic microorganisms.

Gram Negative Bacteremia
In a typical ICU set up

Microorganism profile of short-term
peripheral venous
catheter-associated bloodstream infections

Risk Factors for Bacteremia
Decreased Immune Competency of Selected Patient Populations
 Bacteremias are more frequent among persons with neoplasia
especially those with Hematologic Malignancies,
those receiving Immunosuppressive Chemotherapy, and
those undergoing Bone Marrow Transplantations.
 Persons with other chronic underlying diseases (e.g., Diabetes, Cirrhosis) and
 Those receiving immunosuppressive therapy for
 Rheumatoid arthritis,
Stem cell or solid organ transplant recipients
 Infection with human immunodeficiency virus (HIV) predisposes patients to
increased risk of bacteremias because of the immunosuppression caused by the
virus.

Increased Use of Invasive Procedures
 The increased use of
 indwelling devices,
respirators, and
invasive diagnostic procedures may be a factor in the occurrence of bacteremia.
Semi-permanent vascular catheters ( for chemotherapy or, hemodialysis) increases the risk of
bacteremia.
Indwelling urethral catheters,
suprapubic catheters, and
IV pyelography also predispose patients.
 Surgery involving the
Urinary,
GI, and biliary tracts may also result in bacteremia

Age of the Patient
 Bacteremias are more prevalent in people at the extremes of age
 Infants,
 Young children, and
 Adults older than 55 years being most susceptible.
 The presence of comorbid conditions, such as
 Diabetes,
 Hypertension,
 Chronic obstructive pulmonary disease, and
 Congestive heart failure in older adults, and
 Neoplastic disorders,
 HIV infection, and
 Low and very low birth weights in neonates and infants

Antimicrobial Resistance
 The indiscriminate administration of broad-spectrum antimicrobials reduces susceptible
normal microbiota and favors colonization and invasion by resistant bacteria.
 Data from the SENTRY Antimicrobial Surveillance Program revealed that, on average,
58% of S. aureus isolates in the United States were resistant to methicillin (oxacillin),
30% of Enterococcus isolates were resistant to vancomycin,
19.5% of Klebsiella spp. were extended-spectrum β-lactamase (ESBL) producers, and
19% of E. coli isolates were resistant to ciprofloxacin. These percentages vary widely
from region to region.
 An increasing population of antimicrobial-resistant organisms results in bacteremias that
are harder to treat, leading to increased morbidity and mortality.

Pathogenesis
 The pathogenesis of bacteremia depends
Infecting pathogen,
Portal of initial entry, and
Immune status of the patient.
 Bacteremia occurs because of disruption of normal skin or mucosal
barriers bacterial invasion of the bloodstream. Such disruption may occur
because of 
Trauma,
Burns, or
Ischemia giving rise to breaks in the skin that allow access to the
microvasculature;

Pathogenesis
 Viral infection disrupts the epithelial lining (e.g., influenza virus infection involving the upper
respiratory tract) resident biota to invade the bloodstream via capillaries.
 Iatrogenic disruptions  surgery, instrumentation, or placement of an indwelling device.
 Focal bacterial infection (e.g., bacterial pneumonia)tissue destruction disrupts nearby
vascular structures and allows bloodstream invasion.
 Once bacteremia occurs, the patient’s immune system attempts to control infection via
antibodies opsonize organisms activate complement-mediated killing and by phagocytosis.
 In addition, filtering mechanisms in the lymphatics and large vascular beds in the liver and spleen
may sequester organisms and facilitate their destruction by phagocytic cells.
 If, however, these defences are unsuccessful
 Two major complications may ensue METASTATIC INFECTION
SEPTIC SHOCK

Pathogenesis
 Invasion of the bloodstream may result in spread of organisms throughout the body, causing
seeding of multiple sites and leading to widely disseminated infection.
 Bacteremia caused by S. pneumoniae  Pneumococcal meningitis,
 Other infections associated with a period of bacteremia as part of the disease process include
Salmonellosis,
Infective endocarditis, and
Acute hematogenous osteomyelitis.
 S. aureus bacteremia is particularly likely to cause metastatic infection or abscess formation
Endocarditis,
Osteomyelitis,
Septic arthritis,
Hepatic abscess, or pyomyositis.
 Sepsis and septic shock are also potential consequences of bacteremia.

Pathogenesis
 Although gram-negative bacteremias were once thought to be more likely to cause septic
shock than bacteremias caused by gram-positive organisms, the risk of sepsis, severe
sepsis, septic shock, and death is now known to be similar between these two classes of
bacteremia.
In both cases, a bacterial membrane component
 Lipopolysaccharide [LPS], also known as endotoxin, in gram-negative organisms;
 Lipoteichoic acid and peptidoglycan in gram-positive organisms) interacts with
macrophages and causes the release of
Tumor necrosis factor,
Interleukin (IL)-1,
IL-6, and other proinflammatory cytokines
 Increasing endothelial activation,
 Vascular permeability, blood flow, and recruitment of neutrophils.

