In this issue: Celebrating the 70th Anniversary of the Hungarian Society of Laboratory Medicine

Interpretation of blood microbiology results –

function of the clinical microbiologist
Katalin Kristóf, Júlia Pongrácz
Clinical Microbiology Laboratory, Department of Laboratory Medicine, Semmelweis University,
Budapest, Hungary

ARTICLE INFO ABSTRACT

Corresponding author: The proper use and interpretation of blood microbiol-

Katalin Kristóf, MD
Clinical Microbiology Laboratory
ogy results may be one of the most challenging and
Department of Laboratory Medicine one of the most important functions of clinical micro-
Nagyvárad tér 4, Floor 11, H-1089 biology laboratories. Effective implementation of this
Budapest, Hungary
E-mail:
function requires careful consideration of specimen
[email protected] collection and processing, pathogen detection tech-
niques, and prompt and precise reporting of identi-
Key words:
blood culture, sepsis, rapid diagnostics, fication and susceptibility results. The responsibility
microbiology techniques, laboratory workload of the treating physician is proper formulation of the
analytical request and to provide the laboratory with
complete and precise patient information, which are
inevitable prerequisites of a proper testing and inter-
pretation. The clinical microbiologist can offer advice
concerning the differential diagnosis, sampling tech-
niques and detection methods to facilitate diagno-
sis. Rapid detection methods are essential, since the
sooner a pathogen is detected, the better chance the
patient has of getting cured. Besides the gold-standard
blood culture technique, microbiologic methods that
decrease the time in obtaining a relevant result are
more and more utilized today. In the case of certain
pathogens, the pathogen can be identified directly
from the blood culture bottle after propagation with
serological or automated/semi-automated systems

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 Katalin Kristóf, Júlia Pongrácz
Interpretation of blood microbiology results – function of the clinical microbiologist

or molecular methods or with MALDI-TOF MS result in haematogenous spread, or the patient

(matrix-assisted laser desorption-ionization time has a fever of unknown origin. For sample col-
of flight mass spectrometry). Molecular biology lection they should use national guidelines and
methods are also suitable for the rapid detection recommendations of the local microbiologi-
and identification of pathogens from aseptically cal laboratory (4, 5, 6). Proper formulation of
collected blood samples. Another important the analytical request by the treating physician
duty of the microbiology laboratory is to notify is essential in order to provide the laboratory
the treating physician immediately about all with complete and precise patient information,
relevant information if a positive sample is de- which are inevitable prerequisites of a proper
tected. The clinical microbiologist may provide testing and interpretation. The partner micro-
important guidance regarding the clinical signifi- biological laboratory should prepare useful
cance of blood isolates, since one-third to one- guidelines, which contain every important pre-
half of blood culture isolates are contaminants or analytical rule (timing and sampling of blood
isolates of unknown clinical significance. To fully culture – sample collection, volume of blood re-
exploit the benefits of blood culture and other quired, blood-to-broth ratio, formulation of the
(non- culture based) diagnoses, the microbiolo- analytical request, and transportation. The mi-
gist and the clinician should interact directly. crobiologist should aim to provide the clinician
with proper results as soon as possible, utilizing
 every available diagnostic method when evalu-
ating culture results (7, 8, 9). The clinician and
BLOOD CULTURE – the microbiologist should cooperate during the
PRINCIPLE, INTRODUCTION whole test procedure, but especially during the
Bacteraemia/fungaemia can induce a system- evaluation of the results, to ensure the highest
ic inflammatory response syndrome and as a possible standard of patient care. In this short
clinical continuum can fall into life-threatening summary, as microbiologists, our goal is to pro-
severe sepsis or septic shock. Huge number of vide answers to the clinicians’ most frequent
studies have inferred that clinical outcomes in questions.
severe sepsis and septic shock hinge upon the
optimized selection, dosing, and delivery of WHEN SHOULD THE SAMPLE
highly potent antimicrobial therapy (1, 2, 3). BE COLLECTED FOR BLOOD CULTURE?
With this in mind the recovery of the causative In case of periodical bacteraemia and fungaemia
agent is one of the most important tasks of the blood should be collected at the beginning of
microbiological laboratory. Blood cultures- as a the fever episode, during the chills or at the start
gold standard -, in which a sample of blood is of the fever curve. In case of continuous bacte-
allowed to incubate with a medium that pro- raemia or fungaemia (e.g. suspicion of endocar-
motes bacterial growth, are used to diagnose
ditis) sample collection time is not critical (4).
bacteraemia or fungaemia, confirmed by iso-
lating one or more microorganisms from the
WHAT AMOUNT OF BLOOD SHOULD
blood culture. Clinicians are supposed to collect
BE COLLECTED, AND FROM WHERE?
blood cultures (BC) from patients with clinical
signs and symptoms indicating sepsis, or if the In adults, if local infection is present or suspect-
laboratory or imaging results suggest an infec- ed, or the patient presents with fever of un-
tion, and the presumed infection is known to known origin, an amount of 20-30 ml of blood

