Positive Blood Culture Broths Research Articles

Blood-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Carbapenemase-Producing Enterobacteriaceae Directly from Positive Blood Culture Broths.

A variant of the modified carbapenem inactivation method (mCIM) was developed to detect carbapenemase activity directly from positive blood culture broths. The method, termed "Blood-mCIM," was evaluated using Bactec blood culture bottles (Becton, Dickinson and Company, Franklin Lakes, NJ) inoculated with 27 different carbapenemase-producing Enterobacteriaceae (CPE) isolates and 34 different non-CPE isolates. The assay was positive for all blood culture broths inoculated with CPE isolates and negative for all blood culture broths inoculated with non-CPE isolates, corresponding to a diagnostic sensitivity and specificity of 100%, respectively. This assay is inexpensive using "off the shelf" reagents, does not require centrifugation or mechanical lysis, and can be readily implemented in any clinical microbiology laboratory. The Blood-mCIM should facilitate expedient administration of antimicrobial therapy targeted toward CPE bloodstream infections and assist infection control and public health surveillance.

Processing of Positive Blood Cultures Using a Total Laboratory Automation System: Workflow and Clinical Validation

Abstract Automated systems for culture-based microbiology are in the early phase of implementation in clinical laboratories. Here, our objective was to evaluate the performance of the BD Kiestra Total Laboratory Automation (TLA) System for inoculation, incubation, and imaging of positive blood culture broth specimens. To optimize parameters for clinical testing, 56 clinical specimens were processed using both TLA and manual standard-of-care (SOC) methods. For TLA processing, 3 mL positive blood culture broth (35 VersaTREK: 19 aerobic, 16 anaerobic; 21 BD BACTEC: 15 aerobic, 6 anaerobic) was transferred to a no-additive vacutainer using a safety adapter and syringe. This aliquot was then placed on TLA for fully automated processing: 10 µL was inoculated to blood, chocolate, and MacConkey agar (Remel) and, for anaerobic bottles only, Brucella blood agar (Hardy Diagnostics). Kiestra cross-streak pattern 5 was optimal for obtaining isolated colonies and was superior to quadrant-streaking methods. Additional media types were added based on Gram stain results of the positive blood specimen: CandiSelect (Bio-Rad) and Sabouraud Dextrose (Remel) were added if yeast were identified, and Colistin Nalidixic Acid agar (Remel) for Gram stains with mixed Gram-positive and Gram-negative morphology. Plates were imaged at 6, 8, 10, 12, 18, 38, and 62 hours. Anaerobic media were placed by the system in a media stacker, incubated off-line, and then imaged on the TLA at 24, 48, and 72 hours. SOC cultures were evaluated after overnight incubation and on days 2 and 3. Organism identification was performed using MALDI-TOF MS (Bruker). Based on our evaluation, optimal parameters for clinical implementation of TLA were identified. Microbial growth was scant at 6 hours of incubation, but by 8 hours, small discrete colonies were observed for most aerobes. Thus, imaging parameters selected for routine clinical testing were 8, 18, and 38 hours for aerobic media and one image taken at 38 hours for anaerobic media. TLA and SOC culture results had 96% agreement (29 Gram positive, 12 Gram negative, 5 mixed, 6 yeast, 1 no growth). Two specimens with a second low-abundance bacterial population were observed on TLA that were not observed with SOC. Reproducibility of TLA was tested by processing 6 positive samples in triplicate and found to be 100%. There was no carryover or contamination noted between specimens processed simultaneously on TLA. To evaluate if implementation of TLA impacted epidemiology of positive blood cultures, we compared the 10 most common organisms recovered pre- and post-TLA implementation (August-November 2017 compared to August-November 2018). We found these organisms were not significantly impacted by TLA processing. In conclusion, automated processing of positive blood cultures improves laboratory workflows and facilitates faster workup at 8 hours of incubation. These results demonstrate that automation is a viable avenue for the processing of positive blood cultures.

