Improvement of a rapid direct blood culture microbial identification protocol using MALDI-TOF MS and performance comparison with SepsiTyper kit

Journal of Microbiological Methods 155 (2018) 1–7

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Improvement of a rapid direct blood culture microbial identification protocol using MALDI-TOF MS and performance comparison with SepsiTyper kit

T

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Francesca Di Gaudioa, , Serena Indelicatob, Sergio Indelicatoc, Maria Rita Tricolic, Giuseppe Stamponec, David Bongiornob a b c

Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Università degli Studi di Palermo, Via del Vespro, I-90127 Palermo, Italy Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, I-90123 Palermo, Italy Quality Control Laboratories and Chemical Risk, Ospedali riuniti Villa Sofia Cervello, Via del Vespro, I-90127 Palermo, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacteria Pathogens identification Genus and species identification Matrix- assisted laser desorption ionization time of flight mass spectrometry Clinical diagnostic laboratory

Fast diagnosis of pathogens is critical to guarantee the most adequate therapy for infections; bacterial culture methods, which constitute the actual gold standard, are precise and sensitive but rather slow. Today, new methods have been made available to enable faster diagnosis, with the Matrix-Assisted Laser Desorption Ionization–Time Of Flight Mass Spectrometry (MALDI-TOF MS) technique being the most promising. Even if simpler and faster than traditional bacterial culture methods, analysis of positive blood cultures via MALDI-TOF MS requires a preliminary extraction process of samples. In this study, we compared two extraction protocols for bacterial identification directly from positive blood cultures using the Bruker MALDI Biotyper system (Bruker Daltonics, Billerica, MA). In particular, we evaluated the time employed and the overall performance for their accurate identification. In this work, the performances of a commercial extraction kit, named SepsiTyper™ Kit, and those of the protocol developed by Treibmann et al. were evaluated and proven to be similar. However, the SELTERS method represents the best compromise price/performance. Lastly, an in-house developed analysis protocol has been tested, and the introduced optimizations granted a performance level equal if not better than the SepsiTyper kit, a reduced processing time and reduced costs.

1. Introduction Sepsis is one of the leading causes of death of both adults and children worldwide, imposing a heavy human and economic burden in both developed and developing countries (Dellinger et al., 2012). A rapid identification of the infectious agent is crucial to instruct an adequate antimicrobial treatment and for the outcome of the disease. Currently, blood culture represents the “gold standard” for microbiological diagnosis of sepsis because it allows the identification of the etiologic agent of the systemic inflammatory response and the determination of the in vitro sensitivity profile to antimicrobial drugs, providing clinicians with fundamental support for choosing an appropriate therapy. This analysis has been continuously evolving over the years, going from the classic Castaneda method to technologically more advanced solutions, which significantly increased both the sensitivity and the speed of detection. On the other hand, blood culture is scarcely sensitive to some

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pathogens, such as Legionella pneumophila, Chlamydia pneumoniae and Mycoplasma pneumoniae (which are responsible for pulmonary diseases contracted in communities) and to some pathogens that are difficult or impossible to cultivate, such as Bartonella spp., Francisella tularensis, Hiphomycetes, Nocardia spp, Ricketia spp. and Coxiella burnetii (Stathopulos et al., 2004; Fenollar and Raoult, 2007). The principal disadvantage of the blood culture is bound to the time requested in every step of the analytical procedure (bottle incubation, subculture, identification and antibiogram). This implies that in most cases, a reliable result is not available before 18–24 h starting when positive samples are identified and involves the overnight sub-culture on solid medium to obtain isolated colonies for identification and antibiotic susceptibility testing; the analysis can take longer for slow growing bacteria or fungi (Lucignano et al., 2011; Wieser et al., 2012). This is the main reason why any procedure that can take less time and provide reliable answers and thus provide the correct therapeutic approach is actually very welcome and has a positive impact on patients' treatment.

Corresponding author. E-mail address: [email protected] (F. Di Gaudio).

https://doi.org/10.1016/j.mimet.2018.10.015 Received 24 July 2018; Received in revised form 24 October 2018; Accepted 25 October 2018 Available online 26 October 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved.