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 Katalin Kristóf, Júlia Pongrácz
Interpretation of blood microbiology results – function of the clinical microbiologist

collected from 2 venipuncture sights (the to- infection can be diagnosed (12). If the catheter
tal amount of blood should be at least 40 ml) is removed because of suspected catheter-as-
is sufficient. The samples should be collected sociated infection, the catheter end should be
strictly aseptically. If endocarditis is suspect- sent for culture as well.
ed, at least 3 samples are necessary because
of the low bacterial count in the blood. In WHAT TYPE OF BLOOD CULTURE BOTTLE
case of children, an amount decreased pro- SHOULD BE USED?
portionally with body mass should be collect- IS THE ANAEROBIC BOTTLE OR THE
ed (see guidelines). Venipuncture should be SPECIALIZED FUNGI BOTTLE NECESSARY?
performed on intact peripheral veins; except
if catheter-associated infection is suspect- The blood collected from one sampling is
ed, which case will be addressed separately. usually distributed into two (an aerobic and
Theoretically, it is possible to distribute a suffi- an anaerobic) commercially available blood
cient amount of blood sampled from one sight culture bottles in the amount specified by the
to four different bottles, but in this case, the manufacturer. An anaerobic bottle is recom-
microbiology laboratory findings cannot aid in mended in patients with neutropenia, in case
the evaluation of the clinical relevance of cer- of complications following abdominal sur-
tain potential pathogens which colonize the gery, patients with diabetes and in patients
skin (and may contaminate the sample), but with complicated wound infections. Small
can also cause infection in case of certain risk amounts of blood samples (1-5 ml) collected
factors. Since the number of pathogens in the from children should be distributed into spe-
blood during bacteraemia/fungaemia is very cial childrens’ bottles containing a smaller
low (0.1-300/ml depending on the patient’s amount of media. Fungi usually grow well in
age and the pathogen), the sensitivity of BC aerobic BC media prepared for the culture
is mostly determined by the amount of blood of bacteria, but certain studies showed that
collected. Usually, BC containing samples the TTP is shorter when special fungi bottles
from two or three venipuncture sights is suf- are used. In case of patients under antibiotic
ficient to support or rule out sepsis; however, treatment, bottles containing agents that in-
a single sample is insufficient (4, 10, 11). activate antibiotics (activated carbon, resin)
are recommended (4, 13).
WHAT IF CATHETER-ASSOCIATED
INFECTION IS SUSPECTED? HOW SHOULD INOCULATED
BOTTLES BE STORED?
In this case, blood samples should be taken
through the catheter and a peripheral vein at The inoculated BC bottles – if it is possible –
the same time. Two pairs of BC is not neces- should be sent to the microbiology laboratory
sary (one sample is enough), but if the cath- immediately, otherwise the bottles should be
eter has multiple lumens, a sample should stored at room temperature. Several studies
be taken through each lumen. If the time to have demonstrated (and it is included in the
positivity (TTP) of the sample taken through references of the commercially available bot-
the catheter is at least 2 hours shorter than tles) that a certain time (12-16 h) of storage at
that of the sample from the peripheral vein, room temperature, otherwise called delayed
and the cultured microbe and its antimicrobial time vial entry, does not impact the BC result
susceptibility is the same, catheter-associated significantly (4, 14).

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Interpretation of blood microbiology results – function of the clinical microbiologist