Rapid species identification of Candida directly from blood culture broths by Sepsityper-MALDI-TOF mass spectrometry: impact on antifungal therapy.

We applied Sepsityper matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF-MS) technology on positive blood culture broths for rapid species identification. Of the 74 patients with candidaemia, 25 had the species identified on the day of the positive blood culture directly from the broth (rapid identification group) while the remaining 49 had the species identified from culture (conventional identification group). Three (13.6%) out of 22 treated patients in the rapid identification group received echinocandin compared to 20/45 (44.4%) in the conventional identification group. The appropriateness of therapy was 90.9% in the rapid identification group and 62.2% in the conventional identification group (p = 0.01). Cost savings were more than £10,000 in the first three days of treatment. Sepsityper-MALDI-TOF-MS is a useful tool in supporting antifungal stewardship programmes.

An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).

Sepsis is the leading cause of death among patients in intensive care units (ICUs) requiring an early diagnosis to introduce efficient therapeutic intervention. Rapid identification (ID) of a causative pathogen is key to guide directed antimicrobial selection and was recently shown to reduce hospitalization length in ICUs. Direct processing of positive blood cultures by MALDI-TOF MS technology is one of the several currently available tools used to generate rapid microbial ID. However, all recently published protocols are still manual and time consuming, requiring dedicated technician availability and specific strategies for batch processing. We present here a new prototype instrument for automated preparation of Vitek®MS slides directly from positive blood culture broth based on an “all-in-one” extraction strip. This bench top instrument was evaluated on 111 and 22 organisms processed using artificially inoculated blood culture bottles in the BacT/ALERT® 3D (SA/SN blood culture bottles) or the BacT/ALERT VirtuoTM system (FA/FN Plus bottles), respectively. Overall, this new preparation station provided reliable and accurate Vitek MS species-level identification of 87% (Gram-negative bacteria = 85%, Gram-positive bacteria = 88%, and yeast = 100%) when used with BacT/ALERT® 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo® instruments, respectively. The prototype was then evaluated in a clinical microbiology laboratory on 102 clinical blood culture bottles and compared to routine laboratory ID procedures. Overall, the correlation of ID on monomicrobial bottles was 83% (Gram-negative bacteria = 89%, Gram-positive bacteria = 79%, and yeast = 78%), demonstrating roughly equivalent performance between manual and automatized extraction methods. This prototype instrument exhibited a high level of performance regardless of bottle type or BacT/ALERT system. Furthermore, blood culture workflow could potentially be improved by converting direct ID of positive blood cultures from a batch-based to real-time and “on-demand” process.

Same-Day Identification and Antimicrobial Susceptibility Testing of Bacteria in Positive Blood Culture Broths Using Short-Term Incubation on Solid Medium with the MicroFlex LT, Vitek-MS, and Vitek2 Systems.

BackgroundEarly and appropriate antibiotic treatment improves the clinical outcome of patients with septicemia; therefore, reducing the turn-around time for identification (ID) and antimicrobial susceptibility test (AST) results is essential. We established a method for rapid ID and AST using short-term incubation of positive blood culture broth samples on solid media, and evaluated its performance relative to that of the conventional method using two rapid ID systems and a rapid AST method.MethodsA total of 254 mono-microbial samples were included. Positive blood culture samples were incubated on blood agar plates for six hours and identified by the MicroFlex LT (Bruker Daltonics) and Vitek-MS (bioMeriéux) systems, followed by AST using the Vitek2 System (bioMeriéux).ResultsThe correct species-level ID rates were 82.3% (209/254) and 78.3% (199/254) for the MicroFlex LT and Vitek-MS platforms, respectively. For the 1,174 microorganism/antimicrobial agent combinations tested, the rapid AST method showed total concordance of 97.8% (1,148/1,174) with the conventional method, with a very major error rate of 0.5%, major error rate of 0.7%, and minor error rate of 1.0%.ConclusionsRoutine implementation of this short-term incubation method could provide ID results on the day of blood culture-positivity detection and one day earlier than the conventional AST method. This simple method will be very useful for rapid ID and AST of bacteria from positive blood culture bottles in routine clinical practice.

