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

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

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    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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