HOW LONG UNTIL A RESULT WHY IS THE RESULT OF THE BLOOD

IS AVAILABLE? CULTURE NEGATIVE (NO POSITIVE
SIGNAL DURING INCUBATION),
Continuous monitoring systems have revolu- WHEN THE CLINICAL DIAGNOSIS OF
tionized blood culture practices, because the BACTERAEMIA/FUNGAEMIA IS CERTAIN?
time to detection of microbial growth is sig-
Blood culture is a microbiological test that is
nificantly shorter by continuously agitating the
heavily dependent on the clinician’s procedures
bottles and checking them every 10 minutes.
(timing of sample collection, the amount of the
Depending on the system, the detection meth- collected sample, the number of BC bottles
od can be an indirect measurement of the CO2 used), and the evaluation of the clinical symp-
produced by the microorganism in the bottles toms (estimating the likelihood of bacteraemia/
(at the bottom of the bottles there is an inte- fungaemia and sepsis, the correct assumption
grated CO2 sensor containing a pH indicator or of the probable etiological agent, and the prop-
the level of fluorescence change because of the er evaluation of the results) (4, 7). Based on
reduction in pH). If the system signals a positive the literature. the preanalytics and analytics of
bottle, the microbiology laboratory should ini- blood culture testings are performed properly
tiate analytical tests immediately, according to if 8-14% of the total number of blood cultures
laboratory protocols based on international and is positive. The assessment of this parameter is
recommended in every medical institute/clinic
national guidelines, and all relevant information
with the help of the microbiology laboratory. If
is documented and communicated to the clini-
a significantly different percent is determined,
cian as soon as possible. The result of a Gram the whole procedure should be revised and cor-
stained mount is available in 30 minutes, the re- rected (with the cooperation of the clinician
sult of presumptive or definite identification in and the microbiologist).
4-48 h (depending on the type of microbe), and Sensitivity is basically determined by the type
the preliminary or final antimicrobial suscepti- of sepsis. The BC is positive e.g. in endocarditis
bility report is available in 16-48 h (depending in 53-99%, in S. pneumoniae pneumonia in 25-
on the type of microbe). 30%, in neutropenic fever in 10-20%, in abdom-
The usual incubation time of BC is 5-7 days at 35- inal infection in 30-40%, and in disseminated
37°C. Positive bottles usually signal in the first fungal disease in nearly 50%.
24-48 hours of incubation. The latest guidelines If the symptoms of sepsis still subsist, and the
do not recommend longer incubation time in BC from the day before is not positive, anoth-
certain cases as previous recommendations did er 2-3 sample collections are recommended
(21 days for the detection of Brucella, Legionella, in the next 24 hours. If infective endocarditis
is suspected, and the 3 pairs of BC collected
the fastidious HACEK group bacteria that cause
on the first day are negative, another 2 pairs
endocarditis (Haemophilus, Aggregatibacter,
should be collected the next day. If an infection
Cardiobacterium, Eikenella, Kingella), and in caused by a fastidious microorganism requiring
case of fever of unknown origin). The isolation special culture conditions is suspected despite
of clinically relevant pathogens after 7 days of negative BC results, consultation should be per-
incubation is improbable (except for dimorphic/ formed with the microbiologist before taking a
filamentous fungi) (4, 7, 8, 9). new sample (recommendations for specialized

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Interpretation of blood microbiology results – function of the clinical microbiologist

BC bottles, longer incubation time, alternative β-haemolytic, Haemophilus spp, Neisseria men-
microbiological testing methods – e.g. serology, ingitidis, Listeria monocytogenes, Enterococcus
molecular diagnostics.) (8, 9). spp., Salmonella spp., Brucella spp., Pasteurella
spp., Campylobacter spp., HACEK group, anaer-
WHY IS THE CULTURE RESULT NEGATIVE obes, Candida spp. These microbes are always
WHEN THE BLOOD CULTURE SYSTEM clinically significant, even if they are cultured
YIELDS A POSITIVE SIGNAL? from only one of the (properly collected) four-
Non-conformity with preanalytical methods, six bottles.
namely overfilling the bottles may lead to a false The following microbes are considered sig-
positive signal in systems based on CO2 detec- nificant in only certain cases: Streptococcus
tion, which is caused by the CO2 contained by α-haemolyticus (40-60%), Staphylococcus coag-
the excess amount of RBCs in the blood sample. ulase-negative (20-40%). If a coagulase-negative
A false positive signal may also be detected in Staphylococcus or α-haemolytic Streptococcus
BCs of ventilated patients (elevated partial CO2 is cultured from only one of the ≥2 bottles from
pressure), and blood samples containing high
a set of BC, the isolate is probably a contami-
amounts of WBCs (haematology patients). The
nant. However, an α-haemolytic Streptococcus
microbiologist can immediately confirm this to
cannot be considered as a contaminant if there
the clinician by assessment of BC bottle moni-
was only one bottle. In this case, repeated sam-
toring and the Gram stained mount.
pling is recommended: if ≥2 bottles are positive,
In some cases, the bacterium in the blood may the α-haemolytic Streptococcus is more prob-
start to multiply, it may be seen in the mount ably a significant pathogen. Some studies say
from the positive BC bottle, but it does not that a bacterium is more probably a contami-
grow in subculture. Streptococcus pneumoniae,
nant if it is cultured after a longer than usual
for example, grows well in the rich BC media,
incubation time. However, this observation can-
but also produces a large amount of autoly-
not be used in the assessment of the positive
sin enzyme, which causes the bacteria to die.
results of an individual patient, because there
However, the antigens of the bacteria can be
is significant overlapping in the growth rate of
detected with antigen detection kits. B6 vita-
min-dependent streptococci also propagate contaminants and real pathogens. Further, par-
in BC media containing pyridoxal, but may not allel microbiologic sampling/testing from the
grow on media usually applied for the culture of source of the suspected bloodstream infection
streptococci. Media containing pyridoxal should (e.g. urine, lower respiratory samples, removed
be used for subculturing such strains (4, 7, 8). catheter, etc.) complements and supports the
interpretation of the relevance of the microbes
HOW SHOULD A POSITIVE CULTURE cultured from the blood, and aids in identifying
RESULT BE INTERPRETED? the etiology of the infection.
DOES EVERY MICROBE CULTURED The following microbes are usually consid-
HAVE CLINICAL RELEVANCE? ered contaminants: Staphylococcus coagulase-
The following microorganisms are considered negative, Micrococcus spp., Corynebacterium
significant: Staphylococcus aureus, Entero­ spp. Propionibacterium spp. and Bacillus spp.
bacteriaceae spp., Pseudomonas aeruginosa, However, there is no general rule that they are
Streptococcus pneumoniae, Streptococcus contaminants in all cases.