Pathogenesis
 These responses are directed at controlling infection and are normally
counter regulated by anti-inflammatory mediators to prevent a destructive
systemic inflammatory reaction.
 In sepsis and septic shock, however, an imbalance in regulation leads to an
unopposed proinflammatory state, leading to microvascular abnormalities
and endothelial injury; in turn,
These derangements lead to
Decreased tissue perfusion,
Complement activation, and
Disseminated intravascular coagulation (DIC) multiorgan dysfunction
septic shock and death.

The complex interactions
between the invading
microorganism and the
host defense mechanism
ending in bacteremia.
These interactions present
some unique features in
pathogenesis and they
are under the influence of
the genetic make-up of the
host.
SNPs- Single Nucleotide
Polymorphism

Pathogenesis
Central phenomena in sepsis pathogenesis.
Pattern recognition receptors (PRRs)
 Toll-like receptors,
Nucleotideoligomerization domain leucine-rich repeat proteins) initiate
intracellular signaling after binding to
Pathogen-associated molecular patterns (PAMPs) and
Danger-associated molecular patterns (DAMPs)(i.e. hyaluran, high mobility
group box-1 (HMGB-1), heat shock protein).
 PRRs, expressed by macrophages, neutrophils, dendritic cells activate
intracellular transduction pathways, i.e. via nuclear factor kappa (NF- kb).

Pathogenesis
Activated NF- kb moves from the cell cytoplasm to the nucleus, binds to transcription
sites induces activation of an array of genes for
Acute-phase proteins,
Proinflammatory cytokines,
Inducible nitric oxide synthase (iNOS),
Coagulation factors, and
Enzymatic activation of cellular proteases.
Once activated, the innate immune system initiates the inflammatory response by
Secreting cytokines and chemokines,
Inducing the expression of costimulatory molecules in order to recruit immune cells to the
site of infection
Trigger the adaptive immune response.

Pathogenesis
 The magnitude of clinical sepsis syndrome results from complex
interactions between the microbe and the host response to it.
 A sustained response leads to organ damage.
 Immunoparalysis is associated with the release of anti-inflmmatory
cytokines, lymphocyte apoptosis, decreased antigen presentation, T cell
function and immunosuppression.
 Although proinflammation (T helper 1-driven response with the release of
proinflammatory cytokines) has been thought to precede
 Anti-inflammation (T helper 2-driven response with the release of anti-
inflammatory cytokines), the timing of these events is not well established.

Syndromes Associated
with Bacteremia
Catheter-Related Bloodstream Infections (CRBSIs)
 Intravascular catheters have become indispensable for modern medical and
surgical therapy
Short-term use in the administration of medications and fluids.
Semi-permanent catheters administer chemotherapy and/or parenteral nutrition
Hemodialysis in patients with end-stage renal disease.
Catheters are placed in the pulmonary artery or right-sided chambers of the
heart Hemodynamic monitoring.
 Colonization and Biofilm formation CoNS, S. aureus, and Enterococcus
(Frequently MDR Strains), which are often found on the surface of the patient’s
skin bacteremia.
 Substantial progress has been made in the last 15 years, however, in reducing
these infections: CDC data revealed a 58% reduction since 2001.

with Bacteremia
 At least 50% of these infections are caused by CoNS.
 Production of a polysaccharide biofilm by CoNS is one mechanism that has been
associated with CRBSIpatients with compromised immune systems (e.g.,
patients with cancer becoming neutropenic through chemotherapy) Mortality
rates of 13% to 18%.
 Bacteremias caused by S. aureus are associated with higher mortality rates.
 Infusion-associated bacteremias Typically gram-negative organisms, such as
P. aeruginosa
Enterobacter cloacae.
 Nontuberculous mycobacteria Intravascular catheters Immunosuppressed
hosts, most notably Mycobacterium avium complex in HIV-positive individuals.

with Bacteremia
Urinary Tract Infections
 Infection of the upper urinary tract (acute pyelonephritis)bacteremia in as many
as 40% of affected patients.
 E. coli is the most common cause most common in older patients.
Pneumonias
 Pneumonia that produce a concurrent bacteremia include
S. pneumoniae,
S. aureus,
P. aeruginosa, and
Klebsiella/Enterobacter spp.
 Of patients with pneumococcal pneumonia, 20% to 25% will have positive blood
cultures; the risk of death is 20% to 30%.

with Bacteremia
Intraabdominal Infections
 Primary peritonitis, which frequently occurs in patients with cirrhosis, is associated with
bacteremia in 75% of cases involving aerobic bacteria.
 Common pathogens include
 E. coli,
 K. pneumoniae, and
 Enterococci.
 Secondary peritonitis Perforation of the GI tract intraabdominal abscesses Intermittent
bacteremia caused by E. coli, anaerobes, and enterococci.
Skin Infections
 Cellulitis
 S. aureus,
 Streptococcus pyogenes, or Streptococcus agalactiae

with Bacteremia
 Bedridden patients (bed sores) or peripheral vascular disease from diabetes Polymicrobial
bacteremia.
Proteus mirabilis,
E. coli,
S. aureus,
B. fragilis,
Pseudomonas spp.,
Clostridium spp., and
Peptostreptococcus.
 Patients with severe burns 
 P. aeruginosa
 Klebsiella pneumoniae.

