The ultra-rapid antimicrobial susceptibility test FASTinov® flow cytometry kit was directly evaluated on positive blood cultures (BC) at two sites: i) FASTinov® in Porto (Portugal) using spiked BC with well-characterized bacteria and ii) Ramon y Cajal University hospital in Madrid (Spain) using patients positive BC. Two kits were evaluated, FAST gramneg ( Enterobacterales , Pseudomonas , Acinetobacter ) and FAST grampos ( Staphylococcus , Enterococcus ). A dedicated software for cytometric data analysis and interpretative reporting, both using CLSI and EUCAST criteria, were used. The FAST gramneg kit also provides information about the presence of resistant mechanisms, including ESBLs and carbapenemases. After 1-h incubation at 37°C bacteria were analysed by CytoFLEX® cytometer (Beckman, CA). Disk diffusion was performed as reference susceptibility method. Overall, 447 positive BC were included, 100 from hospitalized patients. Categorical agreement for FAST gramneg panel was 96.8% for EUCAST and 96.4% for CLSI. For FAST grampos panel it was 98.6% when using both criteria. Using EUCAST criteria the percentage of errors for FAST gramneg panel was 2.1% minor errors (mE), 1.3% major errors (ME) and 0.6% very major errors (VME). Concerning CLSI, 2.9% mE, 0.9% ME and 0.4% VME were found. VMEs were mainly observed with amoxicillin-clavulanate, cefotaxime, ceftazidime and gentamicin. FAST grampos panel showed 0.3% mE, 1.4% ME and 0.4% VME using EUCAST criteria (VME regarded gentamicin and Staphylococcus ) while 0.4% mE, 1.4% ME and no VME when using CLSI criteria. FASTinov® flow cytometry kits represent a rapid alternative for direct antimicrobial susceptibility testing from positive BC, showing time-to-results <2-h, which can be used to personalized antibiotics and stewardship practices.

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 β-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.

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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.

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