Journal of Microbiological Methods 155 (2018) 1–7

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SepsiTyper™ MALDI Kit with a protocol described by Treibmann et al., named SELTERS (Treibmann et al., 2015), for bacterial identification directly from positive blood cultures using a MALDI TOF/TOF (Bruker Daltonics AutoFlex). Finally, a new in-house-developed protocol has been tested to reduce the time required for sample treatment and the cost of each analysis, and performance levels were obtained comparable to the SepsiTyper kit. For the new protocol, a deeper evaluation has been performed, comparing its identification results with those obtained from SELTERS protocol using the same instrumentation: a MALDI TOF/TOF (Bruker Daltonics AutoFlex) and validating the recognition by both biochemical (BD Phoenix) and molecular (bioMérieux VITEK Maldi MS) methods.

Indeed, the time spent from triage and qualification to antibiotic administration are critical determinants of mortality incidence (Harbarth et al., 2003; Kollef, 2003): each hour of delay in administration of appropriate antibiotics is associated with a decrease in survival of 7.6% over 6 h, while administration of inappropriate antibiotics has been associated with an approximately fivefold decrease in the survival rate (Kumar et al., 2009). In recent years, many laboratories have relied on molecular diagnostics that allow the identification of the microbiota based on one or more specific gene sequences. Molecular diagnostics are often (but not always) more sensitive and/or specific than traditional methods and are increasingly used when the microorganism is difficult or impossible to cultivate or when its growth in vitro is very slow or when the cost of a traditional diagnosis is higher than a molecular diagnosis. From this point of view, recent developments in mass spectrometry (MS) in clinical analysis constituted a real breakthrough in various fields and brought molecular diagnosis to higher levels of sensitivity and accuracy. MS is an analytical technique that is based on an ionization process and the conversion of neutral compounds into charged ions, allowing the measurement of their mass-to-charge (m/z) ratio. Although MS is an early 1900s technique, its use was limited to chemical analysis due to electron ionization process limitations. However, in recent years, the development of soft ionization sources such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and mostly matrix-assisted laser desorption ionization (MALDI) has increased the applicability of MS to delicate and large molecules such as polymers, proteins, and supramolecular aggregates up to microorganisms (Marie et al., 2000; Di Stefano et al., 2017; Bongiorno et al., 2014; Di Donna et al., 2017; Vogeser et al., 2007; Fenselau and Demirev, 2001). MatrixAssisted Laser Desorption Ionization–Time Of Flight Mass Spectrometry (MALDI-TOF MS) is a currently used approach for rapid and accurate pathogen identification, which is critical for sepsis treatment in patients (Huang et al., 2013). Analysis of bacterial and fungal colonies using MALDI-TOF MS produces reproducible genus-species and species-specific spectral profiles, which can be used to identify species-level microorganisms. This system allows the rapid identification of Gram-positive, Gram-negative bacteria, mycobacteria, yeasts and molds (Hettick et al., 2004; Pignone et al., 2006; Carbonnelle et al., 2007; Mellmann et al., 2008; Qian et al., 2008; Degand et al., 2008; Seng et al., 2009). Early analytical approaches required subculture colony growth on microbiological media, extraction and MS analysis. Currently, the increased instrumentation sensitivity and selectivity allows faster diagnosis (Singhal et al., 2015) and, conversely, growing interest in this technique. In an attempt to address the lack of a standardized protocol for the preparation of blood cultures prior to the MS analysis (Ferreira et al., 2010), a commercial extraction kit, the SepsiTyper™ Kit (Bruker Daltonics, Bremen, Germany), has been developed. The performances of the SepsiTyper are very reliable, but the high reagent cost also hindered a wider adoption of this kit in diagnostics (Kok et al., 2011). In this study, we compared the performances of the commercial

