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The Accelerate PhenoTest BC kit is a multiplexed in vitro diagnostic test utilizing both qualitative nucleic acid fluorescence in situ hybridization (FISH) identification and quantitative, antimicrobial susceptibility testing (AST) methods and is intended for use with the Accelerate Pheno system. The Accelerate PhenoTest BC kit is capable of simultaneous detection and identification of multiple microbial targets followed by susceptibility testing of the appropriate detected bacterial organisms. The Accelerate PhenoTest BC kit is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system. Results are intended to be interpreted in conjunction with Gram stain results. The Accelerate PhenoTest BC kit identifies the following Gram-positive and Gram-negative bacteria and yeasts utilizing FISH probes targeting organism-specific ribosomal RNA sequences: Staphylococcus aureus, Staphylococcus lugdunensis, Coagulase-negative Staphylococcus species (i.e., Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus lugdunensis, Staphylococcus warneri, not differentiated), Enterococcus faecalis, Enterococcus faecium, Streptococcus spp. (i.e., Streptococcus mitis, Streptococcus oralis, Streptococcus gallolyticus, Streptococcus agalactiae, Streptococcus pneumoniae, not differentiated), Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated), Serratia marcescens, Candida albicans and Candida glabrata. The Accelerate PhenoTest BC kit tests the following antimicrobial agents with the specific target organisms identified below: Amikacin: Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Ampicillin: Enterococcus faecalis and Enterococcus faecium Ampicillin/Sulbactam: Escherichia coli, Klebsiella spp. (i.e., Klebsiella pneumoniae, . Klebsiella oxytoca, not differentiated), and Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated) Aztreonam: Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated). Proteus spp. (i.e., Proteus mirabilis. Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Ceftazidime: Pseudomonas aeruginosa, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Ceftaroline : Staphylococcus aureus . Cefepime: Pseudomonas aeruginosa, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Ceftriaxone: Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Ciprofloxacin: Pseudomonas aeruginosa, Klebsiella spp. (i.ess, Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Daptomycin: Staphylococcus aureus, Coagulase-negative Staphylococcus species (i.e., Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus lugdunensis, Staphylococcus warneri, not differentiated), Enterococcus faecalis and Enterococcus faecium Erythromycin: Staphylococcus aureus Ertapenem: Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Gentamicin: Pseudomonas aeruginosa, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Linezolid: Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium . Meropenem: Pseudomonas aeruginosa, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis. Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii. Citrobacter koseri, not differentiated) and Serratia marcescens Piperacillin/Tazobactam: Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Tobramycin: Pseudomonas aeruginosa, Klebsiella spp. (i.e., Klebsiella pneumoniae, Klebsiella ● oxytoca, not differentiated), Escherichia coli, Enterobacter spp. (i.e., Enterobacter cloacae, Enterobacter aerogenes, not differentiated), Proteus spp. (i.e., Proteus mirabilis, Proteus vulgaris, not differentiated), Citrobacter spp. (i.e., Citrobacter freundii, Citrobacter koseri, not differentiated) and Serratia marcescens Vancomycin: Staphylococcus aureus, Staphylococcus lugdunensis, Coagulase-negative . Staphylococcus species (i.e., Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus lugdunensis, Staphylococcus warneri, not differentiated), Enterococcus faecalis and Enterococcus faecium The following resistance phenotypes are reported based on qualitative tests: Methicillin-resistance (S. aureus S. lugdunensis, coagulase negative staphylococci) and macrolide-lincosamide-streptogramin B resistance (MLSb) (S. lugdunensis and coagulase negative staphylococci). The Accelerate PhenoTest BC kit is indicated as an aid in the diagnosis of bacteremia and fungemia. It is also indicated for susceptibility testing of specific pathogenic bacteria as identified above commonly associated with or causing bacteremia. Results are intended to be used in conjunction with other clinical and laboratory findings. Standard laboratory protocols for processing positive blood cultures should be followed to ensure availability of isolates for supplemental testing as needed. Additionally, subculture of positive blood culture is necessary for the identification and susceptibility testing of: organisms not identified by the Accelerate PhenoTest BC kit, organisms present in polymicrobial samples, organisms for which species identification is critical for patient care (e.g., speciation of Streptococcus spp.), samples for which an "indeterminate" result for any probe was obtained, for testing antimicrobial agents not included on the Accelerate panel and for epidemiologic testing.

