Accelerate Pheno system, Accelerate Phenotest BC Kit
Device Facts
| Record ID | DEN160032 |
|---|---|
| Device Name | Accelerate Pheno system, Accelerate Phenotest BC Kit |
| Applicant | Accelerate Diagnotics |
| Product Code | PRH · Microbiology |
| Decision Date | Feb 23, 2017 |
| Decision | DENG |
| Submission Type | Direct |
| Regulation | 21 CFR 866.1650 |
| Device Class | Class 2 |
Intended Use
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 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.
Device Story
Device performs multiplexed in vitro diagnostic testing on positive blood culture samples; utilizes FISH probes for organism identification and quantitative AST methods. Operates within Accelerate Pheno system. Input: positive blood culture samples. Process: simultaneous detection/identification of microbial targets via ribosomal RNA-specific FISH probes; subsequent quantitative susceptibility testing of identified bacteria. Output: identification of specific pathogens and antimicrobial susceptibility/resistance profiles. Used in clinical laboratory settings by trained personnel. Results interpreted alongside Gram stain and other clinical findings to guide antimicrobial therapy. Benefits: rapid identification and susceptibility results to aid in timely, appropriate treatment of bacteremia/fungemia.
Clinical Evidence
Multi-center clinical study (13 sites, 1850 samples: 793 fresh, 65 fresh seeded, 477 challenge, 515 stock). ID assay sensitivity/PPA >95% and specificity/NPA >99% for most targets. AST performance evaluated against CLSI broth microdilution; EA/CA >90% for most antimicrobial/organism combinations. Mean time to ID: 1.38 hours; mean time to AST: 6.69 hours.
Technological Characteristics
Multiplexed in vitro diagnostic test; utilizes FISH probes targeting ribosomal RNA sequences for identification; quantitative AST methods. System-based (Accelerate Pheno). Requires positive blood culture samples. Includes software for analysis and interpretation. Subject to 21 CFR 820 design controls.
Indications for Use
Indicated for patients with suspected bacteremia or fungemia. Used with positive blood culture samples to identify specific Gram-positive/negative bacteria and yeasts and perform antimicrobial susceptibility testing (AST) on identified pathogens.
Regulatory Classification
Identification
A cellular analysis system for multiplexed antimicrobial susceptibility testing is a multiplex qualitative and/or quantitative in vitro diagnostic device intended for the identification and determination of the antimicrobial susceptibility results of organisms detected in samples from patients with suspected microbial infections. This device is intended to aid in the determination of antimicrobial susceptibility or resistance when used in conjunction with other laboratory findings.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) Design verification and validation must include: (i) Detailed device description documentation, including the device components, ancillary reagents required but not provided, a detailed explanation of the methodology, including primer/probe sequence, design, rationale for sequence selection, and details of the antimicrobial agents, as applicable. (ii) Detailed documentation from the following analytical and clinical performance studies: limit of detection, inclusivity, precision, reproducibility, interference, cross-reactivity, carryover, and cross-contamination, quality control and additional studies, as applicable to specimen type and assay intended use. (iii) Detailed documentation from an appropriate clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted reference methods. (iv) Detailed documentation for device software, including software applications and hardware-based devices that incorporate software. (2) The labeling required under § 809.10(b) of this chapter must include: (i) Limitations and protocols regarding the need for correlation of results by standard laboratory procedures, as applicable. (ii) A detailed explanation of the interpretation of results and acceptance criteria. (iii) A detailed explanation of the principles of operation and procedures for assay performance and troubleshooting.
Related Devices
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- K221688 — ASTar BC G- Kit and ASTar Instrument · Q-Linea AB · Apr 26, 2024
Submission Summary (Full Text)
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## EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Accelerate PhenoTest BC Kit
## DECISION SUMMARY
### A. DEN Number:
DEN160032
### B. Purpose for Submission:
De novo request for evaluation of automatic class III designation for the Accelerate PhenoTest BC Kit.
