Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture

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article review.
ARTICLE
Reducing time to identification of positive blood cultures
with MALDI-TOF MS analysis after a 5-h subculture
A. Verroken & L. Defourny & L. Lechgar & A. Magnette &
M. Delmée & Y. Glupczynski
Received: 15 July 2014 /Accepted: 31 August 2014 /Published online: 25 September 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Speeding up the turn-around time of positive blood
culture identifications is essential in order to optimize the
treatment of septic patients. Several sample preparation techniques
have been developed allowing direct matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) identification of positive blood cultures.
Yet, the hands-on time restrains their routine workflow. In this
study, we evaluated an approach whereby MALDI-TOF MS
identification without any additional steps was carried out on
short subcultured colonies from positive blood bottles with the
objective of allowing results reporting on the day of positivity
detection. Over a 7-month period in 2012, positive blood
cultures detected by 9 am with an automated system were
inoculated onto a Columbia blood agar and processed after a
5-h incubation on a MALDI-TOF MicroFlex platform
(Bruker Daltonik GmbH). Single-spotted colonies were coveredwith
1 µl formic acid and 1 µlmatrix solution. The results
were compared to the validated identification techniques. A
total of 925 positive blood culture bottles (representing 470
bacteremic episodes) were included. Concordant identification
was obtained in 727 (81.1 %) of the 896 monomicrobial
blood cultures, with failure being mostly observed with anaerobes
and yeasts. In 17 episodes of polymicrobic bacteremia,
the identification of one of the two isolates was achieved
in 24/29 (82.7 %) positive cultures. Routine implementation
of MALDI-TOF MS identification on young positive blood
subcultures provides correct results to the clinician in more
than 80 % of the bacteremic episodes and allows access to
identification results on the day of blood culture positivity
detection, potentially accelerating the implementation of
targeted clinical treatments.
Introduction
Sepsis is a frequent and severe infection, requiring early,
appropriate, and targeted antibiotic treatment to reduce the
patient’s morbidity and mortality. Speeding up the turnaround
time of positive blood culture identification results
becoming available to the clinician is, therefore, of major
importance [1–3].
Various rapid molecular techniques have been developed in
order to allow the identification of pathogens growing from
blood cultures within 2 h, but also for the direct detection of
pathogens in blood samples without any requirement of culture
[4, 5]. Associated with a high rule-in diagnostic value but
a suboptimal sensitivity, polymerase chain reaction (PCR)-
based pathogen detection is, at this time, only recommended
as an addition to conventional culture techniques [5]. Their
availability remains, furthermore, restricted to a limited number
of laboratories, as they are very costly as well as labor and
time demanding.
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) has emerged as a
new technology for species identification analyzing the protein
composition of a bacterial cell. Through the improvement
of the technique,MALDI-TOF MS has proved over the recent
This work was presented, in part, at the 113th General Meeting of the
American Society for Microbiology (ASM), Denver, CO, May 2013.
A. Verroken (*) : M. Delmée
Institut de recherche expérimentale et clinique (IREC), pôle de
microbiologie (MBLG), Université catholique de Louvain, Brussels,
Belgium
e-mail: [email protected]
A. Verroken : L. Defourny : L. Lechgar : A. Magnette :M. Delmée
Laboratoire de microbiologie, Cliniques universitaires
Saint-Luc—Université catholique de Louvain, Brussels, Belgium
Y. Glupczynski
National Reference Centre for Monitoring of Antimicrobial
Resistance in Gram-negative bacteria, CHU Dinant Godinne | UCL
Namur, Yvoir, Belgium
Eur J Clin Microbiol Infect Dis (2015) 34:405–413
DOI 10.1007/s10096-014-2242-4
years to be a rapid, accurate, easy-to-use, and inexpensive
universal method for the identification of microorganisms
[6]. Subsequently, various purification and extractionmethods
have been developed with MALDI-TOF MS for the direct
identification of positive blood cultures, allowing the
reporting of species results within 1 h after the detection of
blood culture positivity [7–15]. However, directMALDI-TOF
MS identification protocols include several washing and extraction
steps, requiring additional hands-on time. When considering
their workflow implementation, most authors process
the positive blood culture specimens in batches, thereby reducing
the major time gain advantage on the reporting of the
identification results [9, 10, 16, 17].
In this study, we validated an identification process
consisting of the MALDI-TOF MS analysis of positive
blood subcultures after a shortened 5-h incubation without
any preparation steps. This process required much reduced
hands-on time, while also allowing results reporting on the
same day of blood culture positivity detection. In a second
step, a work scheme integrating this process into the identification
of positive blood cultures in daily routine practice
was defined.
Materials and methods
Positive blood cultures
The study was conducted at the Cliniques universitaires
Saint-Luc (UCL), Brussels, Belgium, a 964-bed tertiary
hospital. Positivity of all patients’ blood culture bottles
(BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Peds
Plus/F Medium, Becton Dickinson, Franklin Lakes, NJ,
USA) was detected with the BACTEC FX automated system
(Becton Dickinson, Franklin Lakes, NJ, USA). Specific
blood culture bottles for the recovery of yeast were
not used in our hospital.
During a 7-month period in 2012, all overnight and
early morning weekdays (from 10 pm until 9 am)
positive-detected blood cultures were inoculated by 9 am
on a Columbia agar plate with 5 % sheep blood (COL;
Becton Dickinson, Franklin Lakes, NJ, USA). Anaerobic
positive blood culture bottles were inoculated on a Brucella
agar plate with 5 % sheep blood (BRU; Becton Dickinson,
Franklin Lakes, NJ, USA). COL and BRU agar plates
were incubated at 37 °C in a 5 % supplemented CO2
atmosphere and in an anaerobic atmosphere, respectively.
Blood cultures detected positive during periods outside this
time frame were not included in the study and were processed
according to our standard routine identification procedures
only.
The internal ethics committee of the hospital approved the
anonymous use of remaining patient material.
Identification method
At 2 pm, plates were removed from the incubators for
MALDI-TOF MS identification. A thin layer of growing
colonies was scraped from each plate in order to fill
one-third of a 10-µl plastic loop and was single-spotted
on a steel target, overlaid with 1 µl of 100 % formic
acid, and after drying overlaid with 1 µl matrix, a
saturated solution of a-cyano-4-hydroxycinnamic acid
dissolved in a basic organic solvent composed of 50 %
acetonitrile and 2.5 % trifluoroacetic acid. MALDI-TOF
MS measurements were realized on a MicroFlex LT
platform (Bruker Daltonik, Bremen, Germany). Spectra
were recorded in the positive linear method in a mass
range from 2,000 to 20,000 Da, according to the manufacturer’s
settings. The acquired bacterial spectra with
MALDI-TOF MS were analyzed in the MALDI
Biotyper 3.0 software with database version 3.1.2 and
bearing the spectra of 4,111 cellular organisms. Score
results were interpreted according to a defined cut-off of
1.7 for acceptable identification to the species level. A
score <1.7 was considered unreliable for identification.
No threshold for acceptance to the genus level was
defined.
The results were compared with the routine identification
procedure including optochin susceptibility testing
for Streptococcus pneumoniae suspected strains and standard
MALDI-TOF MS identification from overnight culture
colonies (18-h subculture) for other species isolates
[18]. In this procedure, a single colony was directly
plated onto a steel target and overlaid with 1 µl of
matrix. According to the specifications of the manufacturer,
a high log score =2 was required for identification
to the species level and an intermediate log score lying
between <2 and =1.7 for identification to the genus level.
A low score <1.7 was considered unreliable for
identification.
All result discrepancies were resolved by 16S rRNA
gene sequencing according to a previously published method
[19].
Bacteremic episodes
Review of the patients’ medical records allowed the classification
of all included positive blood culture episodes
into true bloodstream infections (bacteremia/septicemia) or
contaminations according to the Centers for Disease Control
and Prevention/National Healthcare Safety Network
(CDC/NHSN) surveillance definitions of specific infection
types [20]. Positive blood culture bottles originating from
the same patient were considered to belong to a single
bacteremic episode when the difference in the sampling
dates was less than 7 days.
406 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
Results
A total of 925 blood culture bottles were collected over the 7-
month study period, comprising 483 aerobic broths, 377 anaerobic
broths, and 65 pediatric broths.
A single microorganism grew in 896 (96.9 %) blood culture
bottles, while the 29 (3.1 %) remaining bottles yielded
growth of two different microorganisms (Table 1).
These 925 positive blood culture bottles corresponded to
347 bloodstream infections and 123 contaminations, and
accounted for 70 % of the total positive blood cultures, excluding
weekends.
Monomicrobial positive blood cultures
Of the 896 monomicrobial positive blood cultures, species
identification could be obtained in 727 cases (81.1 %), including
433/527 (82.2 %) Gram-positive isolates and 292/323
(90.4 %) Gram-negative isolates (Table 2). Among the
Gram-positive bacteria, staphylococci, enterococci, and streptococci
were correctly identified in 351/410 (85.6 %), 45/52
(86.5 %), and 34/52 (65.4 %) positive blood cultures, respectively.
For Gram-negative bacteria, 255/275 (92.7 %) of the
Enterobacteriaceae and 32/34 (94.1 %) of the non-fermenters
were correctly identified. On the other hand, the identification
of 5-h subcultures growing with anaerobes and yeasts led to
poor results, as only 2/10 and 0/36 isolates, respectively, could
be identified.
In 166 positive blood cultures, the causative organisms
remained unidentified by MALDI-TOF MS due either to an
insufficient score for identification proposal (98 isolates) or
because no peaks were detected (68 isolates). Poor growth at
the 5-h subculture accounted for insufficient scores mainly in
non-identified Gram-positive isolates (coagulase-negative
staphylococci, viridans group streptococci, and the group of
other Gram-positive organisms), while the absence of peaks
could be linked to the absence of growth of yeast and anaerobes
after a 5-h subculture (data not shown).
In three cases with discordant identification results compared
to the routine identification procedure, 16S rRNA PCR
confirmed that we had erroneously identified one
Acinetobacter baumannii isolate as Acinetobacter pittii with
a log score of 1.742, while two Streptococcus isolates (Streptococcus
salivarius and Streptococcus peroris) had been
misidentified as S. pneumoniae with log scores of 1.903 and
1.888, respectively.
Polymicrobial positive blood cultures
Rapid MALDI-TOF MS identification of the polymicrobial
positive blood cultures never allowed the concomitant identification
of both isolates from the same 5-h subcultured plate
(Table 3). One of the two isolates was identified to the species
level in 23 of the 29 blood cultures with mixed bacterial
growth. One S. peroris strain was erroneously identified as
S. pneumoniae with a log score of 1.998, while the scores of
the five remaining positive blood cultures were insufficient to
consider the identification result.
Bloodstream infections versus contamination
Among the 453 monomicrobial blood cultures, 333
corresponded to bloodstream infections, while 120 were
deemed to correspond to contaminations. Species identification
could be obtained for 287/333 (86.2 %) bloodstream
infections and for 82/120 (68.3 %) contaminations,
encompassing, respectively, 640/766 (83.6 %) and 87/130
(66.9 %) identified positive blood culture bottles.
Among the 17 mixed bacterial growths, ten
polymicrobial bloodstream infections, three polymicrobial
contaminations, and four monomicrobial bloodstream infections
associated with a contaminating strain were defined.