2. Materials and methods 450 BACTEC® Plus Aerobic/F, Anaerobic/F, and Pediatric Plus/F blood cultures identified as positive by the automated Bactec incubation system (BD, Franklin Lakes, NJ) and mono microbial blood cultures of patients with suspected sepsis were analyzed. When a bottle signaled positive, a sample of 1 mL was taken to be submitted to MALDI MS recognition analysis on the Bruker AutoFlex instrument. A Gram stain and overnight subculture were performed on the residual sample. Any sample that appeared to be poly-microbial (by Gram stain) was excluded from the present study, in order to get more homogeneus samples. The final identification of the samples was obtained using subcultures processed by BD Phoenix and/or 16S rRNA gene sequencing for bacteria and API 20C AUX bioMérieux for yeasts. In particular, 16S rRNA sequencing has been performed in the case of conflicting results between molecular (MALDI) and biochemical approaches, or in the case of lack of recognition. Identification results of the in-house developed method were also compared to the identification results obtained from a further MALDI MS system (bioMérieux VITEK MS) located in the Hospital laboratory, which independently performed the microorganism determination on subculture isolated colonies. The MALDI-TOF identification results and the time required to obtain a definitive identification have been compared with conventional culture methods based on testing isolated colonies from overnight culture. The SELTERS protocol and SepsiTyper™ MALDI Kit have been applied as the initial clean-up step necessary to remove the hemoglobin and the serum matrix that can affect the quality of the MS spectra generated via MALDI-TOF. Then, a final common protein extraction step was applied to all the analyzed samples. The SELTERS and SepsiTyper™ MALDI Kit protocols are described in detail below and briefly summarized in Table 1. 2.1. Protocol 1 – SELTERS All reagents used for SELTERS protocol Triton X-100 (0.2%) and sodium dodecyl sulfate (SDS 0.1%) were purchased from Sigma Aldrich

Table 1 Schematic description of the SELTERS and SepsiTyper™ protocols used for bacteria identification. Step

SELTERS protocol

MALDI SepsiTyper™ protocol

1

1 mL of positive blood culture in 2 mL capped tube and add 1 mL of Triton X-100 0.2% Vortex mixing for 30 s and centrifugation at 13000 rpm for 5 min Eliminate the supernatant Add 1 mL of 0.1% SDS, vortex mixing, centrifugation at 13000 rpm for 5 min

1 mL of positive blood culture in 2 mL capped tube, add 200 μL of “Lysing Buffer”

2 3 4 5 6 7 8 9

Eliminate the supernatant Add 1 mL di H2O, Vortex mixing for 30 s and centrifugation at 13000 rpm for 5 min Eliminate the supernatant Protein extraction according to the classical protocol Deposit onto target slides 1 μL of supernatant and 1 μL of matrix solution

Vortex mixing for 30 s and centrifugation at 13.000 rpm for 1 min Eliminate the supernatant Wash the pellet with 1 mL of “Washing Buffer” and centrifugation at 13.000 rpm for 1 min Eliminate the supernatant Protein extraction according to the classical protocol

Deposit onto target slides 1 μL of supernatant and 1 μL of matrix solution

2

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2.5. MALDI TOF MS analyses

(Milan, Italy). Deionized water (18 Mohm) was obtained by NANO PURE Diamond Thermo apparatus. In the sample preparation step, we proceeded as follows: 1 mL of positive blood culture was withdrawn with a sterile syringe, and 1 mL Triton X-100 (0.2%) buffer was added to a 2 mL capped tube (Eppendorf, Germany). After vortexing for 30 s, the sample is centrifuged at 13000 rpm for 5 min. Once the supernatant layer is removed, the residue pellet is added to 1 mL of SDS (0.1%), vortexed for 30 s and centrifuged at 13000 rpm for 5 min. The supernatant layer is removed, and the residue pellet, added to 1 mL of deionized water, is vortexed for 30 s and centrifuged again at 13000 rpm for 5 min. The supernatant layer is finally removed. The extraction of proteins is carried out according to the classical protocol in common with both analytical approaches and is detailed below.