English

This study aimed to identify yeasts directly from positive blood cultures by MALDI-TOF mass spectrometry. This was a 5-month prospective study (February to June, 2015) carried out on bottles of blood cultures from in-patients at the Hopital de l’ARCHET at the CHU, Nice. Positive blood culture broth was analysed by MALDI-TOF mass spectrometry using Sodium Dodecyl Sulphate (SDS) as the lysis buffer. Out of 64 samples, 51 (79.6%) were identified by MALDI-TOF mass spectrometry at the species level and 12 strains (18.7%) at the genus level. The isolated yeasts fell into six species: Candida albicans 37.2%, Candida glabrata 31.4%, Candida parapsilosis 15.7%, Candida tropicalis 5.9%, Candida krusei 5.9%, and Candida lusitaniae 3.9%. In comparison, 95.3% of species were identified from cultured colonies, the main ones being C. albicans (37.1%), C. glabrata (30.7%) and C. parapsilosis (16.1%). Identification of yeasts from blood culture bottles by MALDI-TOF mass spectrometry is a fast, reliable technique. However, analysis of colonies remains the best technique for identification and for antifungal imaging in order to refer the patient to the most appropriate therapy. Key words: Yeasts, blood cultures, identii¬cation, MALDI-TOF, mass spectrometry.

Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI- TOF MS) for Early Identification of Septic Patients.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful technique for the rapid identification of bacteria from growing colonies in routine cultures. In this study, we evaluated the feasibility of a 5-hour incubation on solid medium after sub-cultivation of positive blood culture broth without any preparation steps in order to speed up the identification of bacteria. In addition to standard laboratory protocols, a Columbia agar plate with 5% sheep blood was inoculated with 1 drop from the blood culture broth. After a 5-hour incubation period, a colony from the culture plate was submitted to MALDI-TOF MS. A total of 1351 positive blood cultures (1299 monomicrobial and 51 polymicrobial) were analyzed. When compared to routine identification procedure results for positive blood cultures, 79.3% of isolates were correctly identified to the species level. When manufacturer-recommended score values were taken into account, MALDITOF MS correctly identified 98.4% of the isolates to the species level with a score of > 2.0, 89.1% with a score between 1.7 and 2.0, and 75.4% with a score of < 1.7. ln our evaluation, a large majority of the S. aureus (91.5%) and Enterobacteriaceae (87.6%) were correctly identified at the species level. A 5-hour incubation period was found to be associated with moderate identification results for CoNS, Enterococcus spp., and nonfermentative gram negative bacilli, with failure being mostly observed with Streptococcus spp., Candida spp., and other gram positive bacteria. We believe that the performance of MALDI-TOF MS identification after short-term culture is directly related to the sufficient growth of microorganisms at 5 hours.

Evaluation of direct sensitivity testing as a method for early initiation of treatment in Gram-negative sepsis

Purpose: For antibiotic susceptibility results, conventional culture and sensitivity methods take 48 h after a blood culture are flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. The objective of this study was to evaluate direct sensitivity test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 h earlier. Materials and Methods: Blood cultures that flagged positive from 1st January 2015 to 30th June 2015 by BacT/ALERT were stained by Gram stain. All blood cultures with only one kind of Gram-negative bacteria by Gram stain were simultaneously cultured and sensitivity tests put up directly (DST) from the broth using disk-diffusion method according to the British Society of Antimicrobial Chemotherapy guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST). Results of DST (test method) and AST (reference method) were compared for agreements or errors. Results: Of the fifty Gram-negative isolates tested, we observed 91.5% categorical agreement or no error (κ = 0.407, P < 0.001). Conclusions: DST using disk diffusion from positive blood culture broths helps initiate early targeted antibiotic therapy. There is a high concordance between DST and AST.

Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2

The study addresses the utility of Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS) using VITEK MS and the VITEK 2 antimicrobial susceptibility testing (AST) system for direct identification (ID) and timely AST from positive blood culture bottles using a lysis-filtration method (LFM). Between July and December 2014, a total of 140 non-duplicate mono-microbial blood cultures were processed. An aliquot of positive blood culture broth was incubated with lysis buffer before the bacteria were filtered and washed. Micro-organisms recovered from the filter were first identified using VITEK MS and its suspension was used for direct AST by VITEK 2 once the ID was known. Direct ID and AST results were compared with classical methods using solid growth. Out of the 140 bottles tested, VITEK MS resulted in 70.7 % correct identification to the genus and/ or species level. For the 103 bottles where identification was possible, there was agreement in 97 samples (94.17 %) with classical culture. Compared to the routine method, the direct AST resulted in category agreement in 860 (96.5 %) of 891 bacteria-antimicrobial agent combinations tested. The results of direct ID and AST were available 16.1 hours before those of the standard approach on average. The combined use of VITEK MS and VITEK 2 directly on samples from positive blood culture bottles using a LFM technique can result in rapid and reliable ID and AST results in blood stream infections to result in early institution of targeted treatment. The combination of LFM and AST using VITEK 2 was found to expedite AST more reliably.

Preliminary Evaluation of the Research-Use-Only (RUO) iCubate iC-GPC Assay for Identification of Select Gram-Positive Bacteria and Their Resistance Determinants in Blood Culture Broths

The iC-GPC assay (iCubate, Huntsville, AL) provides a molecular option for the rapid, on-demand analysis of positive blood cultures. A preliminary evaluation of the iC-GPC assay using 203 clinical or seeded specimens demonstrated a sensitivity of 93.8% to 100% and a specificity of 98.0% to 100% for the identification of five Gram-positive bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) and three associated genetic resistance determinants (mecA, vanA, and vanB) in positive blood culture broths.

A method for early detection of antibiotic resistance in positive blood cultures: Experience from an oncology centre in eastern India

Purpose: For antibiotic susceptibility results, conventional culture and sensitivity methods takes 48 hours after a blood culture is flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. Objective of this study was to evaluate Direct Sensitivity Test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 hours earlier. Materials and Methods: Blood cultures flagged positive between May 2011 to December 2012 by BacT/ALERT were Gram stained. All uni-microbial gram-negative blood cultures were simultaneously cultured and processed for DST from broth using disk diffusion method using British Society of Antimicrobial Chemotherapy (BSAC) guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST) performed by Vitek-2 on isolated colonies. Results of DST (test method) and AST (reference method) were compared for agreements or errors. Results: Of the 840 antibiotic gram-negative organism combinations tested, Categorical and essential agreements were 83.7% and 96.2% respectively. Minor, major and very major errors were 12.5%, 3.33% and 0.47%, respectively. Conclusions: DST using disk diffusion from positive blood culture broths helps to initiate early targeted antibiotic therapy. There is high concordance between DST and AST.

Multiplex Identification of Gram-Positive Bacteria and Resistance Determinants Directly from Positive Blood Culture Broths: Evaluation of an Automated Microarray-Based Nucleic Acid Test

A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria. 1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., Listeria spp., S. lugdunensis, vanB-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%-100% and 95.4%-100%, respectively. Identification of the mecA gene in 599 cultures containing S. aureus or S. epidermidis was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the vanA gene in 81 cultures containing Enterococcus faecium or E. faecalis was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant S. aureus and vancomycin resistant Enterococcus spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign mecA to a specific organism in cultures containing more than one Staphylococcus isolate and does not identify common blood culture contaminants such as Micrococcus, Corynebacterium, and Bacillus. The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures.

Evaluation of MALDI‐TOF Mass Spectrometry and Sepsityper Kit™ for the Direct Identification of Organisms from Sterile Body Fluids in a Canadian Pediatric Hospital

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.