In two polymicrobial episodes, both strains were
identified from distinct subcultured blood bottles of the
same episode. In the first episode, Staphylococcus aureus
was identified from three bottles and Staphylococcus
Table 1 Performance of the rapid
(5-h) matrix-assisted laser desorption/
ionization time-of-flight
mass spectrometry (MALDI-TOF
MS) identification process among
monomicrobial and
polymicrobial bacteremic
episodes
No. of
episodes
No. (%) of identified
episodes with rapid
MALDI-TOF MS
No. of positive
blood culture
isolates
No. (%) of identified
isolates with rapid
MALDI-TOF MS
Monomicrobial, total 453 369 (81.5) 896 727 (81.1)
Bacteremia 333 287 (86.2) 766 640 (83.6)
Contamination 120 82 (68.3) 130 87 (66.9)
Polymicrobial, total 17 0 29 0
Bacteremia 10 0 22 0
Bacteremia + contamination 4 0 4 0
Contamination 3 0 3 0
Total 470 369 (79) 925 727 (78.6)
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 407
epidermidis from a fourth bottle. Similarly, in the second
episode, Staphylococcus aureus was identified from the
first bottle and Proteus mirabilis from the second bottle.
In 11 of the 17 mixed bacterial episodes, rapid MALDITOF
MS identified only one of the two organisms. Nevertheless,
the concomitant presence of two different isolates
could be anticipated by Gram staining at the time of blood
culture positivity in 6 out of 17 episodes.
Table 2 Identification results of monomicrobial positive blood cultures with the rapid MALDI-TOF MS process
Final identification of monomicrobial
positive blood cultures with Grampositive
bacteria
n Correct species
identification
Final identification of monomicrobial
positive blood cultures with Gramnegative
bacteria, anaerobes, or yeast
n Correct species
identification
n % n % n %
Gram-positive bacteria 527 433 82.2 Gram-negative bacteria 323 292 90.4
Staphylococci 410 351 85.6 Enterobacteriaceae 275 255 92.7
Staphylococcus aureus 179 171 95.5 Citrobacter braakii 1 0 0.0
Staphylococcus capitis 21 16 76.2 Enterobacter aerogenes 10 10 100.0
Staphylococcus cohnii 3 0 0.0 Enterobacter cloacae 17 15 88.2
Staphylococcus epidermidis 161 132 82.0 Escherichia coli 166 156 94.0
Staphylococcus haemolyticus 11 8 72.7 Hafnia alvei 7 7 100.0
Staphylococcus hominis 28 21 75.0 Klebsiella oxytoca 16 16 100.0
Staphylococcus lugdunensis 1 0 0.0 Klebsiella pneumoniae 45 39 86.7
Staphylococcus pettenkoferi 4 2 50.0 Morganella morganii 1 1 100.0
Staphylococcus sciuri 1 1 100.0 Proteus mirabilis 5 5 100.0
Staphylococcus warneri 1 0 0.0 Salmonella sp. 2 2 100.0
Enterococci 52 45 86.5 Serratia marcescens 3 3 100.0
Enterococcus avium 1 1 100.0 Serratia rubidaea 2 1 50.0
Enterococcus faecalis 21 17 81.0 Non-fermenters 34 32 94.1
Enterococcus faecium 30 27 90.0 Acinetobacter baumannii 5 4 80.0
Streptococci 52 34 65.4 Acinetobacter pittii (Acinetobacter
genomospecies 3)
3 3 100.0
Pyogenic group 10 8 80.0 Acinetobacter lwoffii 2 2 100.0
Streptococcus pyogenes 4 2 50.0 Pseudomonas aeruginosa 23 22 95.7
Streptococcus agalactiae 3 3 100.0 Stenotrophomonas maltophilia 1 1 100.0
Streptococcus dysgalactiae 3 3 100.0 Other Gram-negative organisms 14 5 35.7
Viridans group 41 26 63.4 Capnocytophaga sputigena 1 0 0.0
S. anginosus group 3 1 33.3 Haemophilus influenzae 4 1 25.0
S. bovis group 6 2 33.3 Moraxella catarrhalis 1 1 100.0
S. mitis group 23 20 87.0 Moraxella lacunata 3 1 33.3
S. salivarius group 2 0 0.0 Moraxella osloensis 2 0 0.0
S. sanguinis group 7 3 42.9 Neisseria meningitidis 3 2 66.7
Other streptococci 1 0 0.0 Anaerobes 10 2 20.0
Granulicatella adiacens 1 0 0.0 Actinomyces sp. 2 1 50.0
Other Gram-positive organisms 13 3 23.1 Bacteroides fragilis 2 0 0.0
Aerococcus urinae 1 0 0.0 Clostridium perfringens 1 1 100.0
Bacillus cereus 1 1 100.0 Leptotrichia sp. 1 0 0.0
Corynebacterium aurimucosum 1 0 0.0 Propionibacterium acnes 4 0 0.0
Corynebacterium durum 1 0 0.0 Yeasts 36 0 0.0
Corynebacterium jeikeium 1 0 0.0 Candida albicans 17 0 0.0
Gordonia sputi 3 0 0.0 Candida dubliniensis 2 0 0.0
Micrococcus luteus 3 1 33.3 Candida glabrata 1 0 0.0
Rothia aeria 1 0 0.0 Candida tropicalis 10 0 0.0
Rothia mucilaginosa 1 1 100.0 Fusarium spp. 5 0 0.0
Trichosporon inkin 1 0 0.0
408 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
Discussion
We evaluated here a practical approach for the rapid identification
of microorganisms growing from positive blood cultures in
daily routine clinical practice. Numerous studies have already
assessed the performance of MALDI-TOF MS procedures for
rapid microorganism identification when directly applied on
culture-positive blood specimens. Accurate species identification
rates were found to vary between 50.5 % and 91 %,
depending both on the distribution of microbial isolates and
on the applied pretreatment/extraction methods, as well as on
the definitions of the cut-off threshold log scores [7, 9–15].
However, direct bacterial identification by MALDI-TOF MS
from positive blood cultures is time- and labor-intensive, since
it requires at least 30 min hands-on time for the washing,
centrifugation, and extraction steps that are necessary to discard
blood cells and reveal the bacterial proteins. Despite the possibility
of obtaining, in theory, a result within 60 min from the
time a positive blood culture is detected, the proposed workflow
is difficult to integrate in the routine workflow of a clinical
microbiology laboratory. Hence, directMALDI-TOFMS identification
of positive blood cultures is most usually realized in
batches, for instance, every 2 h, as suggested by Loonen et al.,
or twice a day, according to Martiny et al., thereby extending
the time to obtaining identification results [9, 10].
In this study, we investigated an identification procedure
not requiring any additional time- or labor-consuming sample
preparation steps and leading to identification results available
to the clinician within the same day as blood culture positivity.
Our MALDI-TOF MS processing algorithm after a 5-h subculture
frompositive blood bottles with formic acid overlay as
the only preparation step could be considered as an intermediate
method between the direct MALDI-TOF MS identification
process and the “next-day” MALDI-TOF MS identification
from an 18-h subculture. McElvania TeKippe et al. previously
evaluated the formic acid overlay process for the
MALDI-TOF MS identification of Gram-positive cultured
organisms and showed a significant improvement of genusand
species-level identification (by 20 %) and higher scores
compared to the direct smear deposit [21]. Ford and Burnham
similarly demonstrated the added value of the formic acid
overlay versus the direct smear method for the identification
of Gram-negative bacterial colonies by the reduction of unidentified
organisms [22].
Table 3 Rapid (5-h)MALDI-TOF MS identification results of the 17 polymicrobial bacteremic episodes. Clinically relevant pathogens are reported in
bold
Episode Gram staining lecture Rapid MALDI-TOF MS identification results Final identification results
Identified/
total BCB
Identified strain(s)
Polymicrobial bloodstream infection
1 GPC 3/6 and 1/6 Staphylococcus aureus/
Staphylococcus epidermidis
Staphylococcus aureus + Staphylococcus epidermidis
2 GPC in chains + GNB 2/2 Escherichia coli Enterococcus gallinarum + Escherichia coli
3 GPC in chains + GNB 2/2 Escherichia coli Enterococcus faecium + Escherichia coli
4 GPC in clusters 2/2 Enterococcus faecalis Enterococcus faecalis + Enterococcus faecium
5 GPC in clusters 2/2 Enterococcus faecalis Enterococcus faecalis + Acinetobacter baumannii
6 GPC in clusters 2/2 Staphylococcus aureus Staphylococcus aureus + Staphylococcus epidermidis
7 GPC in clusters + GNB 2/2 Escherichia coli Streptococcus pyogenes + Escherichia coli
8 GPC in clusters + GNB 1/2 and 1/2 Staphylococcus aureus/
Proteus mirabilis
Staphylococcus aureus + Proteus mirabilis
9 GNB 1/1 Escherichia coli Escherichia coli + Proteus mirabilis
10 GNB 1/1 Klebsiella oxytoca Escherichia coli + Klebsiella oxytoca
Monomicrobial bloodstream infection + contamination
11 GPC in chains 0/1 – Staphylococcus epidermidis + Enterococcus faecalis
12 GPC in chains 1/1 Streptococcus agalactiae Staphylococcus epidermidis + Streptococcus agalactiae
13 GPC in chains + clusters 1/1 Streptococcus oralis Staphylococcus aureus + Streptococcus oralis
14 GPC in clusters 0/1 – Staphylococcus epidermidis + Escherichia coli
Polymicrobial contamination
15 GPC 1/1 Staphylococcus hominis Staphylococcus hominis + Staphylococcus capitis
16 GPC in chains + clusters 0/1 Streptococcus pneumoniae Streptococcus peroris + Staphylococcus capitis
17 GPC in clusters 0/1 – Staphylococcus epidermidis + Aerococcus urinae
BCB blood culture bottles; GNB Gram-negative bacilli; GPC Gram-positive cocci
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 409
In our evaluation, the processing time of subcultures was
set at 5 h after preliminary MALDI-TOF MS identification.
Experiences following 3 and 4 h of incubation were found to
be associated with very poor identification results for Grampositive
isolates and only moderate results for Gram-negative
strains (data not shown). Idelevich et al. similarly evaluated
rapid MALDI-TOF MS identification of microorganisms
from positive blood cultures subsequent to a 1.5-, 2-, 3-, 4-,
5-, 6-, 7-, 8-, 12-, and 24-h incubation on solid medium [23].
The mean incubation time needed to achieve species-level
identificationwas 5.9 and 2 h forGram-positive aerobic cocci
(n=86) and Gram-negative aerobic rods (n=42), respectively.
For monomicrobial positive blood cultures, species identification
results could be achieved for 81.1 % of the isolates,
which can be considered as a very satisfactory result when
compared to other recent studies using direct MALDI-TOF
MS, in which correct identification ranged between 64.8 and
81.8%[11, 14, 15]. In line with these authors, we also noticed
a higher identification percentage for Gram-negative organisms
(90.4 %) compared to Gram-positive organisms
(82.2 %). As defined in the Materials and methods section,
identification results were accepted to the species according to
a cut-off score =1.7. Using a cut-off score =2 for species
identification allowed correct identification results for
69.6 % of the monomicrobial positive blood cultures and
identified one of the two strains in 21 of the 29 polymicrobial
positive blood cultures. No isolates were erroneously identified.
A less stringent cut-off score =1.5 for species identification
allowed results for 84 % of the monomicrobial positive
blood cultures and identified one of the two strains in 24 of the
29 polymicrobial positive blood cultures. Three isolates were
erroneously identified, as observed with the cut-off score at
1.7. The final choice to set the cut-off at 1.7 in our study was
taken in accordance with the abundant publications using this
scoring system when directMALDI-TOF MS identification is
applied [9, 17, 24].