MS experiments were carried out in linear mode on a MALDI TOF/ TOF instrument (AutoFlex speed Bruker Daltonics) acquired in a mass range from 2000 to 20,000 m/z. MS spectra were analyzed using Bruker Biotyper 3.1.56 software and library version 6.0.0. The identification was considered correct with a high-confidence level when the scores reported by the Bruker Biotyper were ≥ 2.0. The software compares the acquired spectra with those stored in the database, creating a list of the best matches. The spectral concordance degree is evaluated by a proprietary algorithm and generates a logarithmic value (score), ranging from 0.0 to 3.0. According to the manufacturer's instructions, a score ranging from 2.000–3.000 indicates a confident identification of genus and species; a score ranging from 1.700–1.999 indicates a confident identification of genus only; a score below 1.700 is considered unreliable identification. For the evaluation and validation of the in house developed protocol a further MALDI MS (bioMérieux VITEK MS) recognition system has been used. The analysis procedure is briefly summarized below. Isolated colonies from one overnight subculture are transferred to an individual spot on the 48well Vitek MS-DS disposable target slide using 1 μL inoculating loop. Each spot is covered with 1 μL of ready-to-use Vitek MS alpha-cyano-4hydroxycinnamic acid (CHCA) matrix (bioMérieux, France). Fungal samples were covered with 0.5 μL of 28.9% formic acid (bioMérieux, France) before deposing the CHCA matrix. Each sample is prepared in duplicate. The protein profile of each specimen with a m/z of 3000 to 15,000 is generated based on 100 measurements. The profiles are further matched with the Vitek MS reference CE-IVD certified database, which returned the best identification match along with confidence percentages from 0% to 99.9%. According to the manufacturer's recommendation, spot results of 90 to 98% confidence are considered high at the genus level, while results of > 98% confidence are considered high at the species level. Spot results of < 90% confidence are considered unacceptable identifications.

2.2. Protocol 2 – SepsiTyper Kit The MALDI™ SepsiTyper Kit contains the following reagents: lysing buffer and washing buffer. The biological sample was treated as follows: 1 mL of positive blood culture was taken using a sterile syringe and transferred to a 2 mL capped tube (Eppendorf, Germany), 200 μL of lysing buffer was added, and the sample was vigorously shaken in the vortex mixer for 10 s. It follows a centrifugation (13,000 rpm for 1 min) step. The supernatant was removed, the pellet was re-suspended in 1 mL of wash buffer, and the sample was centrifuged at 13000 rpm for another minute. After the removal of the supernatant, the residue pellet is treated for the extraction of proteins according to the classical protocol, as detailed below. 2.3. Common procedure for protein extraction For the protein extraction protocol, the solvents and reagents were purchased from Sigma Aldrich (Milan, Italy). In this procedure, pellets were suspended in 300 μL of water, vortexed, mixed and added to 900 μL of ethanol. The solvent is removed after a centrifugation step (13.000 rpm, 2 min), and the residue pellet, incubated at 37 °C (for 15 min), is finally extracted with 70% formic acid and acetonitrile (from 20 μL to 50 μL on the basis of the pellet size). After centrifugation, supernatants (1 μL) were deposited onto a MALDI target plate and air dried. A second layer of 1 μL of matrix solution, consisting of α-cyano-4hydroxycynnammic acid (CHCA) at a concentration of 10 μg/μL, was then deposited onto the first layer and air dried. Each sample was prepared in duplicate.

3. Results and discussion Among the 450 blood culture-positive specimens, 169 (37.55%) contained Gram-positive organisms, 257 (57.11%) contained Gramnegative organisms, and 24 (5.33%) contained fungi. The microorganisms were identified through routine hospital identification methods: BD Phoenix and API 20C AUX bioMérieux for yeasts. These biochemical methods were chosen to add an external comparator to the MALDI identification protocols. The analysis through 16S rRna sequencing should be applied for cases of conflicting results between MALDI MS and biochemical approaches or in the case of lack of microorganism recognition by both methods. The microorganisms were identified by biochemical tests and automated microbial systems within 3 working days, whereas specimens containing Brevibacterium ravenspurgense and Arthrobacter ramosus species that required 16S rRNA gene sequencing for identification, took 2 additional working days to be processed. In terms of analytical performance, the SELTERS method provided similar results to the SepsiTyper™ kit overall. Indeed, considering the 450 samples cited so far, both methods SELTERS and SepsiTyper generated identifications of genus and species with a similar efficiency: SELTERS 388 identifications corresponding to an efficiency of 86.3%, SepsiTyper 392 identifications corresponding to an efficiency of 87.2%. The identification of genus only was obtained in 60/450 (13.3%) cases for the SELTERS method and 56/450 (12.4%) cases for the SepsiTyper kit. In just two cases, no identification was obtained with both methods (see Table 2). While a chi-squared statistical analysis of results evidence that differences between the protocols in terms of correct recognition of the sample at genus and species levels are not significant, there are