with Bacteremia
Infective Endocarditis
 Organisms associated with acute, sudden-onset endocarditis include virulent
bacteria
S. aureus,
Enterococci, and
S. pneumoniae,
 Slowly progressing subacute endocarditis
Viridans streptococci;
Nutritionally variant streptococci, including Abiotrophia and
Granulicatella
CoNS (particularly in prosthetic heart valves).

with Bacteremia
Musculoskeletal Infections
 Acute osteomyelitis Transient bacteremia caused by S. aureus.
 The organisms seed end loop capillaries in bone, where blood flow is slow,
and begin to multiply Intermittent bacteremia in about 50% of cases.
 Prosthetic joints in the hip can be hematogenously seeded by such
organisms as S. aureus and CoNS bacteremia.
 Bacteremia caused by S. aureus or Neisseria gonorrhoeae can result in
seeding of joints Acute septic arthritis, an infectious disease emergency
that requires prompt drainage of pus from the joint and aggressive
antimicrobial therapy.

Signs and Symptoms
 The classic signs and symptoms of bacteremia include
Abrupt onset of shaking chills, fever, or
Hypothermia.
 About 14% of patients Hypotension
 About 40% of patients Prostration and Diaphoresis (profuse sweating).
 Tachypnea (abnormal rapid breathing) is an early sign of bacteremia
 Adult respiratory distress syndrome occurs in 18% of patients with culture-positive septic
shock.
Other symptoms may include
Delirium,
Stupor, or
Agitation (evidence of decreased central nervous system perfusion), along with
Nausea and vomiting.

Signs and Symptoms
 As many as 38% of patients  Acute renal failure Oliguria or Anuria.
 Ecthyma gangrenosum, a central necrotic area surrounded by an erythematous base, is typically associated
with Pseudomonas bacteremia.
 The failure of the body to mount an elevated temperature is associated with increased mortality in newborns
and older adults.
 Clinical conditions with altered laboratory values that may be indicative of bacteremia include the following:
 Thrombocytopenia
 Leukocytosis or leukopenia
 Lactic acidosis
 Hypoglycemia or hyperglycemia
 Abnormal liver function test results (especially hyperbilirubinemia)
 Coagulopathy
 DIC
 Elevations in C-reactive protein (CRP), haptoglobin, and fibrinogen levels

Laboratory Diagnosis
The need for Urgency
 Because of the potential of a serious negative outcome of a septicemia
or bacteremia, a blood culture is one of the most important cultures to
obtain rapid accurate results.
 As such, a positive blood culture is a critical value.
 Critical values are those laboratory test results that are outside the
reference range and indicate a potentially fatal outcome.
 The primary care provider needs to be notified immediately of all
critical values to provide immediate treatment.
 In the case of a positive blood culture, the Gram stain result should be
called for immediately.

The need for Asepctic Technique
 It is important to remember that even though antiseptic technique is used in the
collection of blood, somewhere between 1% and 3% of blood cultures become
contaminated with such organisms
CoNS,
Corynebacterium spp.,
Bacillus spp. (not B. anthracis),
α-hemolytic streptococci, and
Cutibacterium (Propionibacterium) acnes which are ordinarily skin colonizers,
resulting in pseudobacteremia.
 However, in some patients, such as those undergoing cancer chemotherapy, such
organisms can represent true pathogens,
 It is essential to distinguish between blood culture results that reflect true
bacteremia and those that represent pseudobacteremia.

 Therefore it is most important to prepare the skin properly before venipuncture for blood
culture.
 Palpation for the vein can be checked with a gloved finger.
 Cleansing the skin with 70% to 95% ethanol or isopropyl alcohol, followed by 2%
chlorhexidine (preferred) or 2% tincture of iodine, scrubbed in a concentric fashion
around the venipuncture site is recommended.
 For decontamination to be effective, the antiseptic should be left on skin for at least 30
seconds.
 After the venipuncture, the disinfecting agent should be removed with an alcohol pad.
 Because of systemic absorption and their effect on thyroid function, iodinated antiseptics
should not be used on low-birth-weight infants.
 Similarly, in infants less than 2 months of age, chlorhexidine should not be used;
instead, use alcohol swabs.

 Blood for blood culture should be obtained by venipuncture and not
from indwelling IV or intraarterial lines.
 There is more risk of isolating skin biota from indwelling lines rather than
via venipuncture.
 If blood is collected from an indwelling line, a second sample collected via
venipuncture should be cultured for comparison.
 For patients who have IV lines through which they are getting fluids
and/or medications, blood must be drawn below the line because blood
drawn above the line will be diluted with the fluid being transfused.
 The best practice, however, is not to draw blood from the extremity that
has an IV line but to perform venipuncture on an extremity that does
not have an indwelling line.