One drawback of our rapid MALDI-TOF MS process was
that it failed to yield correct identification results for yeast and
anaerobes most probably related to the insufficient growth of
these microorganisms on the agar plates after a 5-h subculture.
Pondering the high rates of morbidity and mortality as well as
the growing incidence of candidemia and anaerobic septicemia,
a prompt identification result is essential [25, 26]. Hence,
to overcome this flaw in our process, direct analysis from
positive blood culture samples by MALDI-TOF MS should
be considered systematically when Gram staining suggests the
presence of yeast or anaerobes. This procedure preceded by
defined blood lysing protocols using sodium dodecyl sulfate
detergent or Tween 80 and formic acid extraction respectively
enabled Pulcrano et al. [27] to identify 19/21 Candida nonalbicans
bloodstream infections and Leli et al. to identify 7/7
anaerobic septicemia [15. The erroneous identification of two
streptococci (S. salivarius and S. peroris) as S. pneumoniae in
our study confirmed the inability of MALDI-TOF MS to
distinguish oral streptococci strains from S. pneumoniae [18,
28, 29]. On the basis of these observations, we decided not to
consider any S. pneumoniae result through our rapidMALDITOF
MS identification process. The third erroneously identified
strain was an A. baumannii isolate that was misidentified
to the species level as Acinetobacter pittii (formerly
Acinetobacter genomospecies 3). In the MALDI Biotyper
3.0 software, Acinetobacter species identification results are
accompanied with a comment informing about the close relatedness
of several species and the difficulty in differentiating
them.
Themain weakness of ourMALDI-TOFMS protocol was
its inability to identify all organisms in the setting of
polymicrobial bloodstream infections. Various studies also
underlined the lack of ability of MALDI-TOF MS to detect
all microorganisms in mixed cultures through direct identification,
as none or, at best, one single isolate could be identified
[10–12, 15]. Ferroni et al. managed the identification of blood
cultures containing mixed bacteria through the use of Gramspecific
databases selected according to the obtained Gram
result [7]. In our setting, Gram staining of all positive blood
culture bottles and rapidMALDI-TOFMS identification of all
subcultured isolates included in the polymicrobial episodes
were essential elements that partially overcame this limitation
of our algorithm. Regarding the possibility of detecting
polymicrobial bacteremia, visualization of all plated blood
subcultures was systematically repeated the day following
positivity considering that the presence of more than one
organism could go undetected on young subcultures.
The ultimate objective of this study was to speed up the
identification process for improving the management of the
patient and to assist the clinician in deciding whether the
growing microorganisms were to be considered as clinically
relevant and associated with a bloodstreaminfection or, rather,
whether they should be considered as contaminants. Overall,
86.2% of the monomicrobial bloodstream infections could be
identified, thereby potentially allowing an earlier diagnosis
and adaptation of therapy to the documented pathogens. In
parallel, 68.3 % of all organisms regarded as clinically nonsignificant
contaminants could be identified and reported on
the same day of blood culture positivity, possibly leading to
restriction and/or earlier stop of antimicrobial therapy.Martiny
et al. measured the clinical impact of rapid microbial identification
(direct MALDI-TOF MS preceded by an in-house
purification protocol) on the management of septic patients.
An accelerated modification of the treatment regimen was
observed in 13.4 % and 2.5 % of the adult and pediatric
patients, respectively. In other cases, the tool was helpful to
rapidly confirm suspected cases of contamination, thereby
avoiding the administration of unnecessary antibiotics [16].
Vlek et al., likewise, observed an 11.3 % increase in the
proportion of patients receiving appropriate antibiotic
410 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
treatment 24 h after blood culture positivity with direct
MALDI-TOF MS performed twice a day [17].
These results emphasize the benefit of the rapid identification
of positive blood cultures compared toMALDI-TOF MS
analysis on 18-h incubated colonies the day after blood culture
positivity detection.
Considering the satisfactory identification results and the
potentially favorable clinical impact on patientmanagement, a
routine-applicable positive blood culture work scheme integrating
MALDI-TOF MS identification on young positive
blood subcultures was implemented as presented in Fig. 1.
Three time frames were defined according to the time of day
during which growth-positive blood cultures were detected by
the automated culture system. MALDI-TOF MS analysis on
short subcultures was applied at 5 pm for all bottles detected
positive between 0 am and 12 am, thereby allowing the report
of the results to clinicians at 5.30 pm. A direct MALDI-TOF
MS identification (Sepsityper, Bruker Daltonik, Bremen, Germany)
was executed for positive-detected blood cultures between
12 am and 5 pm. This commercial method had been
previously validated in our university hospital, allowing
65.3%correct identifications (data not shown). Positive blood
culture bottles detected between 5 pm and 0 am were
subcultured but only identified on the following day according
to the standard MALDI-TOF MS identification process. During
weekends, the short subculture MALDI-TOF MS identification
was applied once daily at 2 pm, allowing results
reporting of all blood cultures detected positive until 9 am.
Gram staining was systematically performed on all positivedetected
blood bottles and immediately communicated to the
clinicians between 9 am and 00 am every day of the week.
We believe that a major strength of this algorithm is the
gain in hands-on time and cost if compared with systematic
direct MALDI-TOF MS analysis while preserving the gain
in time in positive blood culture identification result
reporting. On weekdays, the results were systematically
communicated by phone at 5.30 pm to the infectious diseases
physicians team, potentially allowing faster antimicrobial
treatment modifications, as previously demonstrated
by several authors [16, 17].
Time frame of growth detecon Day 0 in blood culture by automated culture system
0 AM 12 AM
MALDI-TOF MS ID
on young subculture*
Direct MALDI-TOF MS ID No Rapid ID tesng
ID result with
log score = 1.7
ID result with
log score

Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture

Order Description
article review.
ARTICLE
Reducing time to identification of positive blood cultures
with MALDI-TOF MS analysis after a 5-h subculture
A. Verroken & L. Defourny & L. Lechgar & A. Magnette &
M. Delmée & Y. Glupczynski
Received: 15 July 2014 /Accepted: 31 August 2014 /Published online: 25 September 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Speeding up the turn-around time of positive blood
culture identifications is essential in order to optimize the
treatment of septic patients. Several sample preparation techniques
have been developed allowing direct matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) identification of positive blood cultures.
Yet, the hands-on time restrains their routine workflow. In this
study, we evaluated an approach whereby MALDI-TOF MS
identification without any additional steps was carried out on
short subcultured colonies from positive blood bottles with the
objective of allowing results reporting on the day of positivity
detection. Over a 7-month period in 2012, positive blood
cultures detected by 9 am with an automated system were
inoculated onto a Columbia blood agar and processed after a
5-h incubation on a MALDI-TOF MicroFlex platform
(Bruker Daltonik GmbH). Single-spotted colonies were coveredwith
1 µl formic acid and 1 µlmatrix solution. The results
were compared to the validated identification techniques. A
total of 925 positive blood culture bottles (representing 470
bacteremic episodes) were included. Concordant identification
was obtained in 727 (81.1 %) of the 896 monomicrobial
blood cultures, with failure being mostly observed with anaerobes
and yeasts. In 17 episodes of polymicrobic bacteremia,
the identification of one of the two isolates was achieved
in 24/29 (82.7 %) positive cultures. Routine implementation
of MALDI-TOF MS identification on young positive blood
subcultures provides correct results to the clinician in more
than 80 % of the bacteremic episodes and allows access to
identification results on the day of blood culture positivity
detection, potentially accelerating the implementation of
targeted clinical treatments.
Introduction
Sepsis is a frequent and severe infection, requiring early,
appropriate, and targeted antibiotic treatment to reduce the
patient’s morbidity and mortality. Speeding up the turnaround
time of positive blood culture identification results
becoming available to the clinician is, therefore, of major
importance [1–3].
Various rapid molecular techniques have been developed in
order to allow the identification of pathogens growing from
blood cultures within 2 h, but also for the direct detection of
pathogens in blood samples without any requirement of culture
[4, 5]. Associated with a high rule-in diagnostic value but
a suboptimal sensitivity, polymerase chain reaction (PCR)-
based pathogen detection is, at this time, only recommended
as an addition to conventional culture techniques [5]. Their
availability remains, furthermore, restricted to a limited number
of laboratories, as they are very costly as well as labor and
time demanding.
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) has emerged as a
new technology for species identification analyzing the protein
composition of a bacterial cell. Through the improvement
of the technique,MALDI-TOF MS has proved over the recent
This work was presented, in part, at the 113th General Meeting of the
American Society for Microbiology (ASM), Denver, CO, May 2013.
A. Verroken (*) : M. Delmée
Institut de recherche expérimentale et clinique (IREC), pôle de
microbiologie (MBLG), Université catholique de Louvain, Brussels,
Belgium
e-mail: [email protected]
A. Verroken : L. Defourny : L. Lechgar : A. Magnette :M. Delmée
Laboratoire de microbiologie, Cliniques universitaires
Saint-Luc—Université catholique de Louvain, Brussels, Belgium
Y. Glupczynski
National Reference Centre for Monitoring of Antimicrobial
Resistance in Gram-negative bacteria, CHU Dinant Godinne | UCL
Namur, Yvoir, Belgium
Eur J Clin Microbiol Infect Dis (2015) 34:405–413
DOI 10.1007/s10096-014-2242-4
years to be a rapid, accurate, easy-to-use, and inexpensive
universal method for the identification of microorganisms
[6]. Subsequently, various purification and extractionmethods
have been developed with MALDI-TOF MS for the direct
identification of positive blood cultures, allowing the
reporting of species results within 1 h after the detection of
blood culture positivity [7–15]. However, directMALDI-TOF
MS identification protocols include several washing and extraction
steps, requiring additional hands-on time. When considering
their workflow implementation, most authors process
the positive blood culture specimens in batches, thereby reducing
the major time gain advantage on the reporting of the
identification results [9, 10, 16, 17].
In this study, we validated an identification process
consisting of the MALDI-TOF MS analysis of positive
blood subcultures after a shortened 5-h incubation without
any preparation steps. This process required much reduced
hands-on time, while also allowing results reporting on the
same day of blood culture positivity detection. In a second
step, a work scheme integrating this process into the identification
of positive blood cultures in daily routine practice
was defined.
Materials and methods
Positive blood cultures
The study was conducted at the Cliniques universitaires
Saint-Luc (UCL), Brussels, Belgium, a 964-bed tertiary
hospital. Positivity of all patients’ blood culture bottles
(BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Peds
Plus/F Medium, Becton Dickinson, Franklin Lakes, NJ,
USA) was detected with the BACTEC FX automated system
(Becton Dickinson, Franklin Lakes, NJ, USA). Specific
blood culture bottles for the recovery of yeast were
not used in our hospital.
During a 7-month period in 2012, all overnight and
early morning weekdays (from 10 pm until 9 am)
positive-detected blood cultures were inoculated by 9 am
on a Columbia agar plate with 5 % sheep blood (COL;
Becton Dickinson, Franklin Lakes, NJ, USA). Anaerobic
positive blood culture bottles were inoculated on a Brucella
agar plate with 5 % sheep blood (BRU; Becton Dickinson,
Franklin Lakes, NJ, USA). COL and BRU agar plates
were incubated at 37 °C in a 5 % supplemented CO2
atmosphere and in an anaerobic atmosphere, respectively.