The Accelerate Pheno system is comprised of the Accelerate Pheno instrument, software, host computer, analysis computer, and the Accelerate PhenoTest BC kit. The Accelerate PhenoTest BC Kit contains a sample vial, a 48-channel disposable test cassette and a reagent cartridge needed to test samples from a blood culture bottle that has been flagged as positive by a continuous monitoring blood culture system. All identification (ID) and antimicrobial susceptibility testing (AST) is performed in individual flowcells of the test cassette. The reagent cartridge contains gel electrofiltration (GEF) stations, fluorescence in situ hybridization (FISH) probes, antibiotics, and reagents for automated sample preparation, identification of bacterial and fungal target organisms (Table 1), and antimicrobial susceptibility testing and phenotypic resistance detection testing for bacterial target organisms (Tables 2 and 3). The user loads an aliquot of the positive blood culture into the sample vial, places the test cassette, reagent cartridge and sample vial into an Accelerate Pheno System module, and then presses the module button to close the module door and start the run. The remainder of the operations are automated as described below. Automated Sample Preparation Automated sample preparation is performed using gel electro-filtration (GEF) which is based on gel electrophoresis principles. The sample is automatically transferred to a gel containing pores smaller than bacterial or yeast cells. Application of an electric field causes lysed blood cells and/or other sample debris to pass into the gel while bacterial/yeast cells remain inside the gel well. The electric field is briefly reversed to dislodge bacterial/yeast cells from the gel wall prior to removal. Cell Capture Following sample preparation, recovered cells are automatically pipetted into multiple flowcell channels of the test cassette. Conductive layers of transparent indium tin oxide (ITO) coat the top and bottom inner surfaces of each flowcell channel and act as electrodes. An additional cationic poly-Llysine layer on the bottom of each flowcell acts as a capture surface. When a voltage is applied, the negatively-charged bacterial/yeast cells migrate to the positively-charged capture surface where they are captured prior to imaging. Fluorescence in situ Hybridization (FISH) for Identification Cocktails of ATTO-532 (green) fluorescently-labeled DNA probes bind to the ribosomal RNA of target organisms following permeabilization. Each cocktail also includes ATTO-647 (red) labeled universal bacterial probe that binds to the ribosomal RNA of all clinically relevant bacterial ID channels) or universal eukaryotic probe that binds to the ribosomal RNA of all clinically relevant yeast (yeast ID channels). The system images each flowcell using an epifluorescence microscope with camera at 532 nm, 647 nm and in dark-field. To exclude debris, only dark-field objects that are colocalized with universal probe signal are included in analysis. Colocalization of target probe signal and universal probe signal identifies a target organism. The software also quantitates the total number of organisms present in a sample using a nucleic acid stain in a separate control flowcell. Comparing the relative numbers of each target organism to the number of objects lit up by the universal probes and universal nucleic acid stain allows for non-target organism and polymicrobial sample detection. FISH ID results are reported approximately 90 minutes after loading the sample, and the ID result determines the selection of appropriate antibiotics for subsequent antimicrobial susceptibility testing. Morphokinetic Cellular Analysis (MCA) for Antimicrobial Susceptibility Testing (AST) Sample remaining after the identification assay is initiated is combined with growth media and organisms contained in the sample undergo a pre-growth step during the FISH ID assay to normalize growth rates prior to AST. Following automated sample preparation and cell capture, growth media containing single concentrations of each test antibiotic are added to separate flowcell channels; antibiotics are selected based on the identification provided by the FISH identification (Tables 2 and 3). The bacteria in each flowcell are imaged every 10 minutes for up to 4.5 hours, creating a timelapse record of bacterial growth from individual progenitor cells into clones of daughter cells. During this period. several microscopic features are measured through morphokinetic cellular analysis, such as cell morphology and the light intensity of a growing clone over time, and used for analysis. The precise quantitative measurement of individual clone growth rate over time is an indicator of antimicrobial efficacy. Onboard software algorithms derive minimum inhibitory concentration (MIC) values from the measured features, and apply appropriate expert rules for proper interpretation and reporting of categorical interpretations - S, I or R (susceptible, intermediate, or resistant) for MIC determinations and positive or negative for phenotypic resistance markers. AST results are reported in approximately 5 hours after ID results. The reportable ranges for each antimicrobial and phenotypic resistance markers are listed in Tables 4 and 5.