The Importance of Blood Culture Bottles
 When blood is collected for culture, it is critical that the blood not be allowed to clot.
 The formation of a clot will trap the bacteria and reduce the ability to detect them.
 Thus blood should be inoculated directly into blood culture bottles or, if necessary, into a
tube containing anticoagulants.
 Tubes containing heparin, ethylenediaminetetraacetic acid (EDTA), and sodium citrate
have been shown to inhibit the growth of many different organisms and should not be
used.
 Tubes containing 0.025% to 0.050% sodium polyanetholsulfonate (SPS) are better
tubes to use for collecting blood for culture,
 SPS can inhibit a few organisms as well, notably
Peptostreptococcus anaerobius and
some strains of Neisseria spp. and Streptobacillus moniliformis.
 Intermediate collection tubes are therefore not recommended.

Determining the Volume, Frequency, and
Number of Blood Cultures
 Bacteremia may involve a large number of microorganisms in the blood; however, more
commonly, only a relatively small number of bacteria per unit volume of blood (as low as
one bacterium per milliliter) is seen in patients with clinically significant bacteremia.
 The detection methods commonly used in most laboratories produce positive results in adult
patients with bacteremias if organisms are present in the range of 10 to 15 bacteria per
milliliter mL of blood
 It has seen that the detection of bacteria by culture depends on the optimal amount of blood
collected for culture in a blood culture bottle.
 Less or more amount of blood than the recommended optimal amount reduces the sensitivity of
culture.
 In general, higher quantities of blood gives a better result but too high a quantity actually
reduces the culture sensitivity.
 It is due to the presence of many bacterial growth inhibitors like serum, complements, WBCs etc.
in blood that will be present in high quantity if the quantity of blood is increased.

 Thus blood and the inhibitors present in blood need to be diluted by the blood
culture medium.
 The optimal ratio of blood to culture medium is about 1 : 5 to 1 : 10. The
dilution aids in negating the bactericidal effect of normal serum.
 The recommended volume of blood for adults in most cases is 10 ml per blood
culture bottle.
 In new borns, sepsis tends to be more severe because of their immature immune
defense mechanisms and generally higher numbers of microorganisms per
milliliter of blood.
 It is not uncommon, however, to see low-level bacteremia in children as seen in
adults.
 Because newborns and children have a smaller volume of total blood, it is not safe
to take as much blood from a new born or child as that which can be taken from an
adult.

 A protocol published by the American Society for Microbiology (ASM) in
2005 used the relationship between weight and blood volume to determine
how much blood can be drawn for culture in this age group, assuming that
similarly to adults, up to 4% of blood volume can be safely obtained.
In this scheme
2 mL of blood weighing less than 1 kg (<2.2 lb),
4 mL total  up to 2 kg (up to 4.4 lb),
6 mL total  up to 12.7 kg (27 lb), and
10 mL total  up to 36.3 kg (up to 80 lb).
A child who weighs more than 36.3 kg (>80 lb) can have as much blood
drawn as an adult (20 to 30 mL).

 Because less blood is typically drawn from newborns and children
compared with adults, most commercial blood culture systems have
pediatric bottles that contain less blood culture medium into which less
blood is inoculated.
 Some pediatric bottles may be supplemented with X and V factors to
enhance the recovery of H. influenzae, which is more likely (or was more
likely before implementation of the Hib vaccine) to be isolated from
children than from adults.
 The frequency of bacteremic episodes is another factor to consider when
determining the timing and frequency of blood collection for culture.
 Because bacteremias can be transient, intermittent, or continuous with
respect to the presence of microorganisms in the peripheral circulation,
collection of samples may depend on the type of bacteremia suspected.

 In patients with transient bacteremia, organisms are immediately cleared from the
peripheral system by the reticuloendothelial system.
 In such patients, clinical symptoms, especially fever, may not occur until after
bacteria have been cleared from the bloodstream.
 Because these symptoms serve as the signal to obtain blood cultures, bacteremia
may go undetected because of delays in obtaining blood cultures relative to the
time of peak concentration of circulating bacteria.
 Therefore in suspected cases of Intermittent bacteremia, it is recommended
that blood culture specimens be collected before an anticipated temperature
rise to ensure maximum recovery.
 Continuous bacteremia the organisms are constantly released into the
bloodstream and therefore are likely to be isolated whenever the blood culture
specimen is taken.

 Although a single set of blood culture bottles may yield the causative agent, two,
three, and even four sets of blood cultures are recommended.
 A set consists of one bottle for recovery of aerobic organisms and a second bottle
for recovery of anaerobic organisms.
One study revealed
 About 80% of bacteremias are discovered in the first set of blood culture
specimens taken,
 90% are detected if two sets of specimens are taken, and
 As many as 99% are detected if a third set is taken.
 Blood culture sets may be obtained simultaneously or consecutively as long as
they are from separate venipunctures.
 The exception is in suspected infective endocarditis, for which 30- to 60-
minute intervals are recommended to document continuous bacteremia.