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Interpretation of blood microbiology results – function of the clinical microbiologist

The specificity of BC is determined by the per- fungaemia is the clinician’s task. So-called “fol-
centage of false positive isolates. The interpre- low-up” BC is not necessary, except for some
tation of contaminants depends to a certain de- special cases. In infective endocarditis, it is
gree on patient characteristics. The spectrum of recommended to guide treatment (the anti-
real pathogens and contaminants can be easily microbial susceptibility of the pathogen may
determined in the case of community acquired change after prolonged treatment). In every
infection. In nosocomial infections, however, case of bacteraemia caused by Staphylococcus
bacteria that are considered contaminants in aureus, when isolation of the pathogen in the
“healthy” (immunocompetent) people may be repeated BC taken after 2-3 days may indicate
real pathogens in immunocompromised pa- complicated sepsis caused by S. aureus (e.g.
tients. Specificity can be improved primarily by secondary metastatic infection), and the need
strictly abiding to sample collecting guidelines, for a change in therapy. Several recommenda-
mainly the methods to ensure asepsis, and to tions contain “follow-up” BC in case of fungae-
have multiple samples collected in cases of sep- mia to determine the necessary duration of
sis in which potential pathogens are the same treatment (4, 16).
as potential contaminants (e.g. catheter or oth-
er indwelling device associated infection, neu- ARE THERE ANY METHODS
tropenic fever). The number of positive blood THAT SIGNIFICANTLY DECREASE
cultures containing a contaminant can be as- THE TIME TO IDENTIFICATION
sessed at a certain medical institute. If the rate OF THE PATHOGEN OF SEPSIS?
of these bottles is significantly more than 3%,
Timely initiation of adequate therapy signifi-
the situation should be remedied by education
cantly affects the patient’s life expectancy;
and consultation (4, 5, 8, 9, 10).
therefore microbiologic methods that decrease
the time to obtaining a relevant result are more
WHAT DOES A POLYMICROBIAL BLOOD
and more utilized today.
CULTURE RESULT SIGNIFY?
WHAT IS ITS CLINICAL SIGNIFICANCE? In the case of certain pathogens, the pathogen
can be identified directly from the BC bottle af-
In about 15 % of cases multiple microorgan- ter propagation with antigen detection or rapid
isms are grown from blood cultures. The rate identification methods (e.g. Streptococcus pneu-
of polymicrobial blood cultures ranges from moniae, Neisseria meningitidis, Streptococcus
10% to 30% in immuno-compromised patients agalactiae – antigen detection by latex aggluti-
and in nosocomial BSI of patients treated at in- nation) (4, 7, 8).
tensive care units. Polymicrobial BSI often indi-
Identification and susceptibility testing per-
cates catheter-related or intraabdominal infec-
formed with automated/semi-automated sys-
tions (15).
tems can identify Gram-negative sepsis patho-
gens in 92-99% of cases, while Gram-positives
SHOULD PATIENTS WITH POSITIVE
are identified in 43-75% of cases. The advan-
BLOOD CULTURE RESULTS
tage of these systems is that the most frequent
BE RE-SAMPLED FOR FOLLOW-UP?
pathogens in routine microbiology can be
The blood may not become sterile even after 2-4 identified in 4-16 hours. Susceptibility results
days of adequate treatment; the assessment show 95% correlation with conventional meth-
of the recovery of patients with bacteraemia/ ods (17, 18).