2.4. New protocol development To improve the SELTERS method and reduce the time required for sample treatment, a new in-house developed protocol was tested. A volume of 1 mL of positive blood culture was taken using a sterile syringe and transferred to a 2 mL Eppendorf tube. Triton X-100 buffer (1 mL at 0.2%) was added to the tube, and the sample was vigorously shaken in the vortex mixer for 20 s. It follows a centrifugation (5000 rpm for 3 min) step. The supernatant was removed, the pellet was re-suspended in 1 mL of SDS solution (1.8%), vortexed for 20 s, and the sample was centrifuged at 5000 rpm for another 3 min. After the removal of the supernatant, the residue pellet was washed with 1 mL of H2O deionized water, vortexed for 20 s and centrifuged again at 5000 rpm for 3 min. The supernatant layer was finally removed, and the pellet was extracted with 70% formic acid (10 μL to 50 μL on the basis of the pellet size). The samples are spotted onto reusable stainless steel MALDI target plate and air dried. A second layer of 1 μL of CHCA matrix (bioMérieux, France) is then deposited onto the first layer and air dried. Each sample was prepared in duplicate. 3

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Table 2 Comparison between SELTERS and SepsiTyper protocols identification results. SELTERS Final identification

Gram positive Staphylococcus haemolyticus Staphylococcus epidermidis Proprionibacterium acnes Staphylococcus hominis Enterococcus faecalis Brevibacterium ravenspurgense Staphylococcus aureus Streptococcus anginosus Enterococcus faecium Staphylococcus chonii Staphylococcus simulans Staphylococcus warneri Arthobacter ramosus Streptococcus gordonii Total Percentages % Gram negative Klebsiella pneumoniae Acinetobacter baumannii Escherichia coli Pseudomonas aeruginosa Total Percentages % Bacteria identification Percentages % Mycetes Candida parapsilosis Candida albicans Total Percentages % Total Percentages %

SepsiTyper

N°

Correct genus and species ID

Correct genus ID

30

25

26 5 17 21 1

21

29 3 32 1 1 1 1 1 169 37,6

27 1 31

72 59 78 48 257 57,1 94,7

67 44 78 44 233 90,7 87,56

5 15

6 18 24 5,33 450 100,00

5 10 15 62,50 388 86,22

1 8 9 37,50 60 13,33

15 20

NO ID

Correct identification

N°

Correct genus and species ID

Correct genus ID

5

30

30

27

3

30

5 5 2 1

26 5 17 21 0

26 5 17 21 1

23 3 16 19

3 2 1 2

26 5 17 21 0

29 3 32 1 1 1 1 1 169 37,6

23 2 29

2 1,2

29 3 32 1 1 1 0 1 167 98,8

72 59 78 48 257 57,1 94,7

69 54 78 40 241 93,8 90,14

3 5

0 0,0 0,47

72 59 78 48 257 100,0 99,53

0 0,00 2 0,44

6 18 24 100,00 448 99,5

6 18 24 5,33 450 100,00

1 7 8 33,33 392 87,11

5 11 16 66,67 56 12,44

1 2 2 1 1 1 1 1 140 82,8

1 27 16,0

4 24 9,3 11,97

NO ID

1 6 1 3 1

2 1,2

29 3 32 1 1 1 0 1 167 98,8

0 0,0 0,47

72 59 78 48 257 100,0 99,53

0 0,00 2 0,44

6 18 24 100,00 448 99,56

1 1 1 143 84,6

1 24 14,2

8 16 6,2 9,39

Correct identification

Finally, in this work, a further simplification of the sample treatment has been made and proposed. The main difference between the new protocol and the SELTERS procedure lies in the extraction step, which has been modified, reducing the sample treatment to a single extraction step with formic acid (70%). This reduces the overall sample manipulation time to 10 min. A brief comparison of the three protocols adopted is reported in Table 4. The proposed procedure has been used on 60 positive blood culture samples, and the results of the identification obtained with this in house-made protocol have been confirmed using the usual biochemical methods, in addition to a MALDI MS identification system constituted by a bioMérieux VITEK MS coupled with MYLA and a large mass spectral database. This procedure can be used as a control and is currently a validated method for identifying pathogens from blood culture samples using MALDI-TOF MS. The results of the comparison of our new method and SELTERS protocol results are reported in Table 5. They confirm that the overall efficiency is similar or even better than the reference method, while in statistical terms, the new protocol gave better results for the SELTERS and SepsiTyper approaches with respect to the recognition of bacteria and similar efficiency in fungi determination. It should be noted that the simplified SELTERS protocol developed here matches both the reduced sample price of the SELTERS approach and the reduced manipulation time of the SepsiTyper protocol with high reliability (100% of correctly identified samples).