 Best practices for timing and blood volume collection that yield the highest percentage of
positive cultures,
At least 60 mL of blood
Collected in a 24-hour period,
With 10 mL of blood inoculated into each bottle;
Three sets of two bottles each
 Another reason for inoculating multiple bottles with blood collected from multiple separate
venipunctures is to aid in the determination of whether an isolated organism is a true pathogen
or a contaminant.
 The collection of one single sample should be strongly discouraged because the volume of
blood cultured is not sufficient for detecting some infections.
 The significance of the isolation of CoNS or other skin microbiota from one culture is hard to
interpret.
 It is not until there have been repeated isolations of the same organism from multiple cultures
collected from separate venipunctures that the significance can be determined.

 Multiple blood cultures are recommended to document bacteremia, but
there is a limit to the number that should be collected.
 Repeated blood cultures or daily collections of blood for culture are not
necessary.
 If at least 40 mL of blood has been cultured, and antimicrobial therapy
has begun, it is best to wait for the results of the initial sets of cultures
before collecting more blood.
 Most organisms grow within 3 to 5 days in the continuous monitoring
systems used in most laboratories, so it is best to wait at least 3 days
before collecting additional blood for culture.
 If no organisms grow in the blood cultures, it is more reasonable to consider
other potential causes of patient symptoms, rather than culturing more
blood.

Blood Culture Methods
Culture Media Used in Conventional Broth Systems
 A blood culture set typically includes a bottle designed for recovery of aerobic microorganisms and another
bottle for recovery of anaerobic microorganisms.
 Aerobic culture bottle contains a medium that is nutritionally enriched, such as
 Soybean casein digest broth,
 Peptone broth,
 Tryptic or trypticase soy broth,
 Brain-heart infusion broth,
 Brucella broth, or
 Columbia broth base.
 Anaerobic broth contain the same types of basic media as the aerobic culture systems, 0.5% cysteine
growth of certain thiol-requiring organisms, pre-reduced to decrease the oxidation reduction potential 
growth of anaerobes.
 Newer anaerobic broth media (e.g., F/X; Becton, Dickinson, Sparks, MD) contain blood-lysing agents that
reduce the time to detection of anaerobes by lysing red blood cells (RBCs), which provides added nutrients,
and WBCs, which releases phagocytized organisms.

 Many commercially available automated blood culture systems have blood culture bottles
containing an Antimicrobial Removal Device (ARD) A resin that non-specifically absorbs
any antimicrobial agent present in the patient’s blood, whereas other systems incorporate
activated charcoal for this purpose.
 The yield of bacteria and yeasts increases with the incorporation of these inhibitors into the
culture medium.
SPS, one of the commonly used additives, performs the following functions:
Anticoagulation (effective at a 0.03% concentration)
Neutralization of the bactericidal activity (i.e., complement and lysozyme) of human serum
Prevention of phagocytosis
Inactivation of certain antimicrobial agents (e.g., streptomycin, kanamycin, gentamicin,
polymyxin B)

 Despite its usefulness in blood culture media, SPS inhibits the growth of certain organisms,
notably P. anaerobius, N. gonorrhoeae, N. meningitidis, and Gardnerella vaginalis.
 If these organisms are suspected, 1.2% gelatin added to the blood culture bottle may help
neutralize the inhibitory effect of SPS.
 In automated systems, blood culture bottles are typically incubated at 35°± 2° C for 5 days and
may be held for 7 days for manual systems.
 During incubation the aerobic bottles and, in some systems, the anaerobic bottles as well are
agitated during incubation Rocking motion, BacT/ALERT (bioMérieux, Durham, NC) and
BACTEC (Becton, Dickinson, Sparks, MD)/ Rotary motion with the formation of a vortex,
VersaTREK (TREK Diagnostic Systems, Cleveland, OH) Increases oxygenation of the
broth enhancing the detection of microorganisms.
 The head space of the Aerobic bottles ambient air + increased concentration of carbon dioxide
(CO2)/ Anaerobic bottles Nitrogen (N2) and CO2.

Blood Culture Systems
Manual Systems
Only three manual blood culture systems are currently marketed and used.
 They are easy to use, inexpensive, and generally adequate for the detection of common bacteria
and fungi.
 A broth-slide system (Septi-Chek, Becton, Dickinson, Sparks, MD) was designed from the
original biphasic (solid agar and broth combination) blood culture medium called the
Castañeda culture bottle.
 Septi-Chek consists of a slide paddle containing chocolate (CHOC), MacConkey (MAC), and
malt extract agars (selective for yeast and fungi) attached to the top of a standard broth bottle.
 Once these bottles have been inoculated, they should be tipped daily or at least twice weekly to
bathe the slide paddle with the broth culture medium, thereby allowing frequent blind
subcultures without the use of needles and syringes.
 Bacterial growth appears as small discrete colonies or as a confluent growth on the slide paddle.
 Most organisms will grow within 48 hours of inoculation, but the bottles are incubated for 7
days before they are discarded and reported as negative.