Blood cultures detected positive during periods outside this
time frame were not included in the study and were processed
according to our standard routine identification procedures
only.
The internal ethics committee of the hospital approved the
anonymous use of remaining patient material.
Identification method
At 2 pm, plates were removed from the incubators for
MALDI-TOF MS identification. A thin layer of growing
colonies was scraped from each plate in order to fill
one-third of a 10-µl plastic loop and was single-spotted
on a steel target, overlaid with 1 µl of 100 % formic
acid, and after drying overlaid with 1 µl matrix, a
saturated solution of a-cyano-4-hydroxycinnamic acid
dissolved in a basic organic solvent composed of 50 %
acetonitrile and 2.5 % trifluoroacetic acid. MALDI-TOF
MS measurements were realized on a MicroFlex LT
platform (Bruker Daltonik, Bremen, Germany). Spectra
were recorded in the positive linear method in a mass
range from 2,000 to 20,000 Da, according to the manufacturer’s
settings. The acquired bacterial spectra with
MALDI-TOF MS were analyzed in the MALDI
Biotyper 3.0 software with database version 3.1.2 and
bearing the spectra of 4,111 cellular organisms. Score
results were interpreted according to a defined cut-off of
1.7 for acceptable identification to the species level. A
score <1.7 was considered unreliable for identification.
No threshold for acceptance to the genus level was
defined.
The results were compared with the routine identification
procedure including optochin susceptibility testing
for Streptococcus pneumoniae suspected strains and standard
MALDI-TOF MS identification from overnight culture
colonies (18-h subculture) for other species isolates
[18]. In this procedure, a single colony was directly
plated onto a steel target and overlaid with 1 µl of
matrix. According to the specifications of the manufacturer,
a high log score =2 was required for identification
to the species level and an intermediate log score lying
between <2 and =1.7 for identification to the genus level.
A low score <1.7 was considered unreliable for
identification.
All result discrepancies were resolved by 16S rRNA
gene sequencing according to a previously published method
[19].
Bacteremic episodes
Review of the patients’ medical records allowed the classification
of all included positive blood culture episodes
into true bloodstream infections (bacteremia/septicemia) or
contaminations according to the Centers for Disease Control
and Prevention/National Healthcare Safety Network
(CDC/NHSN) surveillance definitions of specific infection
types [20]. Positive blood culture bottles originating from
the same patient were considered to belong to a single
bacteremic episode when the difference in the sampling
dates was less than 7 days.
406 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
Results
A total of 925 blood culture bottles were collected over the 7-
month study period, comprising 483 aerobic broths, 377 anaerobic
broths, and 65 pediatric broths.
A single microorganism grew in 896 (96.9 %) blood culture
bottles, while the 29 (3.1 %) remaining bottles yielded
growth of two different microorganisms (Table 1).
These 925 positive blood culture bottles corresponded to
347 bloodstream infections and 123 contaminations, and
accounted for 70 % of the total positive blood cultures, excluding
weekends.
Monomicrobial positive blood cultures
Of the 896 monomicrobial positive blood cultures, species
identification could be obtained in 727 cases (81.1 %), including
433/527 (82.2 %) Gram-positive isolates and 292/323
(90.4 %) Gram-negative isolates (Table 2). Among the
Gram-positive bacteria, staphylococci, enterococci, and streptococci
were correctly identified in 351/410 (85.6 %), 45/52
(86.5 %), and 34/52 (65.4 %) positive blood cultures, respectively.
For Gram-negative bacteria, 255/275 (92.7 %) of the
Enterobacteriaceae and 32/34 (94.1 %) of the non-fermenters
were correctly identified. On the other hand, the identification
of 5-h subcultures growing with anaerobes and yeasts led to
poor results, as only 2/10 and 0/36 isolates, respectively, could
be identified.
In 166 positive blood cultures, the causative organisms
remained unidentified by MALDI-TOF MS due either to an
insufficient score for identification proposal (98 isolates) or
because no peaks were detected (68 isolates). Poor growth at
the 5-h subculture accounted for insufficient scores mainly in
non-identified Gram-positive isolates (coagulase-negative
staphylococci, viridans group streptococci, and the group of
other Gram-positive organisms), while the absence of peaks
could be linked to the absence of growth of yeast and anaerobes
after a 5-h subculture (data not shown).
In three cases with discordant identification results compared
to the routine identification procedure, 16S rRNA PCR
confirmed that we had erroneously identified one
Acinetobacter baumannii isolate as Acinetobacter pittii with
a log score of 1.742, while two Streptococcus isolates (Streptococcus
salivarius and Streptococcus peroris) had been
misidentified as S. pneumoniae with log scores of 1.903 and
1.888, respectively.
Polymicrobial positive blood cultures
Rapid MALDI-TOF MS identification of the polymicrobial
positive blood cultures never allowed the concomitant identification
of both isolates from the same 5-h subcultured plate
(Table 3). One of the two isolates was identified to the species
level in 23 of the 29 blood cultures with mixed bacterial
growth. One S. peroris strain was erroneously identified as
S. pneumoniae with a log score of 1.998, while the scores of
the five remaining positive blood cultures were insufficient to
consider the identification result.
Bloodstream infections versus contamination
Among the 453 monomicrobial blood cultures, 333
corresponded to bloodstream infections, while 120 were
deemed to correspond to contaminations. Species identification
could be obtained for 287/333 (86.2 %) bloodstream
infections and for 82/120 (68.3 %) contaminations,
encompassing, respectively, 640/766 (83.6 %) and 87/130
(66.9 %) identified positive blood culture bottles.
Among the 17 mixed bacterial growths, ten
polymicrobial bloodstream infections, three polymicrobial
contaminations, and four monomicrobial bloodstream infections
associated with a contaminating strain were defined.
In two polymicrobial episodes, both strains were
identified from distinct subcultured blood bottles of the
same episode. In the first episode, Staphylococcus aureus
was identified from three bottles and Staphylococcus
Table 1 Performance of the rapid
(5-h) matrix-assisted laser desorption/
ionization time-of-flight
mass spectrometry (MALDI-TOF
MS) identification process among
monomicrobial and
polymicrobial bacteremic
episodes
No. of
episodes
No. (%) of identified
episodes with rapid
MALDI-TOF MS
No. of positive
blood culture
isolates
No. (%) of identified
isolates with rapid
MALDI-TOF MS
Monomicrobial, total 453 369 (81.5) 896 727 (81.1)
Bacteremia 333 287 (86.2) 766 640 (83.6)
Contamination 120 82 (68.3) 130 87 (66.9)
Polymicrobial, total 17 0 29 0
Bacteremia 10 0 22 0
Bacteremia + contamination 4 0 4 0
Contamination 3 0 3 0
Total 470 369 (79) 925 727 (78.6)
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 407
epidermidis from a fourth bottle. Similarly, in the second
episode, Staphylococcus aureus was identified from the
first bottle and Proteus mirabilis from the second bottle.
In 11 of the 17 mixed bacterial episodes, rapid MALDITOF
MS identified only one of the two organisms. Nevertheless,
the concomitant presence of two different isolates
could be anticipated by Gram staining at the time of blood
culture positivity in 6 out of 17 episodes.
Table 2 Identification results of monomicrobial positive blood cultures with the rapid MALDI-TOF MS process
Final identification of monomicrobial
positive blood cultures with Grampositive
bacteria
n Correct species
identification
Final identification of monomicrobial
positive blood cultures with Gramnegative
bacteria, anaerobes, or yeast
n Correct species
identification
n % n % n %
Gram-positive bacteria 527 433 82.2 Gram-negative bacteria 323 292 90.4
Staphylococci 410 351 85.6 Enterobacteriaceae 275 255 92.7
Staphylococcus aureus 179 171 95.5 Citrobacter braakii 1 0 0.0
Staphylococcus capitis 21 16 76.2 Enterobacter aerogenes 10 10 100.0
Staphylococcus cohnii 3 0 0.0 Enterobacter cloacae 17 15 88.2
Staphylococcus epidermidis 161 132 82.0 Escherichia coli 166 156 94.0
Staphylococcus haemolyticus 11 8 72.7 Hafnia alvei 7 7 100.0
Staphylococcus hominis 28 21 75.0 Klebsiella oxytoca 16 16 100.0
Staphylococcus lugdunensis 1 0 0.0 Klebsiella pneumoniae 45 39 86.7
Staphylococcus pettenkoferi 4 2 50.0 Morganella morganii 1 1 100.0
Staphylococcus sciuri 1 1 100.0 Proteus mirabilis 5 5 100.0
Staphylococcus warneri 1 0 0.0 Salmonella sp. 2 2 100.0
Enterococci 52 45 86.5 Serratia marcescens 3 3 100.0
Enterococcus avium 1 1 100.0 Serratia rubidaea 2 1 50.0
Enterococcus faecalis 21 17 81.0 Non-fermenters 34 32 94.1
Enterococcus faecium 30 27 90.0 Acinetobacter baumannii 5 4 80.0
Streptococci 52 34 65.4 Acinetobacter pittii (Acinetobacter
genomospecies 3)
3 3 100.0
Pyogenic group 10 8 80.0 Acinetobacter lwoffii 2 2 100.0
Streptococcus pyogenes 4 2 50.0 Pseudomonas aeruginosa 23 22 95.7
Streptococcus agalactiae 3 3 100.0 Stenotrophomonas maltophilia 1 1 100.0
Streptococcus dysgalactiae 3 3 100.0 Other Gram-negative organisms 14 5 35.7
Viridans group 41 26 63.4 Capnocytophaga sputigena 1 0 0.0
S. anginosus group 3 1 33.3 Haemophilus influenzae 4 1 25.0
S. bovis group 6 2 33.3 Moraxella catarrhalis 1 1 100.0
S. mitis group 23 20 87.0 Moraxella lacunata 3 1 33.3
S. salivarius group 2 0 0.0 Moraxella osloensis 2 0 0.0
S. sanguinis group 7 3 42.9 Neisseria meningitidis 3 2 66.7
Other streptococci 1 0 0.0 Anaerobes 10 2 20.0
Granulicatella adiacens 1 0 0.0 Actinomyces sp. 2 1 50.0
Other Gram-positive organisms 13 3 23.1 Bacteroides fragilis 2 0 0.0
Aerococcus urinae 1 0 0.0 Clostridium perfringens 1 1 100.0
Bacillus cereus 1 1 100.0 Leptotrichia sp. 1 0 0.0
Corynebacterium aurimucosum 1 0 0.0 Propionibacterium acnes 4 0 0.0
Corynebacterium durum 1 0 0.0 Yeasts 36 0 0.0
Corynebacterium jeikeium 1 0 0.0 Candida albicans 17 0 0.0
Gordonia sputi 3 0 0.0 Candida dubliniensis 2 0 0.0
Micrococcus luteus 3 1 33.3 Candida glabrata 1 0 0.0
Rothia aeria 1 0 0.0 Candida tropicalis 10 0 0.0
Rothia mucilaginosa 1 1 100.0 Fusarium spp. 5 0 0.0
Trichosporon inkin 1 0 0.0
408 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
Discussion
We evaluated here a practical approach for the rapid identification
of microorganisms growing from positive blood cultures in
daily routine clinical practice. Numerous studies have already
assessed the performance of MALDI-TOF MS procedures for
rapid microorganism identification when directly applied on
culture-positive blood specimens. Accurate species identification
rates were found to vary between 50.5 % and 91 %,
depending both on the distribution of microbial isolates and
on the applied pretreatment/extraction methods, as well as on
the definitions of the cut-off threshold log scores [7, 9–15].
