An improved protocol for bacteria identification by MALDI-TOF MS directly from positive blood cultures.

FASTinov® developed a rapid antimicrobial susceptibility test that includes the purification of a bacterial suspension directly from positive blood cultures (BC). In order to streamline laboratory workflow, the use of the bacterial suspension obtained through FASTinov® sample prep was tested for identification (ID) by matrix absorption laser deionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker) in 364 positive BC, and its accuracy assessed comparing with the MALDI-TOF MS ID of the next-day subcultured colonies. FASTinov sample prep was highly reliable for rapid ID directly from BC with proportion of agreement of 94.9% for Gram-positive and 96.3% for Gram-negative bacteria.

Rapid Detection of Plasmid AmpC Beta-Lactamases by a Flow Cytometry Assay.

Plasmidic AmpC (pAmpC) enzymes are responsible for the hydrolysis of extended-spectrum cephalosporins but they are not routinely investigated in many clinical laboratories. Phenotypic assays, currently the reference methods, are cumbersome and culture dependent. These methods compare the activity of cephalosporins with and without class C inhibitors and the results are provided in 24-48 h. Detection by molecular methods is quicker, but several genes should be investigated. A new assay for the rapid phenotypic detection of pAmpC enzymes of the Enterobacterales group-I (not usually AmpC producers) based on flow cytometry technology was developed and validated. The technology was evaluated in two sites: FASTinov, a spin-off of Porto University (Portugal) where the technology was developed, and the Microbiology Department of Ramón y Cajal University Hospital in Madrid (Spain). A total of 100 strains were phenotypically screened by disk diffusion for the pAmpC with the new 2 h assay. Molecular detection of the pAmpC genes was also performed on discrepant results. Forty-two percent of the strains were phenotypically classified as pAmpC producers using disk diffusion. The percentage of agreement of the flow cytometric assay was 93.0%, with 95.5% sensitivity and 91.1% specificity. Our proposed rapid assay based on flow cytometry technology can, in two hours, accurately detect pAmpC enzymes.

Evaluation of FASTinov Ultrarapid Flow Cytometry Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures.

The FASTinov flow cytometry kit, an ultrarapid antimicrobial susceptibility test, was directly evaluated on positive blood cultures (BC) at two sites: (i) FASTinov, S.A., in Porto, Portugal, using BC spiked with well-characterized bacteria, and (ii) Ramón y Cajal University Hospital in Madrid, Spain, using positive BC from patients. Two kits were evaluated, FASTgramneg (Enterobacterales, Pseudomonas, Acinetobacter) and FASTgrampos (Staphylococcus, Enterococcus). Dedicated software for cytometric data analysis and interpretative reporting, including both CLSI and EUCAST criteria, was used. The FASTgramneg kit also provides information about the presence of resistant mechanisms, including extended-spectrum beta-lactamases (ESBLs) and carbapenemases. After 1 h of incubation at 37°C, bacteria were analyzed using a CytoFLEX cytometer (Beckman, CA). Disk diffusion was performed as the reference susceptibility method. Overall, 447 positive BC were included, 100 from hospitalized patients. Categorical agreement values for the FASTgramneg panel were 96.8% based on EUCAST criteria and 96.4% based on CLSI criteria. For the FASTgrampos panel, categorical agreement was 98.6% when using both criteria. When EUCAST criteria were used, the percentages of errors for the FASTgramneg panel were 2.1% minor errors (mE), 1.3% major errors (ME), and 0.6% very major errors (VME). When CLSI criteria were used, 2.9% mE, 0.9% ME, and 0.4% VME were found. VME were mainly observed with amoxicillin-clavulanate, cefotaxime, ceftazidime, and gentamicin. The FASTgrampos panel showed 0.3% mE, 1.4% ME, and 0.4% VME when EUCAST criteria were used (VME with respect to gentamicin and Staphylococcus) and 0.4% mE, 1.4% ME, and no VME when CLSI criteria were used. The FASTinov flow cytometry kits represent a rapid alternative for direct antimicrobial susceptibility testing from positive BC, showing time to results of <2 h, and can be used to personalize antibiotic and stewardship practices.

A Rapid Flow Cytometric Antimicrobial Susceptibility Assay (FASTvet) for Veterinary Use - Preliminary Data.

A rapid flow cytometric antimicrobial susceptibility test for bacteria isolated from companion animals - the FASTvet assay, developed by FASTinov®, was evaluated. Bacterial strains isolated from different biological samples of companion animals with infectious diseases in progress were obtained from several veterinary clinical laboratories across the country. A total of 115 strains, comprising 65 Gram-negative and 50 Gram positive isolates, were incubated with 13 antimicrobial drugs (ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefpodoxime, imipenem, enrofloxacin, gentamicin, amikacin for Gram-negative; penicillin, cefoxitin, enrofloxacin, vancomycin and ampicillin for Gram-positive) at breakpoint concentrations following CLSI protocol (CLSI Vet 01, 2018) for 1 h and analyzed by flow cytometry. The overall categorical agreement was 95.6% in case of Gram-negative and of 96.7% in Gram-positive isolates when compared to microdilution. FASTvet kits contribute to reduce the turnaround time (2 vs. 24 h) with early determination of the antimicrobial susceptibility profile. The correct and rapid choice of the target antibiotic therapy, will have a positive impact on animal care, contributing for preventing antimicrobial resistance. In conclusion, FASTinov® vet kits showed an excellent performance, both for Gram-negative and Gram-positive isolates encouraging us to enlarge the sample size and planning multicentric studies.

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii.

OBJECTIVES: Both EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay. METHODS: One hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined. RESULTS: Overall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed. CONCLUSIONS: FASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.

Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures.

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit.

Laboratory assessment of antimicrobial susceptibility is a prerequisite for adequate management of infections. The aim of this research was to evaluate the performance of the novel FASTinov® kit for antimicrobial susceptibility testing (AST) of Gram negative bacilli directly on positive blood cultures. One hundred and two positive blood cultures from patients of a Portuguese University Hospital were included. AST were performed with routine method, Vitek2, with FASTinov® kit, and with the gold standard microdilution. Bacteria directly extracted from blood cultures were used to inoculate the FASTinov® kit. Time-to-result as well as the number of patients receiving initially inappropriate therapy (and those in whom de-escalation would have been done) and length of stay (LOS) was recorded. Seventy percent of patients were over 70 years old and 18.6% were admitted in intensive care units. Regarding the isolates, 88.2% were Enterobacteriaceae, 9.8% Pseudomonas spp. and 1% Acinetobacter spp. Extended spectrum ß-lactamases producing-Enterobacteriaceae were found in 7.8% of cases and 10.8% were multi-drug resistant. Fifty-one hours was the mean of time-to-result for routine test (Vitek2) vs. 2 h response regarding Fastinov® test. The overall agreement between FASTinov® and the reference microdilution method was 98%. According to the susceptibility phenotype, 16.7% of patients received initially inappropriate therapy and the mean hospital LOS of these patients was significantly higher. FASTinov® kit revealed an excellent correlation with the AST standard method and provided much earlier results than Vitek2.