Manual Systems
 Advantage Rapid recovery of facultative anaerobic bacteria and
Isolated colonies for identification and susceptibility
testing.
 Disadvantages Slightly higher cost of materials and
Contamination rate, and they are
Labor-intensive.
 An additional unvented bottle is still required for adequate isolation of
anaerobes.

Manual Systems
 The Oxoid Signal system (Thermo Fisher Scientific, Waltham, MA) is a manual blood culture
system in which blood is inoculated into a bottle containing a liquid medium that will support the
growth of aerobes, anaerobes, and microaerophiles.
 A clear plastic signal device is then attached to the top of the bottle.
 The Signal device has a long needle that extends down into the bottle below the level of the
liquid.
 When microorganisms grow in the bottle, they generate CO2, which accumulates in the head
space of the bottle increases pressure on the liquid, forcing it up through the needle and into the
clear plastic Signal device indicates the growth of bacteria.
 The fluid from the Signal device can then be removed for Gram staining and plating.
 Bottles are held for a total of 7 days, with a terminal blind subculture performed and examined
before the culture is reported as negative.

Manual Systems
 Lysis centrifugation method (Isolator/Isostat; Wampole, Inverness Medical Professional Diagnostics,
Princeton, NJ) has been shown to provide optimal recovery of unusual fastidious bacteria
 Bartonella,
 yeasts,
 filamentous and dimorphic fungi, and
 mycobacteria that are causing systemic infections.
 This method produces a concentrated sample of blood for direct inoculation onto appropriate solid agar
media,
 As opposed to the two systems described earlier, in which blood is inoculated and incubated in a broth
medium.
 The Isolator tube contains a mixture of saponin, propylene glycol, SPS, and EDTA.
 This mixture causes lysis of WBCs and RBCs, releasing intracellular organisms, prevents clotting, and
neutralizes complement.
 Microorganisms are concentrated through high-speed centrifugation (3000g for 30 minutes). The sediment
containing the organisms is directly inoculated onto a solid culture medium that includes fungal and
mycobacterial media.

Manual Systems
 Examination of Blood Culture Bottles in a Manual System Blood culture bottles are examined
macroscopically with transmitted and reflected light for evidence of
Turbidity,
Heamolysis,
Gas production, or
Bacterial colonies in or on the blood layer.
 If visible growth is observed, 0.25 mL of blood should be aspirated with a sterile needle and
syringe.
 A smear is prepared for Gram stain and then plated to a solid medium.
 In addition to examination of the blood culture fluid for evidence of microbial growth,
The Septi-Chek paddle is examined daily for the presence of colonies; the
Oxoid bottle Signal device is examined daily for the presence of fluid, indicating microbial growth
in the medium.
 Any finding of microbial growth should be reported immediately to a physician.

Continuous-Monitoring BloodCulture Systems
 The BACTEC system (BACTEC 460; Becton, Dickinson, Sparks, MD) was
the first automated growth detection system for blood cultures that detected the
growth of microorganisms using radio labeled carbon (14C) in the broth
medium.
 When the organism in the blood culture bottle used the 14C-labeled substrate,
14CO2 was released.
 The instrument monitored 14CO2 production by aspirating gas into an ionization
chamber by using sterile needles injected into the bottle.
 In the ionization chamber, the amount of 14CO2 produced was measured as a
growth index and compared with an established threshold level.
 If the patient’s blood culture showed a growth index that exceeded the threshold
level, the instrument sent a signal indicating that the bottle was positive.

Continuous-Monitoring Blood Culture Systems
 The automated radiometric blood culture system had the advantage of
early detection of bacterial growth, especially of slow-growing
bacterial species (e.g., Mycobacterium tuberculosis).
 The disadvantages included
High initial cost of the instrument,
High contamination rate (the result of inadequate needle sterilization
between bottles)
Hazards and expense associated with radioisotope disposal.
 The development of subsequent automated blood culture instruments
kept the detection of CO2 as an indication of microbial growth.

BACTEC 9000 Series and BD FX
 To eliminate radioactive isotopes as a growth detection mechanism, Becton,
Dickinson (Sparks, MD) introduced the BACTEC 9000 series.
 Three models are currently available—
The 9240 (holds 240 bottles per module),
9120 (holds 120 bottles per module), and
9050 (holds 50 bottles per module).
The FX holds 400 bottles per module.
 These are non-invasive, continuous-monitoring blood culture instruments
that use fluorescence to detect CO2.