some practical points that favor the SELTERS method. The most appealing point is of an economic nature, with the SELTERS approach being cheaper (0.50 vs. 5.50 €/analysis for SELTERS and SepsiTyper kit, respectively). It should be noted that evaluating the effectiveness of both methods on Gram-positive, Gram-negative bacteria and mycetes, there is a slight difference. Specifically, likely due to the different structures of the bacterial wall, identification is better for Gram-negative than for Grampositive bacteria. This observation confirms previous studies reporting that the bacterial wall greatly affects identification scores (De Carolis et al., 2014). Identification scores depend on the number of peptides matching the reference spectra and therefore on the overall peptide extraction efficiency from pathogens. Thus, we tried to insert a further step in the SELTERS method to further improve the genus and species recognition scores. In particular, taking into consideration that ultrasound tissue treatment is currently used to ease protein extraction for various tissues (Fukase et al., 1994; Preece et al., 2017), 5 min of sonication have been added to the first lysis procedure step. This experimental variation has been applied to 17 samples also treated with SELTERS and SepsiTyper methods. The results shown in Table 3 indicate that the sonication procedure led to slightly worse identification results. This could be due to denaturation/modification of proteins due to the cavitation that occurs during the treatment of the samples with ultrasounds (Stathopulos et al., 2004). The evidence is that the trade-off between extraction efficiency and protein denaturation is not advantageous. A point in favor of the SepsiTyper protocol is the smaller amount of time required for sample treatment before MALDI MS acquisition. 4

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Table 3 Genus and species identification results for seventeen samples treated with sonication procedure. Final identification

SELTERS

SELTERS with sonication

N°

Correct genus and species ID

Correct enus ID

3 2

2 2

1

1 1 1 8 47,1

1

Gram negative Klebsiella pneumoniae Escherichia coli Pseudomonas aeruginosa Total Percentages %

2 3 2 7 41,2

Micetes Candida albicans Total Percentages % Total Percentages %

2 2 11,8 17 100,0

Gram positive S. haemolyticus S.taphylococcus epidermidis Enterococcus faecalis Staphylococcus aureus Streptococcus anginosus Totale Percentages %

NO ID

SepsiTyper

Correct genus and species ID

Correct genus ID

1

NO ID

Correct genus and species ID

Correct genus ID

2 2

3 1

1

1

1 12,5

2 25,0

1 1 3 37,5

5 62,5

1 1 1 7 87,5

2 3 1 6 85,7

1 1 14,3

1 1 2 28,6

2 2 1 5 71,4

2 3 2 7 100,0

1 1 50,0 13 76,5

1 1 50,0 4 23,5

5 29,4

1 1 50,0 11 64,7

1 1 6 75,0

4. Conclusions

1 1 50,0 1 5,9

NO ID

2 2 100,0 3 17,6

14 82,4

However, while requiring only some extra processing time, the cost per sample of the SELTERS method was approximately 10 times lower than the cost of the SepsiTyper kit with identical consumption of consumables and leaved margins for further optimizations. In particular, the proposed introduction of a single extraction step greatly reduced the sample manipulation time and gave results comparable if not better than the commercial kit. Our study confirms the advantages of rapid pathogen identification from positive blood cultures: We show that rapid identification might be an additional source of public health information and that it may be particularly helpful when fruitful collaboration and communication between health professionals occur. In terms of running costs and analytical performances, the application of an in-house method proved to be the best options for a diagnostic microbiology setting. Finally, our results suggest that MALDITOF-based identification directly from blood cultures is not much more labor intensive (in particular reducing the extraction steps) but requires lower reagent costs compared to conventional methods or to automated multiplex PCR systems. Subculture analysis of positive blood culture remains the gold standard method for diagnosing sepsis because it allows evaluating the sensitivity of microorganisms to drugs, enabling the administration of the correct therapy.