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Most laboratories have access to MALDI-TOF albicans/Candida parapsilosis, Candida kru-

MS (matrix-assisted laser desorption-ionization sei/Candida glabrata, Candida tropicalis. The
time of flight mass spectrometry) to identify disadvantage of the method is that antimicro-
cultured bacteria and fungi. Since the method bial susceptibility can only be performed after
is based on the mass spectrometry measure- conventional culture (2, 5, 9, 20).
ments of conserved microbial ribosomal and Because of the pronounced significance of sep-
other proteins, the result is precise, mostly sis, more and more manufacturers produce
equivalent to DNA sequencing. Since a test can complex tests based on molecular techniques
be performed from very little sample size (10’4 (PCR), providing identification of the most fre-
- 10’6 CFU/ml), testing of barely visible isolated quent pathogens from positive BC bottles after
colonies after short incubation time can often a simple or more complicated protocol, with ad-
be performed and the species identification equate sensitivity and specificity, in 1-3 h (e.g.
result can be communicated to the treating FilmArray BCID Panel (BioFire): 19 pathogens,
physician. Hyplex BloodScreen (BAG):10 pathogens, Prove-
It should be emphasized that MALDI-TOF MS it Sepsis (MobiDiag):50 pathogens). Molecular
can be used to identify pathogens directly from methods are suitable for the detection of cer-
the blood culture bottles as well. Different sep- tain resistance genes as well e.g. mecA, van-
aration and lysis protocols are available to re- gene detection (20).
move proteins of human origin from the media, Molecular biology methods are also suitable for
and concentrate the bacteria in the sample to the rapid detection and identification of patho-
the appropriate amount, resulting 80-96% cor- gens from aseptically collected blood samples
rect identification results (compared with con- (plasma, serum or EDTA-treated whole blood).
ventional culture and identification methods). Certain pathogens can be detected directly from
However, the method is not always applicable blood with species-specific real-time quantita-
(e.g. BC media containing activated carbon, tive PCR tests (e.g. Neisseria meningitidis DNA
polymicrobial infection) (9, 19). detection). Broad-range real-time PCR tests
Commercial and/or validated “home-made” can be performed directly from blood samples:
molecular methods are also available. Another Gram-positive and Gram-negative bacteria
method is PNA FISH (fluorescent in-situ hy- and fungi can be detected (the clinically most
bridization) which identifies microbes from important species in every group), along with
positive BC bottles with 95-99% sensitivity certain resistance genes e.g. mecA, van-gene
and specificity. It is a quick method, since the detection. Several commercially available multi-
whole procedure takes 90 minutes, but its plex molecular tests (e.g. Septifast Test (Roche),
disadvantage is that it is only able to identify Sepsi Test (Molzym)) are able to detect the most
a small number of microbial species (though frequent bacteria and fungi/the ones included
the most frequent ones) (e.g. S. aureus and in their panel, after more or less complicated
coagulase-negative Staphylococcus (without test protocols in approximately 1-8 hours. The
identification to species level), Enterococcus advantages of PCR testing performed directly
faecalis, Enterococcus faecium, Escherichia from blood are rapid detection, it is not influ-
coli, Klebsiella pneumoniae, Pseudomonas ae- enced by antibiotic treatment administered at
ruginosa). The identification of yeast groups the time of sample collection, and quantitative
is based on intrinsic azole sensitivity: Candida detection is available; its disadvantage is that it

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Interpretation of blood microbiology results – function of the clinical microbiologist

detects bacterial DNA and not viable bacteria. A Guideline. CLSI document M47-A. Wayne, PA: Clinical and
Laboratory Standards Institute 2007.
further disadvantage is that it does not detect
the fastidious HACEK group. Antibiotic suscep- 5. Caliendo A. M., Gilbert D. N., Ginocchio C. C. et al; for
the Infectious Diseases Society of America (IDSA): Better
tibility/resistance detection is limited to certain Tests, Better Care: Improved Diagnostics for Infectious
resistance genes, the sample may be contami- Diseases. CID 2013:57 (Suppl 3) S139-170.
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blood may be troublesome. After review of the on Blood Cultures. J Microbiol Immunol Infect 2010;
currently available diagnostic palette, attention 43(4):347–349.
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Pfaller, M.A.: Manual of Clinical Microbiology. 9th Ed.
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8. Lynne S. Garcia Ed.: Clinical Microbiology Procedures
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