 When microorganisms grow in the bottle, the CO2 that they produce is detected by a gas-
permeable sensor on the bottom of each vial.
 When a bottle is placed into the instrument, a baseline reading of the sensor is taken; this
reading is used as a reference for subsequent readings.
 Carbon dioxide produced by an organism diffuses into the sensor, generating hydrogen
ions.
 The increase in hydrogen ion concentration increases the fluorescence output of the
sensor.
 Using photodetectors, the instrument measures the amount of fluorescence every 10
minutes, which corresponds to the amount of CO2 produced by the microorganism.
 A computer program then interprets these data using several algorithms to determine
when to flag a bottle as positive.
 The instrument alerts the microbiologist to the presence of a positive vial by
displaying a message on the computer monitor and with an audible alarm.

BacT/ALERT 3D System
 A fully automated, nonradiometric blood culture system, the BacT/ALERT 3D system,
consists of aerobic and anaerobic bottles with pH-sensitive membranes placed in the
bottom of the bottles.
 Microbial growth causes a release of CO2, which changes the pH in the sensor, as
indicated by a change in color from gray to yellow.
 The color change is measured by reflected light.
 The instrument measures CO2 production colorimetrically without entering the bottles.
 An advantage of this system is that the changes in the color of the sensor can be detected
and verified, if necessary.
 Another advantage of this system is that in 2003, bioMérieux (Durham, NC) released gas-
impermeable plastic blood culture bottles that are safer and lighter than traditional glass
bottles and do not interfere with microorganism growth or metabolism.

Recovery of Fastidious Organisms from Blood
 There are organisms that can be found in blood that require special conditions to be
detected.
 Communication with the physician is critical in deciding when and what extraordinary
procedures should be performed.
Francisella tularensis.
 Francisella tularensis, the causative agent of tularemia, is best recovered from a liquid
blood culture medium to which L-cysteine and glucose have been added.
 Leptospira spp.
 Leptospira spp. are best recovered early in the disease before onset of symptoms.
 Therefore blood should be collected as soon as this infection is suspected, during the first
week of disease, with one to three drops of freshly drawn blood placed in 5 mL of
Fletcher medium.

 Alternatively, 0.1 mL heparinized blood can be inoculated onto Ellinghausen-McCullough-
Johnson-Harris (EMJH) medium.
 Cultures are incubated at 30° C for 1 to 3 months in the dark and examined weekly by using dark-
field microscopy.
 Other methods of diagnosis (polymerase chain reaction [PCR], serology) are recommended
because they are often more sensitive and timely for detection of this organism because treatment
can be initiated more quickly, resulting in better patient outcomes.
Brucella spp.
 When Brucella is suspected, conventional, manual blood culture medium (Ruiz Castañeda) should
be held for up to 6 weeks at 35°± 2° C and terminally subcultured.
 Automated blood culture systems higher yields and faster recovery 10 to 14 days.
 The manual lysis centrifugation system (Isolator) increased yields risk of exposure during
manipulation and plating should be done using special safety precautions.

Nutritionally Variant Streptococci.
 The nutritionally variant streptococci (NVS) include Abiotrophia and Granulicatella.
 These streptococci are adequately recovered by using standard broth culture bottles
because of the vitamin B6 present in human blood.
 However, a pyridoxal-containing blood agar medium, addition of a pyridoxal disk to
a blood agar subculture plate, or staphylococci streak is necessary for subculture to
recover this group of streptococci.
 The staphylococci streak test is performed with a confluent growth of the test organism
on a blood agar plate.
 A single line of S. aureus is streaked across the middle of the plate. Following 24 hours
of incubation, the plate is observed for satellitism—tiny colonies growing near the S.
aureus.
 These organisms frequently grow well on CHOC agar.

Campylobacter spp.
 Campylobacter lari, Campylobacter fetus, and Campylobacter upsaliensis
can be isolated from blood.
 Although Campylobacter jejuni may grow in blood culture bottles,
subcultures will show no growth unless the broth is subcultured onto
selective media or enriched media, such as CHOC agar, and incubated
in a microaerophilic environment at 42° C for 48 hours.
 A clue that the bottle contains Campylobacter will be the presence of curved
gram-negative rods in the blood culture broth.
 These organisms are often difficult to find in the Gram stain of the blood
bottle; counterstaining with the darker pink basic fuchsin instead of
safranin may help with visualization.
 An alternative stain, such as acridine orange (AO), can also be used.

Bartonella spp.
 Bartonella spp. are best isolated using lysis centrifugation and plating the
concentrated blood onto freshly prepared media containing 5% horse or rabbit
blood.
 Plates must be incubated for at least 3 weeks in a humid atmosphere containing elevated
CO2 levels at 35° to 37° C.
 Because the isolation of Bartonella spp. is difficult, serology and molecular methods of
detection are preferred.
HACEK Group of Gram-Negative Bacilli.
 These bacteria are known causes of endocarditis and will grow in blood culture bottles
within 5 days.
 Extended incubation times and a terminal subculture are recommended only if blood
cultures are negative at 5 days and there is a high clinical index of suspicion for these

Mycobacteria from Blood.
 Using special media, Mycobacterium spp. can be detected with the automated blood culture
systems discussed earlier.
 Medium in the bottles contains a lysing agent and enrichment (Middlebrook based) designed
for long incubation periods, up to 6 weeks.
 The Isolator system can also be used to culture blood for mycobacteria.
 Plated selective media are held for 6 to 8 weeks before mycobacteria can be ruled out.
Fungemia.
 The presence of fungi in the blood is termed fungemia.
 Candida spp. are isolated almost as frequently as are gram-negative bacilli from blood in some
institutions.
 Yeasts grow in the continuous-monitoring blood culture systems.
 The best way to recover filamentous or dimorphic fungi in blood, however, is to use the Isolator
system with fungal agar plates, which are usually held for 4 weeks.

