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

Abstract

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.

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

“…Here, we describe a new, rapid and inhibitors-based assay to detect pAmpC enzymes by flow cytometry technology. Flow cytometry has proved to be an accurate and rapid technique for the evaluation of the antimicrobial susceptibility profiles of bacteria both in human [ 15 ] and veterinary [ 24 ] fields. Flow cytometry also detects the relevant mechanisms of resistance such as ESBLs or carbapenemases [ 16 ].…”

“…The work described here was performed from pure colonies after sub-culturing in broth media to be in an exponential growth phase, as previously described [ 15 ]. Nevertheless, it has the potential to be used directly on positive blood cultures [ 15 ].…”

“…Rapid phenotypic antimicrobial susceptibility assays are needed, and a disruptive technology based on flow cytometry has been proven with results showing it to be an excellent tool [ 15 ]. Instead of looking for their ability to grow in the presence of the drugs, the cell lesions are detected after short periods of incubation.…”

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“…Silva-Dias et al described the performance of a flow cytometric approach to rapid susceptibility determination utilizing flow to measure the response of bacterial cells to a 1-h exposure to various antibiotics. Compared to disk diffusion, using CLSI criteria, they reported 96.4% (Gram negative) and 98.6% (Gram positive) categorical agreement, with <1% VME for each [ 29 ]. The assay uses a fluorescent probe during antimicrobial exposure to flag bacterial viability and permissivity to fluorescent-molecule uptake to correlate with susceptibility or resistance.…”

Real Life Clinical Impact of Antimicrobial Stewardship Actions on the Blood Culture Workflow from a Microbiology Laboratory

“…Molecular techniques are rapid and sensitive, but to date, they are not affordable for all laboratories [ 7 ]. There are also groups working on the use of several techniques, such as flow cytometry, droplet-based microfluidics, calorimetry, or spectroscopy, in order to obtain AST results in less than 2 h [ 8 , 9 , 10 , 11 , 12 , 13 ].…”