### C. Measurand:
The following Gram-positive and Gram-negative bacteria and yeasts are identified using the Accelerate PhenoTest BC kit Staphylococcus aureus, Staphylococcus lugdunensis, Coaeulasenegative Staphylococcus species (i.e., Staphylococcus evidermidis, Staphylococcus haemolvticus, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus lugdunensis, Staphylococcus warnerii, 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 as appropriate for the identified organism (see section H below): amikacin, ampicillin/sulbactam, aztreonam, ceftazidime, ceftaroline, ceftriaxone, ciprofloxacin, daptomycin, erythromycin, ertapenem, gentamicin, linezolid, meropenem, piperacillin/tazobactam, tobramycin and vancomycin.
The following resistance phenotypes are reported based on qualitative tests as appropriate for the identified organism (see section H below): Methicillin-resistance and macrolide-lincosamidestreptogramin B resistance (MLSb).
## D. Type of Test:
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 PhenoTest BC assay is performed directly on positive blood culture samples identified as positive by a continuous monitoring blood culture system.
## E. Applicant:
Accelerate Diagnostics
- F. Proprietary and Established Names: Accelerate PhenoTest BC Kit
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Accelerate Pheno system
## G. Regulatory Information:
- 1. Regulation section: 866.1650
- 2. Classification: Class II
- 3. Product code: PRH, NSU, PEO, PAM, PEN, LON
- 4. Panel: 83 (Microbiology)
## H. Indications for Use:
- 1. Indications for Use:
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
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- . 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.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
- . 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
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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.
- Special conditions for use statement(s): 2. For prescription use only
Limitations:
General Limitations
- This product can only be used with the Accelerate Pheno system.
- . The Accelerate PhenoTest BC kit assay has not been evaluated for specimens other than blood (e.g., sterile body fluids inoculated into blood culture bottles)
- . The performance of this test has only been evaluated using the following blood culture bottles:
- BD BACTEC Standard/10 Aerobic/F Medium, O
- BD BACTEC Standard/10 Anaerobic/F Medium, о
- BD BACTEC Lytic/10 Anaerobic/F Medium O
- BD BACTEC PEDS PLUS/F Medium O
- BD BACTEC Plus Aerobic/F Medium O
- BD BACTEC Plus Anaerobic/F Medium O
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- BioMérieux BacT/ALERT SA Standard Aerobic O
- BioMérieux BacT/ALERT SN Standard Anaerobic O
- o BioMérieux BacT/ALERT FA Plus Aerobic
- BioMérieux BacT/ALERT FN Plus Anaerobic O
- BioMérieux BacT/ALERT PF Plus O
- о Versa TREK REDOX 1 (Aerobic) Medium and
- Versa TREK REDOX 2 (Anaerobic) Medium O
- This product should not be used with blood culture bottles containing charcoal.
- Positive blood culture samples must be run using the Accelerate PhenoTest BC kit on the ● Accelerate Pheno system within 8 hours of sample positivity.
- . Positive blood culture sample must be loaded on the Accelerate Pheno system and the run must be initiated within 15 minutes of pipetting sample vial and within 1 hour of removing the assay kit from refrigerated storage.
- . Failure to observe proper procedures for sample collection, preparation, storage, handling and/or transportation may cause incorrect results.
Identification (ID) Limitations
- Due to the possibility of cross reactivity, all Accelerate PhenoTest BC kit results should ● be interpreted in conjunction with Gram stain.
- . The ability of probes to detect all strains of a target species was not predicted by in-silico analysis.
- . Additional subculture is required for the identification of S. pneumoniae in cases of a positive Streptococcus spp. call.
- . Subculture of positive blood culture is required in the following situations:
- O For the identification and susceptibility testing of off-panel organisms not identified by the Accelerate PhenoTest BC kit,
- For samples that give a polymicrobial result о
- For organisms for which species identification is critical for patient care. (e.g., о speciation of streptococci)
- For testing antimicrobial agents not included on the Accelerate panel O
- For testing certain antimicrobial agents as discussed in AST limitations below O
- For testing samples for which an "indeterminate" result for any probe was O obtained
- To obtain isolates for epidemiologic testing. o
- Accelerate PhenoTest BC kit identification results that are discordant with the result of . the blood culture Gram stain (for example, no organism detection when the Gram stain is positive or detection of a Gram-positive cocci when Gram-positive cocci were not observed in the Gram stain) should be confirmed by an alternate technique prior to reporting the test result. For some polymicrobic calls, false positive results may not be mitigated by Gram stain analysis (for example, detection of two Enterobacteriaceae species with Gram-negative rods observed in the Gram stain). Results of such polymicrobic calls should be verified by subculture and/or an alternative identification method.