Rapid Identification of Microorganisms
Growing in Blood Cultures
 Even after using CBCMS, definitive identification and susceptibility results are typically not available until
there are isolated colonies growing on the sub-cultured plates, which usually takes 24 to 48 hours.
 During this time, the patient is given empiric antimicrobial therapy that may or may not be optimally effective
for the isolate.
 The sooner the patient is treated with an effective antimicrobial agent, the better the outcome.
 To decrease the time it takes to identify a microorganism growing in a blood culture, several rapid methods
are available.
Direct Tube Coagulase Test.
 This classic test can be performed to determine whether gram-positive cocci in clusters growing in a blood
culture are S. aureus (coagulase positive) or CoNS.
 A small portion of the blood culture fluid (100 μL) is inoculated into a tube containing 0.5 mL rabbit
plasma, incubated at 35° C for 3 hours, and then examined for the presence of a clot (positive).
 The result can be communicated to the physician, who will determine the best course of therapy.
 Many microbiology laboratories have eliminated coagulase in favour of latex and other methods of testing for
staphylococci, so this test is not widely performed.

 Other Rapid Diagnostic Tests. Biochemical and enzymatic tests and stains that can be performed directly from
blood culture bottles include
 Thermonuclease (a heat-stable DNase produced by S. aureus),
 Bile solubility & Quellung capsule stain (S. pneumoniae)
 Modified Kinyoun stain (Acid fast organisms)
 These are presumptive, not definitive tests, but can provide the physician with preliminary information.
Fluorescence In Situ Hybridization.
 Fluorescence in situ hybridization targets ribosomal ribonucleic acid (rRNA) in an organism by using an
oligonucleotide or peptide nucleic acid (PNA) probe with a fluorescent label.
 Although this is a molecular method, there is no amplification of nucleic acid involved.
 The procedure takes 1.5 to 3 hours to perform;
 the sensitivity of species-specific probes in one study was reported to be 97%, with 95% specificity.
 Different probes are available that target common organisms isolated from blood cultures, such as S. aureus,
Enterococcus spp., and Candida spp.

 Nucleic acid amplification tests (NAATs), using PCR to directly identify microorganisms growing
in a blood culture, have been approved by the U.S. Food and Drug Administration (FDA) and are
available.
 The Verigene system (Nanosphere, Northbrook, IL) amplifies nucleic acid targets that are then
detected by hybridization of oligonucleotides bound to nanosphere particles with a silver staining
process.
 The FilmArray system (BioFire, Salt Lake City, UT) involves an initial nucleic acid extraction
and purification followed by PCR amplification in a two-step process.
 The Verigene (Nanosphere, Northbrook, IL) and FilmArray (BioFire, Salt Lake City, UT) systems
are both multiplex assays Identify a group of gram-positive and gram-negative organisms and
yeasts, & several resistance genes within a few hours.
 The GeneXpert system also offers assays that detect resistance genes resulting in a great
reduction in time to reporting and appropriate treatment, costs, and hospital stays for patients.
 Disadvantage Do not perform well in the case of polymicrobial infections, and identification of
certain groups of organisms.

Verigene (Nanosphere, NOrthbrook, IL) test
cartridge. Nucleic acid amplification testing
(NAAT) can identify commonly isolated organisms
and some species with resistance genes within 2–4
hours. Isolate from blood culture bottle is
inoculated into wells.

Matrix-Assisted Laser Desorption/Ionization–Time-of- Flight
(MALDI TOF)
 Mass spectrometry identifies a wide variety of organisms, including fungi,
fastidious bacteria, and anaerobes, in a matter of minutes, and is currently
used routinely in many modern microbiology laboratories (Bruker
Microflex is an instrument using this technology)
 The organisms must first be subcultured from the blood and isolated in pure
culture.
 Direct detection from blood is not yet cleared by the FDA
 There is currently no ability to determine antimicrobial susceptibility by
using this method.

Laboratory diagnosis of Blood Stream Infections (BSIs)

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Laboratory diagnosis of Blood Stream Infections (BSIs)

Similar to Laboratory diagnosis of Blood Stream Infections (BSIs) (20)

More from Suprakash Das

More from Suprakash Das (20)

Recently uploaded

Recently uploaded (20)

Laboratory diagnosis of Blood Stream Infections (BSIs)