However, direct bacterial identification by MALDI-TOF MS
from positive blood cultures is time- and labor-intensive, since
it requires at least 30 min hands-on time for the washing,
centrifugation, and extraction steps that are necessary to discard
blood cells and reveal the bacterial proteins. Despite the possibility
of obtaining, in theory, a result within 60 min from the
time a positive blood culture is detected, the proposed workflow
is difficult to integrate in the routine workflow of a clinical
microbiology laboratory. Hence, directMALDI-TOFMS identification
of positive blood cultures is most usually realized in
batches, for instance, every 2 h, as suggested by Loonen et al.,
or twice a day, according to Martiny et al., thereby extending
the time to obtaining identification results [9, 10].
In this study, we investigated an identification procedure
not requiring any additional time- or labor-consuming sample
preparation steps and leading to identification results available
to the clinician within the same day as blood culture positivity.
Our MALDI-TOF MS processing algorithm after a 5-h subculture
frompositive blood bottles with formic acid overlay as
the only preparation step could be considered as an intermediate
method between the direct MALDI-TOF MS identification
process and the “next-day” MALDI-TOF MS identification
from an 18-h subculture. McElvania TeKippe et al. previously
evaluated the formic acid overlay process for the
MALDI-TOF MS identification of Gram-positive cultured
organisms and showed a significant improvement of genusand
species-level identification (by 20 %) and higher scores
compared to the direct smear deposit [21]. Ford and Burnham
similarly demonstrated the added value of the formic acid
overlay versus the direct smear method for the identification
of Gram-negative bacterial colonies by the reduction of unidentified
organisms [22].
Table 3 Rapid (5-h)MALDI-TOF MS identification results of the 17 polymicrobial bacteremic episodes. Clinically relevant pathogens are reported in
bold
Episode Gram staining lecture Rapid MALDI-TOF MS identification results Final identification results
Identified/
total BCB
Identified strain(s)
Polymicrobial bloodstream infection
1 GPC 3/6 and 1/6 Staphylococcus aureus/
Staphylococcus epidermidis
Staphylococcus aureus + Staphylococcus epidermidis
2 GPC in chains + GNB 2/2 Escherichia coli Enterococcus gallinarum + Escherichia coli
3 GPC in chains + GNB 2/2 Escherichia coli Enterococcus faecium + Escherichia coli
4 GPC in clusters 2/2 Enterococcus faecalis Enterococcus faecalis + Enterococcus faecium
5 GPC in clusters 2/2 Enterococcus faecalis Enterococcus faecalis + Acinetobacter baumannii
6 GPC in clusters 2/2 Staphylococcus aureus Staphylococcus aureus + Staphylococcus epidermidis
7 GPC in clusters + GNB 2/2 Escherichia coli Streptococcus pyogenes + Escherichia coli
8 GPC in clusters + GNB 1/2 and 1/2 Staphylococcus aureus/
Proteus mirabilis
Staphylococcus aureus + Proteus mirabilis
9 GNB 1/1 Escherichia coli Escherichia coli + Proteus mirabilis
10 GNB 1/1 Klebsiella oxytoca Escherichia coli + Klebsiella oxytoca
Monomicrobial bloodstream infection + contamination
11 GPC in chains 0/1 – Staphylococcus epidermidis + Enterococcus faecalis
12 GPC in chains 1/1 Streptococcus agalactiae Staphylococcus epidermidis + Streptococcus agalactiae
13 GPC in chains + clusters 1/1 Streptococcus oralis Staphylococcus aureus + Streptococcus oralis
14 GPC in clusters 0/1 – Staphylococcus epidermidis + Escherichia coli
Polymicrobial contamination
15 GPC 1/1 Staphylococcus hominis Staphylococcus hominis + Staphylococcus capitis
16 GPC in chains + clusters 0/1 Streptococcus pneumoniae Streptococcus peroris + Staphylococcus capitis
17 GPC in clusters 0/1 – Staphylococcus epidermidis + Aerococcus urinae
BCB blood culture bottles; GNB Gram-negative bacilli; GPC Gram-positive cocci
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 409
In our evaluation, the processing time of subcultures was
set at 5 h after preliminary MALDI-TOF MS identification.
Experiences following 3 and 4 h of incubation were found to
be associated with very poor identification results for Grampositive
isolates and only moderate results for Gram-negative
strains (data not shown). Idelevich et al. similarly evaluated
rapid MALDI-TOF MS identification of microorganisms
from positive blood cultures subsequent to a 1.5-, 2-, 3-, 4-,
5-, 6-, 7-, 8-, 12-, and 24-h incubation on solid medium [23].
The mean incubation time needed to achieve species-level
identificationwas 5.9 and 2 h forGram-positive aerobic cocci
(n=86) and Gram-negative aerobic rods (n=42), respectively.
For monomicrobial positive blood cultures, species identification
results could be achieved for 81.1 % of the isolates,
which can be considered as a very satisfactory result when
compared to other recent studies using direct MALDI-TOF
MS, in which correct identification ranged between 64.8 and
81.8%[11, 14, 15]. In line with these authors, we also noticed
a higher identification percentage for Gram-negative organisms
(90.4 %) compared to Gram-positive organisms
(82.2 %). As defined in the Materials and methods section,
identification results were accepted to the species according to
a cut-off score =1.7. Using a cut-off score =2 for species
identification allowed correct identification results for
69.6 % of the monomicrobial positive blood cultures and
identified one of the two strains in 21 of the 29 polymicrobial
positive blood cultures. No isolates were erroneously identified.
A less stringent cut-off score =1.5 for species identification
allowed results for 84 % of the monomicrobial positive
blood cultures and identified one of the two strains in 24 of the
29 polymicrobial positive blood cultures. Three isolates were
erroneously identified, as observed with the cut-off score at
1.7. The final choice to set the cut-off at 1.7 in our study was
taken in accordance with the abundant publications using this
scoring system when directMALDI-TOF MS identification is
applied [9, 17, 24].
One drawback of our rapid MALDI-TOF MS process was
that it failed to yield correct identification results for yeast and
anaerobes most probably related to the insufficient growth of
these microorganisms on the agar plates after a 5-h subculture.
Pondering the high rates of morbidity and mortality as well as
the growing incidence of candidemia and anaerobic septicemia,
a prompt identification result is essential [25, 26]. Hence,
to overcome this flaw in our process, direct analysis from
positive blood culture samples by MALDI-TOF MS should
be considered systematically when Gram staining suggests the
presence of yeast or anaerobes. This procedure preceded by
defined blood lysing protocols using sodium dodecyl sulfate
detergent or Tween 80 and formic acid extraction respectively
enabled Pulcrano et al. [27] to identify 19/21 Candida nonalbicans
bloodstream infections and Leli et al. to identify 7/7
anaerobic septicemia [15. The erroneous identification of two
streptococci (S. salivarius and S. peroris) as S. pneumoniae in
our study confirmed the inability of MALDI-TOF MS to
distinguish oral streptococci strains from S. pneumoniae [18,
28, 29]. On the basis of these observations, we decided not to
consider any S. pneumoniae result through our rapidMALDITOF
MS identification process. The third erroneously identified
strain was an A. baumannii isolate that was misidentified
to the species level as Acinetobacter pittii (formerly
Acinetobacter genomospecies 3). In the MALDI Biotyper
3.0 software, Acinetobacter species identification results are
accompanied with a comment informing about the close relatedness
of several species and the difficulty in differentiating
them.
Themain weakness of ourMALDI-TOFMS protocol was
its inability to identify all organisms in the setting of
polymicrobial bloodstream infections. Various studies also
underlined the lack of ability of MALDI-TOF MS to detect
all microorganisms in mixed cultures through direct identification,
as none or, at best, one single isolate could be identified
[10–12, 15]. Ferroni et al. managed the identification of blood
cultures containing mixed bacteria through the use of Gramspecific
databases selected according to the obtained Gram
result [7]. In our setting, Gram staining of all positive blood
culture bottles and rapidMALDI-TOFMS identification of all
subcultured isolates included in the polymicrobial episodes
were essential elements that partially overcame this limitation
of our algorithm. Regarding the possibility of detecting
polymicrobial bacteremia, visualization of all plated blood
subcultures was systematically repeated the day following
positivity considering that the presence of more than one
organism could go undetected on young subcultures.
The ultimate objective of this study was to speed up the
identification process for improving the management of the
patient and to assist the clinician in deciding whether the
growing microorganisms were to be considered as clinically
relevant and associated with a bloodstreaminfection or, rather,
whether they should be considered as contaminants. Overall,
86.2% of the monomicrobial bloodstream infections could be
identified, thereby potentially allowing an earlier diagnosis
and adaptation of therapy to the documented pathogens. In
parallel, 68.3 % of all organisms regarded as clinically nonsignificant
contaminants could be identified and reported on
the same day of blood culture positivity, possibly leading to
restriction and/or earlier stop of antimicrobial therapy.Martiny
et al. measured the clinical impact of rapid microbial identification
(direct MALDI-TOF MS preceded by an in-house
purification protocol) on the management of septic patients.
An accelerated modification of the treatment regimen was
observed in 13.4 % and 2.5 % of the adult and pediatric
patients, respectively. In other cases, the tool was helpful to
rapidly confirm suspected cases of contamination, thereby
avoiding the administration of unnecessary antibiotics [16].
Vlek et al., likewise, observed an 11.3 % increase in the
proportion of patients receiving appropriate antibiotic
410 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
treatment 24 h after blood culture positivity with direct
MALDI-TOF MS performed twice a day [17].
These results emphasize the benefit of the rapid identification
of positive blood cultures compared toMALDI-TOF MS
analysis on 18-h incubated colonies the day after blood culture
positivity detection.
Considering the satisfactory identification results and the
potentially favorable clinical impact on patientmanagement, a
routine-applicable positive blood culture work scheme integrating
MALDI-TOF MS identification on young positive
blood subcultures was implemented as presented in Fig. 1.
Three time frames were defined according to the time of day
during which growth-positive blood cultures were detected by
the automated culture system. MALDI-TOF MS analysis on
short subcultures was applied at 5 pm for all bottles detected
positive between 0 am and 12 am, thereby allowing the report
of the results to clinicians at 5.30 pm. A direct MALDI-TOF
MS identification (Sepsityper, Bruker Daltonik, Bremen, Germany)
was executed for positive-detected blood cultures between
12 am and 5 pm. This commercial method had been
previously validated in our university hospital, allowing
65.3%correct identifications (data not shown). Positive blood
culture bottles detected between 5 pm and 0 am were
subcultured but only identified on the following day according
to the standard MALDI-TOF MS identification process. During
weekends, the short subculture MALDI-TOF MS identification
was applied once daily at 2 pm, allowing results
reporting of all blood cultures detected positive until 9 am.
Gram staining was systematically performed on all positivedetected
blood bottles and immediately communicated to the
clinicians between 9 am and 00 am every day of the week.
We believe that a major strength of this algorithm is the
gain in hands-on time and cost if compared with systematic
direct MALDI-TOF MS analysis while preserving the gain
in time in positive blood culture identification result
reporting. On weekdays, the results were systematically
communicated by phone at 5.30 pm to the infectious diseases
physicians team, potentially allowing faster antimicrobial
treatment modifications, as previously demonstrated
by several authors [16, 17].