Antimicrobial Susceptibility Testing (AST) Limitations
- Due to insufficient number of test isolates, the ability of the Accelerate PhenoTest BC kit to detect inducible MLSb resistance in coagulase-negative staphylococci is unknown when used with the following blood culture bottle types: BacT/Alert SN Standard Anaerobic, BACTEC Peds Plus/F, BACTEC Plus Anaerobic/F, BACTEC Standard Anaerobic, BACTEC Standard/10 Aerobic, VersaTrek Redox 1 Aerobic, VersaTrek
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Redox 2 Anaerobic. Use an alternative method for detection of inducible MLSb resistance when using these blood culture bottle types if critical to patient care.
- . Susceptibility testing of monomicrobial samples will only be performed when on-panel species eligible for susceptibility testing are detected. See intended use.
- Susceptibility testing of polymicrobial samples will only be performed on one organism out of a pair of species that meet all of the following criteria:
- One or both organisms must be on the Accelerate PhenoTest BC kit test panel and eligible for AST testing (except Proteus spp.)
- The two organisms must have distinct growth responses or morphological o differences such that growing clones from each species can be differentiated by the software. These include the following pairs:
Polymicrobial Testing Pairs
| Organism 1 | Organism 2 |
|---------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------|
| • AST-eligible organism<br>(except Proteus spp.) | • AST-ineligible organism |
| • Escherichia coli<br>• Klebsiella spp.<br>• Enterobacter spp.<br>• Citrobacter spp.<br>or<br>• Serratia marcescens | • Staphylococcus aureus<br>• Coagulase-negative Staphylococcus spp.<br>• Enterococcus spp. (E. faecalis, E. faecium)<br>or<br>• Pseudomonas aeruginosa |
| • Acinetobacter baumannii | • Enterococcus spp. (E. faecalis, E. faecium) |
- Only one organism when diluted for AST must be within the required O concentration limits for AST testing (10-130 growing clones per field of view).
If the concentration ratio between organisms is such that only one organism could be diluted to the concentration range required for AST testing, AST testing will only be performed on the higher concentration organism. If one of the organisms in the pair is eligible for AST testing and the other is not, AST testing will only be performed on the on-panel AST-eligible organism. If both organisms are eligible for AST testing and are within the required concentration limits for AST testing, AST results will not be reported.
- . If an AST result is not provided by the Accelerate PhenoTest BC kit, susceptibility testing must be performed using an alternative method.
- Subculturing of positive blood culture is necessary for the identification and . susceptibility testing of organisms not identified by the Accelerate PhenoTest BC kit and for antimicrobial agents not included on the Accelerate panel.
- . Potential interference by antimicrobial agents that may be present in a patient blood specimen has not been established with the Accelerate PhenoTest BC kit.