The rapid identification in blood samples of infecting pathogens is a valuable tool to reduce unnecessarily broad and prolonged antibiotic treatment, mostly in the context of emerging antibiotic resistance (Bonura et al., 2015; Mammina et al., 2012). Previous studies exploring the impact of rapid identification of pathogens directly from positive blood cultures have demonstrated improvements in antibiotic treatment and patient outcomes only in combination with an antimicrobial stewardship program (Beuving et al., 2015). For a clinical diagnostic laboratory, it is attractive to select the convenience of a commercially available and validated kit such as SepsiTyper™, whose performances have been assessed in at least 21 reported studies, as recently reviewed by Morgenthaler and Kostrzewa (Morgenthaler and Kostrzewa, 2015). Thus, the use of the MALDI-TOF MS method can be applied to shortly obtain the exact classification of an isolate and can be considered a valid method to establish a correct diagnosis and appropriate therapy (Fasciana et al., 2015). Using a cut off score of ≥2.000 (i.e., the manufacturer's recommended cut-off value for identification of organisms from culture plates), no differences were found between the rates of obtaining subculture concordant species-level identifications when SELTERS or SepsiTyper protocol was applied to positive blood cultures for direct MALDI-TOF/MS analysis.

Table 4 Comparison of the procedural steps adopted for SepsiTyper, SELTERS and newly developed internal method; (*) lysis time is not included. STEP

SepsiTyper

Manipulation time (min)

SELTERS

Duration (min)

New protocol

Manipulation time (min)

Lysis

Lysis buffer Wash buffer

1 1

5 5 5

-2 Water and ethanol (1:3) Drying F.A. + ACN (1:1) 15 17* 5

Triton 0,2% SDS 1,8% water -9 F.A. 70%

3 3 3

Total lysis time Extraction procedure

Triton 0,2% SDS 0,1% water -15 Water and ethanol (1:3) drying F.A. + ACN(1:1) 15 30* 6

Extraction time Total time Total steps

2 10 3

5

2 10 3 1

10* 4

1

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Table 5 Comparison results obtained from SELTERS and SELTERS simplified protocols. SELTERS Final identification

Gram positive Staphylococcus epidermidis Staphylococcus hominis Staphylococcus capitis Staphylococcus intermedius Staphylococcus infantis Staphylococcus haemolyticus Staphylococcus aureus Staphylococcus capitis Enterococcus faecium Enterococcus faecalis Enterococcus raffinosus Propionibacterium acnes Total Percentages % Gram negative Escherichia coli Klebsiella pneumoniae Rhizobium radiobacter Pseudomonas aeruginosa Acinetobacter baumanni Serratia marcescens Achromobacter xylosoxidans Stenotrophomonas maltophilia Alcaligenes faecalis Total Percentages % Bacteria identification Percentages % Mycetes Candica albicans Candida parapsilosis Total Percentages % Total Percentages %

SELTERS simplified protocol

N°

Correct genus and species ID

Correct genus ID

14 2 2 1 1 2

12 2 2 1

2

2 1 3 1 2 2

2 1 2 1 2 1

1

33 55,93

28 84,85

5 15,15

7 4 1 2 2 1 1

7 4 1 2 2 1

1

1

1 20 33,90 89,83

1 19 95,00 88,68

1 5,00 11,32

4 2 6 10,17 59 100,00

2 1 3 50,00 50 84,75

2 1 3 50,00 9 15,25

NO ID

Correct identification

N°

Correct genus and species ID

Correct genus ID

14 2 2 1 1 2

14 2 2 1 1 2

13 2 1 1 1 1

1

2 1 3 1 2 2 0 33 100,00

2 1 3 1 2 2

2 1 3 1 2 2

33 55,93

30 90,91

7 4 1 2 2 1 1

7 4 1 2 2 1 1

7 4 1 2 1 1

1

1

1

0 0,00 0,00

1 20 100,00 100,00

1 20 33,90 89,83

1 18 90,00 90,57

2 10,00 9,43

0 0,00 0 0,00

4 2 6 100,00 59 100,00

4 2 6 10,17 59 100,00

2 1 3 50,00 51 86,44

2 1 3 50,00 8 13,56

1 2

1

0 0,00

1

References

NO ID

Correct identification

1

14 2 2 1 1 2

3 9,09

2 1 3 1 2 2 0 33 100,00

1

0 0,00

7 4 1 2 2 1 1

1 1

1

0 0,00 0,00

1 20 100,00 100,00

0 0,00 0 0,00

4 2 6 100,00 59 100,00

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