Time frame of growth detecon Day 0 in blood culture by automated culture system
0 AM 12 AM
MALDI-TOF MS ID
on young subculture*
Direct MALDI-TOF MS ID No Rapid ID tesng
ID result with
log score = 1.7
ID result with
log score < 1.7
Streptococcus pneumoniae
ID result
Day 0
5 PM
Day 0
5.30 PM
ID result
reporting
No ID result reporng
Optochin tesng
Day 1
9 AM
MALDI-TOF MS ID
on subculture
ID result
reporting
MALDI-TOF MS ID
on subculture
ID result
reporting
ID result
reporting
12 AM 5 PM 5 PM 0 AM
No ID result
reporng
Optochin reading
Day 1
9.30 AM
BC plang:
8 AM 12 AM
BC plang:
12 AM 5 PM
BC plang:
5 PM 0 AM
Fig. 1 Modified weekday routine workflow scheme for the identification
of blood cultures in accordance with the time of positivity detection by
theautomated incubation system. *Direct MALDI-TOF MS ID when
Gram staining suggestive of yeast or anaerobes. BC blood culture; ID
identification; MALDI-TOF MS matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 411
The large amount of tested isolates, thereby representative
of the routine positive blood culture microorganism proportions
in a tertiary hospital, enabled us to validate the applied
process. All during the study period, testing was carried out by
various technologists and medical junior residents, thereby
highlighting the robustness and reproducibility of the method
in clinical routine practice.
In conclusion, the integration of MALDI-TOF MS identification
on 5-h subcultured colonies in the laboratory
workflow represents an excellent compromise between the
direct blood culture process associated with labor-intensive
steps and the direct smear method of 18-h subcultured colonies,
as it leads to the reporting of correct identification results
on the day of positivity in more than 80 % of the
monomicrobial bacteremic episodes. An ongoing challenge
is the development of rapid tests for the detection of clinically
important resistance mechanisms, since we should keep in
mind that the identification results alone only give partial
microbiological information to the clinicians. Hence, the impact
on the patient’s clinical management of the rapid positive
blood culture identification result “alone” may also be very
dependent on the local epidemiology of bacterial resistance.
Indeed, the increasing trends of resistance of Gram-negative
bacteria to third-generation cephalosporins and to carbapenems,
as well as the high rates of methicillin-resistant Staphylococcus
aureus across Europe, remind us that clinicians can
no longer simply rely on the wild susceptibility profile of the
identified bacteria for therapeutic decision-making [30]. A
study is actually ongoing in our hospital to assess whether
the combination of rapid MALDI-TOF MS identification
associated with rapid detection of resistance mechanisms to
selected antimicrobial agents may favorably impact on antimicrobial
therapy among septic patients with positive blood
cultures.
Acknowledgments This study was supported, in part, by a research
grant from Fondation Saint-Luc, Cliniques universitaires Saint-Luc,
UCL, Bruxelles.
Conflict of interest The authors declare that they have no conflict of
interest.
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Eur J Clin Microbiol Infect Dis (2015) 34:405–413 413

Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture

Order Description
article review.
ARTICLE
Reducing time to identification of positive blood cultures
with MALDI-TOF MS analysis after a 5-h subculture
A. Verroken & L. Defourny & L. Lechgar & A. Magnette &
M. Delmée & Y. Glupczynski
Received: 15 July 2014 /Accepted: 31 August 2014 /Published online: 25 September 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Speeding up the turn-around time of positive blood
culture identifications is essential in order to optimize the
treatment of septic patients. Several sample preparation techniques
have been developed allowing direct matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) identification of positive blood cultures.
Yet, the hands-on time restrains their routine workflow. In this
study, we evaluated an approach whereby MALDI-TOF MS
identification without any additional steps was carried out on
short subcultured colonies from positive blood bottles with the
objective of allowing results reporting on the day of positivity
detection. Over a 7-month period in 2012, positive blood
cultures detected by 9 am with an automated system were
inoculated onto a Columbia blood agar and processed after a
5-h incubation on a MALDI-TOF MicroFlex platform
(Bruker Daltonik GmbH). Single-spotted colonies were coveredwith
1 µl formic acid and 1 µlmatrix solution. The results
were compared to the validated identification techniques. A
total of 925 positive blood culture bottles (representing 470
bacteremic episodes) were included. Concordant identification
was obtained in 727 (81.1 %) of the 896 monomicrobial
blood cultures, with failure being mostly observed with anaerobes
and yeasts. In 17 episodes of polymicrobic bacteremia,
the identification of one of the two isolates was achieved
in 24/29 (82.7 %) positive cultures. Routine implementation
of MALDI-TOF MS identification on young positive blood
subcultures provides correct results to the clinician in more
than 80 % of the bacteremic episodes and allows access to
identification results on the day of blood culture positivity
detection, potentially accelerating the implementation of
targeted clinical treatments.
Introduction
Sepsis is a frequent and severe infection, requiring early,
appropriate, and targeted antibiotic treatment to reduce the
patient’s morbidity and mortality. Speeding up the turnaround
time of positive blood culture identification results
becoming available to the clinician is, therefore, of major
importance [1–3].
Various rapid molecular techniques have been developed in
order to allow the identification of pathogens growing from
blood cultures within 2 h, but also for the direct detection of
pathogens in blood samples without any requirement of culture
[4, 5]. Associated with a high rule-in diagnostic value but
a suboptimal sensitivity, polymerase chain reaction (PCR)-
based pathogen detection is, at this time, only recommended
as an addition to conventional culture techniques [5]. Their
availability remains, furthermore, restricted to a limited number
of laboratories, as they are very costly as well as labor and
time demanding.
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) has emerged as a
new technology for species identification analyzing the protein
composition of a bacterial cell. Through the improvement
of the technique,MALDI-TOF MS has proved over the recent
This work was presented, in part, at the 113th General Meeting of the
American Society for Microbiology (ASM), Denver, CO, May 2013.
A. Verroken (*) : M. Delmée
Institut de recherche expérimentale et clinique (IREC), pôle de
microbiologie (MBLG), Université catholique de Louvain, Brussels,
Belgium
e-mail: [email protected]
A. Verroken : L. Defourny : L. Lechgar : A. Magnette :M. Delmée
Laboratoire de microbiologie, Cliniques universitaires
Saint-Luc—Université catholique de Louvain, Brussels, Belgium
Y. Glupczynski
National Reference Centre for Monitoring of Antimicrobial
Resistance in Gram-negative bacteria, CHU Dinant Godinne | UCL
Namur, Yvoir, Belgium
Eur J Clin Microbiol Infect Dis (2015) 34:405–413
DOI 10.1007/s10096-014-2242-4
years to be a rapid, accurate, easy-to-use, and inexpensive
universal method for the identification of microorganisms
[6]. Subsequently, various purification and extractionmethods
have been developed with MALDI-TOF MS for the direct
identification of positive blood cultures, allowing the
reporting of species results within 1 h after the detection of
blood culture positivity [7–15]. However, directMALDI-TOF
MS identification protocols include several washing and extraction
steps, requiring additional hands-on time. When considering
their workflow implementation, most authors process
the positive blood culture specimens in batches, thereby reducing
the major time gain advantage on the reporting of the
identification results [9, 10, 16, 17].
In this study, we validated an identification process
consisting of the MALDI-TOF MS analysis of positive
blood subcultures after a shortened 5-h incubation without
any preparation steps. This process required much reduced
hands-on time, while also allowing results reporting on the
same day of blood culture positivity detection. In a second
step, a work scheme integrating this process into the identification
of positive blood cultures in daily routine practice
was defined.
Materials and methods
Positive blood cultures
The study was conducted at the Cliniques universitaires
Saint-Luc (UCL), Brussels, Belgium, a 964-bed tertiary
hospital. Positivity of all patients’ blood culture bottles
(BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Peds
Plus/F Medium, Becton Dickinson, Franklin Lakes, NJ,
USA) was detected with the BACTEC FX automated system
(Becton Dickinson, Franklin Lakes, NJ, USA). Specific
blood culture bottles for the recovery of yeast were
not used in our hospital.
During a 7-month period in 2012, all overnight and
early morning weekdays (from 10 pm until 9 am)
positive-detected blood cultures were inoculated by 9 am
on a Columbia agar plate with 5 % sheep blood (COL;
Becton Dickinson, Franklin Lakes, NJ, USA). Anaerobic
positive blood culture bottles were inoculated on a Brucella
agar plate with 5 % sheep blood (BRU; Becton Dickinson,
Franklin Lakes, NJ, USA). COL and BRU agar plates
were incubated at 37 °C in a 5 % supplemented CO2
atmosphere and in an anaerobic atmosphere, respectively.
Blood cultures detected positive during periods outside this
time frame were not included in the study and were processed
according to our standard routine identification procedures
only.
The internal ethics committee of the hospital approved the
anonymous use of remaining patient material.
Identification method
At 2 pm, plates were removed from the incubators for
MALDI-TOF MS identification. A thin layer of growing
colonies was scraped from each plate in order to fill
one-third of a 10-µl plastic loop and was single-spotted
on a steel target, overlaid with 1 µl of 100 % formic
acid, and after drying overlaid with 1 µl matrix, a
saturated solution of a-cyano-4-hydroxycinnamic acid
dissolved in a basic organic solvent composed of 50 %
acetonitrile and 2.5 % trifluoroacetic acid. MALDI-TOF
MS measurements were realized on a MicroFlex LT
platform (Bruker Daltonik, Bremen, Germany). Spectra
were recorded in the positive linear method in a mass
range from 2,000 to 20,000 Da, according to the manufacturer’s
settings. The acquired bacterial spectra with
MALDI-TOF MS were analyzed in the MALDI
Biotyper 3.0 software with database version 3.1.2 and
bearing the spectra of 4,111 cellular organisms. Score
results were interpreted according to a defined cut-off of
1.7 for acceptable identification to the species level. A
score <1.7 was considered unreliable for identification.
No threshold for acceptance to the genus level was
defined.
The results were compared with the routine identification
procedure including optochin susceptibility testing
for Streptococcus pneumoniae suspected strains and standard
MALDI-TOF MS identification from overnight culture
colonies (18-h subculture) for other species isolates
[18]. In this procedure, a single colony was directly
plated onto a steel target and overlaid with 1 µl of
matrix. According to the specifications of the manufacturer,
a high log score =2 was required for identification
to the species level and an intermediate log score lying
between <2 and =1.7 for identification to the genus level.
A low score <1.7 was considered unreliable for
identification.
All result discrepancies were resolved by 16S rRNA
gene sequencing according to a previously published method
[19].
Bacteremic episodes
Review of the patients’ medical records allowed the classification
of all included positive blood culture episodes
into true bloodstream infections (bacteremia/septicemia) or
contaminations according to the Centers for Disease Control
and Prevention/National Healthcare Safety Network
(CDC/NHSN) surveillance definitions of specific infection
types [20]. Positive blood culture bottles originating from
the same patient were considered to belong to a single
bacteremic episode when the difference in the sampling
dates was less than 7 days.
406 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
Results
A total of 925 blood culture bottles were collected over the 7-
month study period, comprising 483 aerobic broths, 377 anaerobic
broths, and 65 pediatric broths.
A single microorganism grew in 896 (96.9 %) blood culture
bottles, while the 29 (3.1 %) remaining bottles yielded
growth of two different microorganisms (Table 1).
These 925 positive blood culture bottles corresponded to
347 bloodstream infections and 123 contaminations, and
accounted for 70 % of the total positive blood cultures, excluding
weekends.