- . The ability of the Accelerate PhenoTest BC kit to detect resistance in the following combinations is unknown because an insufficient number of resistant isolates were encountered at the time of comparative testing:
- o Amikacin: Citrobacter spp., Enterobacter spp., E. coli, Proteus spp., S. marcescens
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- Aztreonam: Proteus spp., S. marcescens O
- Cefepime: Citrobacter spp., Proteus spp., S. marcescens O
- o Ceftazidime: Proteus spp., S. marcescens
- Ceftaroline: S. aureus O
- Ceftriaxone: Citrobacter spp., E. cloacae, S. marcescens O
- O Ciprofloxacin: Citrobacter spp., Proteus spp., S. marcescens
- Daptomycin: S. aureus O
- Ertapenem: Citrobacter spp., Proteus spp., and S. marcescens O
- Gentamycin: Citrobacter spp., Enterobacter spp., Proteus spp., S. marcescens o
- Meropenem: Citrobacter spp., E. coli, Proteus spp., and S. marcescens O
- Piperacillin/Tazobactam: Proteus spp., and S. marcescens о
- Tobramycin: Citrobacter spp., Proteus spp., S. marcescens O
- Cefoxitin for Phenotypic Resistance: S.lugdunensis о
- MLSb: S. lugdunensis O
- The following antimicrobial/organism combinations may produce a resistant result that ● can be found susceptible by the reference method. If critical to patient care confirm these results with an alternate method:
- o Meropenem: Enterobacter
- Ceftazidime: Pseudomonas aeruginosa (Any P. aeruginosa isolate that provides O an MIC ≥16 ug/mL should be retested using an alternate method)
- o Cefepime: Pseudomonas aeruginosa
- Ertapenem: Enterobacter spp. o
- Piperacillin/Tazobactam: Acinetobacter baumannii, Klebsiella spp. o
- The ability of the Accelerate PhenoTest BC kit to provide accurate MICs with amikacin ● resistant strains of A. baumannii has not been established; isolates of this species that provide resistant results should be confirmed by an alternative method.
- Due to a low essential agreement for Serratia marcescens with ceftriaxone, results should . be confirmed with an alternate method if critical to patient care.
- The current absence of data on daptomycin-resistant isolates precludes defining any . categories other than "susceptible." Isolates yielding test results suggestive of a nonsusceptible category should be retested and if the result is confirmed, the isolate should be retested using the reference method.
- . The ability of the Accelerate PhenoTest BC kit to detect vancomycin-intermediate Staphylococcus aureus isolates (VISA) is unknown because insufficient numbers of VISA isolates were evaluated at the time of comparative testing.
- Any S. aureus isolate for which the vancomycin MIC is >= 8 ug/mL should be sent to a ● reference laboratory for reference method testing.
- Any coagulase negative Staphylococcus isolate for which the vancomycin MIC is >= 32 . ug/mL should be sent to a reference laboratory for reference method testing.
- The ability of the Accelerate PhenoTest BC kit to provide accurate results for S. aureus ● with MLSb as compared to the reference method has not been established; isolates of this species should be tested by an alternative method.
## 3. Special instrument requirements:
The Accelerate PhenoTest BC Kit is performed on the Accelerate Pheno System.
## I. Device Description:
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
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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.
| Probe Set | On-panel Species |
|-----------|-------------------------|
| ABA | Acinetobacter baumannii |
| CAL | Candia albicans |
| CGL | Candida glabrata |
| CIT | Citrobacter freundii |
#### Table 1. Probe Sets and Species Identified by Each Probe Set
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| Probe Set | On-panel Species |
|-----------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| CNS | Citrobacter koseri<br>Staphylococcus epidermidis<br>Staphylococcus haemolyticus<br>Staphylococcus hominis<br>Staphylococcus capitis<br>Staphylococcus lugdunensis<br>Staphylococcus warneri |
| ECO | Escherichia coli |
| EFM | Enterococcus faecium |
| EFS | Enterococcus faecalis |
| ENT | Enterobacter aerogenes<br>Enterobacter cloacae |
| KLE | Klebsiella oxytoca<br>Klebsiella pneumoniae |
| PAE | Pseudomonas aeruginosa |
| PRO | Proteus mirabilis<br>Proteus vulgaris |
| SAU | Staphylococcus aureus |
| SLU | Staphylococcus lugdunensis |
| SMA | Serratia marcescens |
| STR | Streptococcus mitis<br>Streptococcus oralis<br>Streptococcus gallolyticus<br>Streptococcus agalactiae<br>Streptococcus pneumoniae |
## 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.
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## Algorithm for Performance of AST
For samples determined to be monomicrobic (only one pathogen detected), susceptibility testing will only be performed when one of the on-panel species eligible for susceptibility testing are detected. The following limitation is included in the device labeling:
Susceptibility testing of monomicrobial samples will only be performed when on-panel species eligible for susceptibility testing are detected.