Rapid identification of Gram-positive pathogens and their resistance genes from positive blood culture broth using a multiplex tandem RT-PCR assay

The early initiation of targeted antibiotic therapy in patients with bacteraemia and septic shock impacts favourably on outcomes. Rapid methods are therefore increasingly employed for bacterial identification directly from positive blood culture bottles, but with variable success. We evaluated the performance of the Gram Positive 12 multiplex tandem PCR (MT-PCR) assay (AusDiagnostics; catalogue no. 6202, version 07) containing targets for the identification of staphylococci including Staphylococcus aureus, streptococci including Streptococcus pneumoniae, enterococci including Enterococcus faecalis and Enterococcus faecium and their common antibiotic resistance genes (mecA, vanA, vanB). A total of 673 aerobic and anaerobic blood culture broths demonstrating Gram-positive cocci on microscopy were analysed in parallel with traditional phenotypic methods. Amplification of the internal control was inhibited in 79/673 (11.7 %) samples; however, MT-PCR identification was in concordance with phenotypic identification to the genus level in 96.6 % (537/556) of the remaining monomicrobial specimens and to the species level, where applicable, in 100 % (172/172) of samples. MT-PCR identification for 94.7 % (36/38) of polymicrobial samples matched traditional phenotypic identification. Meticillin and vancomycin susceptibility results determined by MT-PCR in blood culture broths demonstrated complete agreement with those determined by phenotypic methods in all 143 Staphylococcus aureus isolates and eight E. faecium isolates, respectively. Gram-positive pathogens and their key antibiotic resistance markers were reliably identified with the MT-PCR assay within 3 h of a positive blood culture result.

Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction

In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method.

Direct Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Improves Appropriateness of Antibiotic Treatment of Bacteremia

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01)). Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.

Comparison of an in-house method and the commercial Sepsityper™ kit for bacterial identification directly from positive blood culture broths by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry

The identification of bacteria directly from positive blood cultures using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a new challenge to microbiologists. However, the protocols previously described are often difficult to implement in routine and comparisons are not always possible due to the variability of interpretative criteria. This study evaluated the analytical and practical performances of an in-house (IH) method, adapted from previous protocols, and the Sepsityper™ kit (Bruker Daltonics, Bremen, Germany). Positive blood cultures from 63 different patients were prospectively evaluated by both methods. To enhance the sensitivity of these methods, lowered cut-offs were assessed and validated on 66 additional samples. The IH method produced 86.4% and 73.7% correct genus and species identifications, respectively, when using the lowered cut-offs of 1.4 and 1.6 for correct genus and species identifications. The Sepsityper™ kit showed similar results (78.0% and 68.4% correct genus and species identification, respectively). However, the IH method is ten-fold less expensive than the commercial option (0.72 vs. 7.45 <euro>/analysis) and its turnaround time is approximately 20 min versus the nearly 40 min required for the Sepsityper™ kit, which includes an extraction step. Finally, the IH method was introduced twice-daily in our routine practice.

Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper™ and time of flight mass spectrometry.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper™ Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturer's specified bacterial identification criteria (spectral scores ≥1.700–1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.

Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert(®) SA (bioMérieux), and charcoal-containing BacT/Alert(®) FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert(®) SA, and BacT/Alert(®) FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.

Intact cell MALDI-TOF mass spectrometry-based approaches for the diagnosis of bloodstream infections

MALDI-TOF mass spectrometry (MS) on intact cells is at the forefront of a new revolution in the routine identification of microorganisms in clinical microbiology laboratories. In addition to its high accuracy for identification, it provides results in just a few minutes. As correct and timely diagnoses are pivotal for the management of bloodstream infections, MALDI-TOF MS has the potential to replace or at least complement other methods for microorganism identification in bloodstream infections. With the improvement of databases and software, this method has an accurate identification rate of nearly 100% on nearly all agar-grown microorganisms and more than 80% on positive blood culture broth tests. Even if it is highly efficient now, after a decade of developments, the technique is currently still under improvement. It lacks standardization and exhaustive databases or more efficient softwares that are currently under development. Even now, both the technology itself and the reagents are improving significantly. Thus, it is likely that identifications will become more accurate in the future, especially on more complex fluids, such as blood culture broth.