Monomicrobial positive blood cultures
Of the 896 monomicrobial positive blood cultures, species
identification could be obtained in 727 cases (81.1 %), including
433/527 (82.2 %) Gram-positive isolates and 292/323
(90.4 %) Gram-negative isolates (Table 2). Among the
Gram-positive bacteria, staphylococci, enterococci, and streptococci
were correctly identified in 351/410 (85.6 %), 45/52
(86.5 %), and 34/52 (65.4 %) positive blood cultures, respectively.
For Gram-negative bacteria, 255/275 (92.7 %) of the
Enterobacteriaceae and 32/34 (94.1 %) of the non-fermenters
were correctly identified. On the other hand, the identification
of 5-h subcultures growing with anaerobes and yeasts led to
poor results, as only 2/10 and 0/36 isolates, respectively, could
be identified.
In 166 positive blood cultures, the causative organisms
remained unidentified by MALDI-TOF MS due either to an
insufficient score for identification proposal (98 isolates) or
because no peaks were detected (68 isolates). Poor growth at
the 5-h subculture accounted for insufficient scores mainly in
non-identified Gram-positive isolates (coagulase-negative
staphylococci, viridans group streptococci, and the group of
other Gram-positive organisms), while the absence of peaks
could be linked to the absence of growth of yeast and anaerobes
after a 5-h subculture (data not shown).
In three cases with discordant identification results compared
to the routine identification procedure, 16S rRNA PCR
confirmed that we had erroneously identified one
Acinetobacter baumannii isolate as Acinetobacter pittii with
a log score of 1.742, while two Streptococcus isolates (Streptococcus
salivarius and Streptococcus peroris) had been
misidentified as S. pneumoniae with log scores of 1.903 and
1.888, respectively.
Polymicrobial positive blood cultures
Rapid MALDI-TOF MS identification of the polymicrobial
positive blood cultures never allowed the concomitant identification
of both isolates from the same 5-h subcultured plate
(Table 3). One of the two isolates was identified to the species
level in 23 of the 29 blood cultures with mixed bacterial
growth. One S. peroris strain was erroneously identified as
S. pneumoniae with a log score of 1.998, while the scores of
the five remaining positive blood cultures were insufficient to
consider the identification result.
Bloodstream infections versus contamination
Among the 453 monomicrobial blood cultures, 333
corresponded to bloodstream infections, while 120 were
deemed to correspond to contaminations. Species identification
could be obtained for 287/333 (86.2 %) bloodstream
infections and for 82/120 (68.3 %) contaminations,
encompassing, respectively, 640/766 (83.6 %) and 87/130
(66.9 %) identified positive blood culture bottles.
Among the 17 mixed bacterial growths, ten
polymicrobial bloodstream infections, three polymicrobial
contaminations, and four monomicrobial bloodstream infections
associated with a contaminating strain were defined.
In two polymicrobial episodes, both strains were
identified from distinct subcultured blood bottles of the
same episode. In the first episode, Staphylococcus aureus
was identified from three bottles and Staphylococcus
Table 1 Performance of the rapid
(5-h) matrix-assisted laser desorption/
ionization time-of-flight
mass spectrometry (MALDI-TOF
MS) identification process among
monomicrobial and
polymicrobial bacteremic
episodes
No. of
episodes
No. (%) of identified
episodes with rapid
MALDI-TOF MS
No. of positive
blood culture
isolates
No. (%) of identified
isolates with rapid
MALDI-TOF MS
Monomicrobial, total 453 369 (81.5) 896 727 (81.1)
Bacteremia 333 287 (86.2) 766 640 (83.6)
Contamination 120 82 (68.3) 130 87 (66.9)
Polymicrobial, total 17 0 29 0
Bacteremia 10 0 22 0
Bacteremia + contamination 4 0 4 0
Contamination 3 0 3 0
Total 470 369 (79) 925 727 (78.6)
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 407
epidermidis from a fourth bottle. Similarly, in the second
episode, Staphylococcus aureus was identified from the
first bottle and Proteus mirabilis from the second bottle.
In 11 of the 17 mixed bacterial episodes, rapid MALDITOF
MS identified only one of the two organisms. Nevertheless,
the concomitant presence of two different isolates
could be anticipated by Gram staining at the time of blood
culture positivity in 6 out of 17 episodes.
Table 2 Identification results of monomicrobial positive blood cultures with the rapid MALDI-TOF MS process
Final identification of monomicrobial
positive blood cultures with Grampositive
bacteria
n Correct species
identification
Final identification of monomicrobial
positive blood cultures with Gramnegative
bacteria, anaerobes, or yeast
n Correct species
identification
n % n % n %
Gram-positive bacteria 527 433 82.2 Gram-negative bacteria 323 292 90.4
Staphylococci 410 351 85.6 Enterobacteriaceae 275 255 92.7
Staphylococcus aureus 179 171 95.5 Citrobacter braakii 1 0 0.0
Staphylococcus capitis 21 16 76.2 Enterobacter aerogenes 10 10 100.0
Staphylococcus cohnii 3 0 0.0 Enterobacter cloacae 17 15 88.2
Staphylococcus epidermidis 161 132 82.0 Escherichia coli 166 156 94.0
Staphylococcus haemolyticus 11 8 72.7 Hafnia alvei 7 7 100.0
Staphylococcus hominis 28 21 75.0 Klebsiella oxytoca 16 16 100.0
Staphylococcus lugdunensis 1 0 0.0 Klebsiella pneumoniae 45 39 86.7
Staphylococcus pettenkoferi 4 2 50.0 Morganella morganii 1 1 100.0
Staphylococcus sciuri 1 1 100.0 Proteus mirabilis 5 5 100.0
Staphylococcus warneri 1 0 0.0 Salmonella sp. 2 2 100.0
Enterococci 52 45 86.5 Serratia marcescens 3 3 100.0
Enterococcus avium 1 1 100.0 Serratia rubidaea 2 1 50.0
Enterococcus faecalis 21 17 81.0 Non-fermenters 34 32 94.1
Enterococcus faecium 30 27 90.0 Acinetobacter baumannii 5 4 80.0
Streptococci 52 34 65.4 Acinetobacter pittii (Acinetobacter
genomospecies 3)
3 3 100.0
Pyogenic group 10 8 80.0 Acinetobacter lwoffii 2 2 100.0
Streptococcus pyogenes 4 2 50.0 Pseudomonas aeruginosa 23 22 95.7
Streptococcus agalactiae 3 3 100.0 Stenotrophomonas maltophilia 1 1 100.0
Streptococcus dysgalactiae 3 3 100.0 Other Gram-negative organisms 14 5 35.7
Viridans group 41 26 63.4 Capnocytophaga sputigena 1 0 0.0
S. anginosus group 3 1 33.3 Haemophilus influenzae 4 1 25.0
S. bovis group 6 2 33.3 Moraxella catarrhalis 1 1 100.0
S. mitis group 23 20 87.0 Moraxella lacunata 3 1 33.3
S. salivarius group 2 0 0.0 Moraxella osloensis 2 0 0.0
S. sanguinis group 7 3 42.9 Neisseria meningitidis 3 2 66.7
Other streptococci 1 0 0.0 Anaerobes 10 2 20.0
Granulicatella adiacens 1 0 0.0 Actinomyces sp. 2 1 50.0
Other Gram-positive organisms 13 3 23.1 Bacteroides fragilis 2 0 0.0
Aerococcus urinae 1 0 0.0 Clostridium perfringens 1 1 100.0
Bacillus cereus 1 1 100.0 Leptotrichia sp. 1 0 0.0
Corynebacterium aurimucosum 1 0 0.0 Propionibacterium acnes 4 0 0.0
Corynebacterium durum 1 0 0.0 Yeasts 36 0 0.0
Corynebacterium jeikeium 1 0 0.0 Candida albicans 17 0 0.0
Gordonia sputi 3 0 0.0 Candida dubliniensis 2 0 0.0
Micrococcus luteus 3 1 33.3 Candida glabrata 1 0 0.0
Rothia aeria 1 0 0.0 Candida tropicalis 10 0 0.0
Rothia mucilaginosa 1 1 100.0 Fusarium spp. 5 0 0.0
Trichosporon inkin 1 0 0.0
408 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
Discussion
We evaluated here a practical approach for the rapid identification
of microorganisms growing from positive blood cultures in
daily routine clinical practice. Numerous studies have already
assessed the performance of MALDI-TOF MS procedures for
rapid microorganism identification when directly applied on
culture-positive blood specimens. Accurate species identification
rates were found to vary between 50.5 % and 91 %,
depending both on the distribution of microbial isolates and
on the applied pretreatment/extraction methods, as well as on
the definitions of the cut-off threshold log scores [7, 9–15].
However, direct bacterial identification by MALDI-TOF MS
from positive blood cultures is time- and labor-intensive, since
it requires at least 30 min hands-on time for the washing,
centrifugation, and extraction steps that are necessary to discard
blood cells and reveal the bacterial proteins. Despite the possibility
of obtaining, in theory, a result within 60 min from the
time a positive blood culture is detected, the proposed workflow
is difficult to integrate in the routine workflow of a clinical
microbiology laboratory. Hence, directMALDI-TOFMS identification
of positive blood cultures is most usually realized in
batches, for instance, every 2 h, as suggested by Loonen et al.,
or twice a day, according to Martiny et al., thereby extending
the time to obtaining identification results [9, 10].
In this study, we investigated an identification procedure
not requiring any additional time- or labor-consuming sample
preparation steps and leading to identification results available
to the clinician within the same day as blood culture positivity.
Our MALDI-TOF MS processing algorithm after a 5-h subculture
frompositive blood bottles with formic acid overlay as
the only preparation step could be considered as an intermediate
method between the direct MALDI-TOF MS identification
process and the “next-day” MALDI-TOF MS identification
from an 18-h subculture. McElvania TeKippe et al. previously
evaluated the formic acid overlay process for the
MALDI-TOF MS identification of Gram-positive cultured
organisms and showed a significant improvement of genusand
species-level identification (by 20 %) and higher scores
compared to the direct smear deposit [21]. Ford and Burnham
similarly demonstrated the added value of the formic acid
overlay versus the direct smear method for the identification
of Gram-negative bacterial colonies by the reduction of unidentified
organisms [22].
Table 3 Rapid (5-h)MALDI-TOF MS identification results of the 17 polymicrobial bacteremic episodes. Clinically relevant pathogens are reported in
bold
Episode Gram staining lecture Rapid MALDI-TOF MS identification results Final identification results
Identified/
total BCB
Identified strain(s)
Polymicrobial bloodstream infection
1 GPC 3/6 and 1/6 Staphylococcus aureus/
Staphylococcus epidermidis
Staphylococcus aureus + Staphylococcus epidermidis
2 GPC in chains + GNB 2/2 Escherichia coli Enterococcus gallinarum + Escherichia coli
3 GPC in chains + GNB 2/2 Escherichia coli Enterococcus faecium + Escherichia coli
4 GPC in clusters 2/2 Enterococcus faecalis Enterococcus faecalis + Enterococcus faecium
5 GPC in clusters 2/2 Enterococcus faecalis Enterococcus faecalis + Acinetobacter baumannii
6 GPC in clusters 2/2 Staphylococcus aureus Staphylococcus aureus + Staphylococcus epidermidis
7 GPC in clusters + GNB 2/2 Escherichia coli Streptococcus pyogenes + Escherichia coli
8 GPC in clusters + GNB 1/2 and 1/2 Staphylococcus aureus/
Proteus mirabilis
Staphylococcus aureus + Proteus mirabilis
9 GNB 1/1 Escherichia coli Escherichia coli + Proteus mirabilis
10 GNB 1/1 Klebsiella oxytoca Escherichia coli + Klebsiella oxytoca
Monomicrobial bloodstream infection + contamination
11 GPC in chains 0/1 – Staphylococcus epidermidis + Enterococcus faecalis
12 GPC in chains 1/1 Streptococcus agalactiae Staphylococcus epidermidis + Streptococcus agalactiae
13 GPC in chains + clusters 1/1 Streptococcus oralis Staphylococcus aureus + Streptococcus oralis
14 GPC in clusters 0/1 – Staphylococcus epidermidis + Escherichia coli
Polymicrobial contamination
15 GPC 1/1 Staphylococcus hominis Staphylococcus hominis + Staphylococcus capitis
16 GPC in chains + clusters 0/1 Streptococcus pneumoniae Streptococcus peroris + Staphylococcus capitis
17 GPC in clusters 0/1 – Staphylococcus epidermidis + Aerococcus urinae
BCB blood culture bottles; GNB Gram-negative bacilli; GPC Gram-positive cocci
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 409
In our evaluation, the processing time of subcultures was
set at 5 h after preliminary MALDI-TOF MS identification.