For samples determined to be polymicrobic, susceptibility testing will only be performed on one organism out of a pair of species that meet all of the following criteria:
- One or both organisms must be on the Accelerate PhenoTest BC kit test panel and . eligible for AST testing (except Proteus spp.)
- The two organisms must have distinct growth responses or morphological differences . such that growing clones from each species can be differentiated by the software. These include the following pairs:
| Organism 1 | Organism 2 |
|-----------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| AST-eligible organism (except Proteus spp.) | AST-ineligible organism |
| Escherichia coli Klebsiella spp. Enterobacter spp. Citrobacter spp. or<br>Serratia marcescens | Staphylococcus aureus Coagulase-negative Staphylococcus spp. Enterococcus spp. (E. faecalis, E. faecium or other Enterococcus spp.) or<br>Pseudomonas aeruginosa |
| Acinetobacter baumannii | Enterococcus spp. (E. faecalis, E. faecium) |
- Only one organism when diluted for AST must be within the required concentration . limits for AST testing (10-130 growing clones per field of view).
If the concentration ratio between organisms is such that only one organism could be diluted to the concentration range required for AST testing will only be performed on the higher concentration organism. If one of the organisms in the pair is eligible for AST testing and the other is not, AST testing will only be performed on the on-panel AST-eligible organism. If both organisms are eligible for AST testing and are within the required concentration limits for AST testing. AST results will not be reported.
Because the PhenoTest BC Kit will not be capable of performing AST testing on all organisms present in a positive blood culture, subculture is required to recover the additional organism(s) and to allow performance of AST by an alternate method. In addition, subculture is required to recover organisms not detected by the PhenoTest BC Kit and for testing antimicrobial agents not included in the kit. The following limitations are included in the device labeling:
If an AST result is not provided by the Accelerate PhenoTest BC kit, susceptibility testing must be performed using an alternative method.
{10}------------------------------------------------
Subculture of positive blood culture is required in the following situations:
- o For testing antimicrobial agents not included on the Accelerate Panel
- o For testing certain antimicrobial agents as discussed in AST limitations.
## Table 2. Antimicrobial Agents Tested*, Gram-Positive Organisms
| Organism | Ampicillin | Ceftaroline | Erythromycin | Daptomycin | Linezolid | Vancomycin | Cefoxitin | MLSb |
|-----------------------------------|------------|-------------|--------------|------------|-----------|------------|-----------|------|
| S. aureus | - | X | X | X | X | X | X | - |
| S. lugdunensis | - | - | - | - | - | X | X | X |
| Coagulase negative Staphylococcus | - | - | - | X | - | X | X | X |
| Enterococcus faecalis | X | - | - | X | X | X | - | - |
| Enterococcus faecium | X | - | - | X | X | X | - | - |
*(X) = Tested, (-) = Not tested
## Table 3. Antimicrobial Agents Tested*, Gram-Negative Organisms
| Organism | Ampicillin-<br>sulbactam | Piperacillin-<br>tazobactam | Cefepime | Ceftazidime | Ceftriaxone | Ertapenem | Meropenem | Amikacin | Gentamicin | Tobramycin | Ciprofloxacin | Aztreonam |