Experiences following 3 and 4 h of incubation were found to
be associated with very poor identification results for Grampositive
isolates and only moderate results for Gram-negative
strains (data not shown). Idelevich et al. similarly evaluated
rapid MALDI-TOF MS identification of microorganisms
from positive blood cultures subsequent to a 1.5-, 2-, 3-, 4-,
5-, 6-, 7-, 8-, 12-, and 24-h incubation on solid medium [23].
The mean incubation time needed to achieve species-level
identificationwas 5.9 and 2 h forGram-positive aerobic cocci
(n=86) and Gram-negative aerobic rods (n=42), respectively.
For monomicrobial positive blood cultures, species identification
results could be achieved for 81.1 % of the isolates,
which can be considered as a very satisfactory result when
compared to other recent studies using direct MALDI-TOF
MS, in which correct identification ranged between 64.8 and
81.8%[11, 14, 15]. In line with these authors, we also noticed
a higher identification percentage for Gram-negative organisms
(90.4 %) compared to Gram-positive organisms
(82.2 %). As defined in the Materials and methods section,
identification results were accepted to the species according to
a cut-off score =1.7. Using a cut-off score =2 for species
identification allowed correct identification results for
69.6 % of the monomicrobial positive blood cultures and
identified one of the two strains in 21 of the 29 polymicrobial
positive blood cultures. No isolates were erroneously identified.
A less stringent cut-off score =1.5 for species identification
allowed results for 84 % of the monomicrobial positive
blood cultures and identified one of the two strains in 24 of the
29 polymicrobial positive blood cultures. Three isolates were
erroneously identified, as observed with the cut-off score at
1.7. The final choice to set the cut-off at 1.7 in our study was
taken in accordance with the abundant publications using this
scoring system when directMALDI-TOF MS identification is
applied [9, 17, 24].
One drawback of our rapid MALDI-TOF MS process was
that it failed to yield correct identification results for yeast and
anaerobes most probably related to the insufficient growth of
these microorganisms on the agar plates after a 5-h subculture.
Pondering the high rates of morbidity and mortality as well as
the growing incidence of candidemia and anaerobic septicemia,
a prompt identification result is essential [25, 26]. Hence,
to overcome this flaw in our process, direct analysis from
positive blood culture samples by MALDI-TOF MS should
be considered systematically when Gram staining suggests the
presence of yeast or anaerobes. This procedure preceded by
defined blood lysing protocols using sodium dodecyl sulfate
detergent or Tween 80 and formic acid extraction respectively
enabled Pulcrano et al. [27] to identify 19/21 Candida nonalbicans
bloodstream infections and Leli et al. to identify 7/7
anaerobic septicemia [15. The erroneous identification of two
streptococci (S. salivarius and S. peroris) as S. pneumoniae in
our study confirmed the inability of MALDI-TOF MS to
distinguish oral streptococci strains from S. pneumoniae [18,
28, 29]. On the basis of these observations, we decided not to
consider any S. pneumoniae result through our rapidMALDITOF
MS identification process. The third erroneously identified
strain was an A. baumannii isolate that was misidentified
to the species level as Acinetobacter pittii (formerly
Acinetobacter genomospecies 3). In the MALDI Biotyper
3.0 software, Acinetobacter species identification results are
accompanied with a comment informing about the close relatedness
of several species and the difficulty in differentiating
them.
Themain weakness of ourMALDI-TOFMS protocol was
its inability to identify all organisms in the setting of
polymicrobial bloodstream infections. Various studies also
underlined the lack of ability of MALDI-TOF MS to detect
all microorganisms in mixed cultures through direct identification,
as none or, at best, one single isolate could be identified
[10–12, 15]. Ferroni et al. managed the identification of blood
cultures containing mixed bacteria through the use of Gramspecific
databases selected according to the obtained Gram
result [7]. In our setting, Gram staining of all positive blood
culture bottles and rapidMALDI-TOFMS identification of all
subcultured isolates included in the polymicrobial episodes
were essential elements that partially overcame this limitation
of our algorithm. Regarding the possibility of detecting
polymicrobial bacteremia, visualization of all plated blood
subcultures was systematically repeated the day following
positivity considering that the presence of more than one
organism could go undetected on young subcultures.
The ultimate objective of this study was to speed up the
identification process for improving the management of the
patient and to assist the clinician in deciding whether the
growing microorganisms were to be considered as clinically
relevant and associated with a bloodstreaminfection or, rather,
whether they should be considered as contaminants. Overall,
86.2% of the monomicrobial bloodstream infections could be
identified, thereby potentially allowing an earlier diagnosis
and adaptation of therapy to the documented pathogens. In
parallel, 68.3 % of all organisms regarded as clinically nonsignificant
contaminants could be identified and reported on
the same day of blood culture positivity, possibly leading to
restriction and/or earlier stop of antimicrobial therapy.Martiny
et al. measured the clinical impact of rapid microbial identification
(direct MALDI-TOF MS preceded by an in-house
purification protocol) on the management of septic patients.
An accelerated modification of the treatment regimen was
observed in 13.4 % and 2.5 % of the adult and pediatric
patients, respectively. In other cases, the tool was helpful to
rapidly confirm suspected cases of contamination, thereby
avoiding the administration of unnecessary antibiotics [16].
Vlek et al., likewise, observed an 11.3 % increase in the
proportion of patients receiving appropriate antibiotic
410 Eur J Clin Microbiol Infect Dis (2015) 34:405–413
treatment 24 h after blood culture positivity with direct
MALDI-TOF MS performed twice a day [17].
These results emphasize the benefit of the rapid identification
of positive blood cultures compared toMALDI-TOF MS
analysis on 18-h incubated colonies the day after blood culture
positivity detection.
Considering the satisfactory identification results and the
potentially favorable clinical impact on patientmanagement, a
routine-applicable positive blood culture work scheme integrating
MALDI-TOF MS identification on young positive
blood subcultures was implemented as presented in Fig. 1.
Three time frames were defined according to the time of day
during which growth-positive blood cultures were detected by
the automated culture system. MALDI-TOF MS analysis on
short subcultures was applied at 5 pm for all bottles detected
positive between 0 am and 12 am, thereby allowing the report
of the results to clinicians at 5.30 pm. A direct MALDI-TOF
MS identification (Sepsityper, Bruker Daltonik, Bremen, Germany)
was executed for positive-detected blood cultures between
12 am and 5 pm. This commercial method had been
previously validated in our university hospital, allowing
65.3%correct identifications (data not shown). Positive blood
culture bottles detected between 5 pm and 0 am were
subcultured but only identified on the following day according
to the standard MALDI-TOF MS identification process. During
weekends, the short subculture MALDI-TOF MS identification
was applied once daily at 2 pm, allowing results
reporting of all blood cultures detected positive until 9 am.
Gram staining was systematically performed on all positivedetected
blood bottles and immediately communicated to the
clinicians between 9 am and 00 am every day of the week.
We believe that a major strength of this algorithm is the
gain in hands-on time and cost if compared with systematic
direct MALDI-TOF MS analysis while preserving the gain
in time in positive blood culture identification result
reporting. On weekdays, the results were systematically
communicated by phone at 5.30 pm to the infectious diseases
physicians team, potentially allowing faster antimicrobial
treatment modifications, as previously demonstrated
by several authors [16, 17].
Time frame of growth detecon Day 0 in blood culture by automated culture system
0 AM 12 AM
MALDI-TOF MS ID
on young subculture*
Direct MALDI-TOF MS ID No Rapid ID tesng
ID result with
log score = 1.7
ID result with
log score < 1.7
Streptococcus pneumoniae
ID result
Day 0
5 PM
Day 0
5.30 PM
ID result
reporting
No ID result reporng
Optochin tesng
Day 1
9 AM
MALDI-TOF MS ID
on subculture
ID result
reporting
MALDI-TOF MS ID
on subculture
ID result
reporting
ID result
reporting
12 AM 5 PM 5 PM 0 AM
No ID result
reporng
Optochin reading
Day 1
9.30 AM
BC plang:
8 AM 12 AM
BC plang:
12 AM 5 PM
BC plang:
5 PM 0 AM
Fig. 1 Modified weekday routine workflow scheme for the identification
of blood cultures in accordance with the time of positivity detection by
theautomated incubation system. *Direct MALDI-TOF MS ID when
Gram staining suggestive of yeast or anaerobes. BC blood culture; ID
identification; MALDI-TOF MS matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry
Eur J Clin Microbiol Infect Dis (2015) 34:405–413 411
The large amount of tested isolates, thereby representative
of the routine positive blood culture microorganism proportions
in a tertiary hospital, enabled us to validate the applied
process. All during the study period, testing was carried out by
various technologists and medical junior residents, thereby
highlighting the robustness and reproducibility of the method
in clinical routine practice.
In conclusion, the integration of MALDI-TOF MS identification
on 5-h subcultured colonies in the laboratory
workflow represents an excellent compromise between the
direct blood culture process associated with labor-intensive
steps and the direct smear method of 18-h subcultured colonies,
as it leads to the reporting of correct identification results
on the day of positivity in more than 80 % of the
monomicrobial bacteremic episodes. An ongoing challenge
is the development of rapid tests for the detection of clinically
important resistance mechanisms, since we should keep in
mind that the identification results alone only give partial
microbiological information to the clinicians. Hence, the impact
on the patient’s clinical management of the rapid positive
blood culture identification result “alone” may also be very
dependent on the local epidemiology of bacterial resistance.
Indeed, the increasing trends of resistance of Gram-negative
bacteria to third-generation cephalosporins and to carbapenems,
as well as the high rates of methicillin-resistant Staphylococcus
aureus across Europe, remind us that clinicians can
no longer simply rely on the wild susceptibility profile of the
identified bacteria for therapeutic decision-making [30]. A
study is actually ongoing in our hospital to assess whether
the combination of rapid MALDI-TOF MS identification
associated with rapid detection of resistance mechanisms to
selected antimicrobial agents may favorably impact on antimicrobial
therapy among septic patients with positive blood
cultures.
Acknowledgments This study was supported, in part, by a research
grant from Fondation Saint-Luc, Cliniques universitaires Saint-Luc,
UCL, Bruxelles.
Conflict of interest The authors declare that they have no conflict of
interest.
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