|-------------------|--------------------------|-----------------------------|----------|-------------|-------------|-----------|-----------|----------|------------|------------|---------------|-----------|
| E. coli | X | X | X | X | X | X | X | X | X | X | X | X |
| Klebsiella spp. | X | X | X | X | X | X | X | X | X | X | X | X |
| Enterobacter spp. | - | X | X | X | X | X | X | X | X | X | X | X |
| Proteus spp. | X | X | X | X | X | X | X | X | X | X | X | X |
| Citrobacter spp. | - | X | X | X | X | X | X | X | X | X | X | X |
| S. marcescens | - | X | X | X | X | X | X | X | X | X | X | X |
| P. aeruginosa | - | X | X | X | - | - | X | X | X | X | X | X |
| A. baumannii | - | X | - | - | - | - | - | X | - | - | - | - |
*(X) = Tested, (-) = Not tested
{11}------------------------------------------------
| Antimicrobial | Organisms Tested | Reportable Range in<br>PhenoTest BC<br>(µg/mL) | FDA Breakpoints | | |
|--------------------------|-----------------------------------------------------------------|------------------------------------------------|-----------------|-------|----------|
| | | | S | I | R |
| <b>Gram-Positive</b> | | | | | |
| Ampicillin | Enterococcus spp. | <2 to ≥ 32 | ≤8 | - | ≥ 16 |
| Ceftaroline | <i>S. aureus</i> | ≤0.25 to ≥ 8 | ≤1 | 2 | ≥4 |
| Daptomycin | <i>Staphylococcus spp.</i> | ≤0.25 to ≥ 4 | ≤1 | - | ≥2 |
| | <i>Enterococcus spp.</i> | < 1 to ≥ 16 | ≤4 | - | ≥8 |
| Erythromycin | <i>S. aureus</i> | <0.125 to ≥ 16 | ≤0.5 | 1 - 4 | ≥8 |
| Linezolid | <i>S. aureus</i> | < 1 to ≥ 16 | ≤4 | - | >8 |
| | <i>Enterococcus spp.</i> | ≤0.5 to ≥ 16 | ≤2 | 4 | ≥8 |
| | <i>S. aureus</i> | ≤0.5 to ≥ 32 | ≤2 | 4 - 8 | ≥16 |
| Vancomycin | <i>S. lugdunensis and Coagulase<br/>Negative Staphylococcus</i> | < 1 to ≥ 64 | ≤4 | 8-16 | ≥32 |
| <b>Gram-Negative</b> | | | | | |
| Amikacin | <i>Enterobacteriaceae</i> | < 4 to ≥ 128 | ≤16 | 32 | ≥ 64 |
| | <i>P. aeruginosa</i> | | | | |
| | <i>A. baumannii</i> | | | | |
| Ampicillin/Sulbactama | <i>Enterobacteriaceae</i> | ≤ 2 to ≥ 64 | ≤8 | 16 | ≥32 |
| Aztreonam | <i>Enterobacteriaceae</i> | < 1 to ≥ 32 | ≤4 | 8 | ≥ 16 |
| | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | ≤ 2 to ≥ 64 | ≤8 | 16 | ≥ 32 |
| Cefepimeb | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | < 1 to ≥ 64 | <2 | 4 - 8 | ≥ 16 |
| | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | ≤ 2 to ≥ 64 | ≤8 | - | ≥ 16 |
| Ceftazidimec | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | < 1 to ≥ 32 | ≤4 | 8 | ≥ 16 |
| | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | ≤ 2 to ≥ 64 | ≤8 | - | ≥ 16 |
| Ceftriaxoned | <i>P. aeruginosa</i> | | | | |
| Ceftriaxoned | <i>Enterobacteriaceae</i> | ≤0.25 to ≥ 8 | ≤1 | 2 | ≥4 |
| Ciprofloxacine | <i>Enterobacteriaceae</i> | ≤0.25 to ≥ 8 | ≤1 | 2 | ≥4 |
| | <i>P. aeruginosa</i> | | | | |
| Ertapenem | <i>Enterobacteriaceae</i> | ≤0.125 to ≥4 | ≤0.5 | 1 | ≥2 |
| Gentamycin | <i>Enterobacteriaceae</i> | < 1 to ≥ 32 | ≤4 | 8 | ≥ 16 |
| | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | ≤0.25 to ≥ 8 | ≤1 | 2 | ≥4 |
| Meropenemf | <i>P. aeruginosa</i> | | | | |
| | <i>Enterobacteriaceae</i> | ≤0.5 to ≥ 16 | ≤2 | 4 | ≥8 |
| Meropenemf | <i>P. aeruginosa</i> | | | | |
| Piperacillin/Tazobactamg | <i>Enterobacteriaceae</i> | < 4 to ≥ 256 | ≤16 | 32- | ><br>128 |
| | <i>P. aeruginosa</i> | | | 64 | |
| | <i>A. baumannii</i> | | | | |
| Tobramycin | <i>Enterobacteriaceae</i> | < 1 to ≥ 32 | ≤4 | 8 | ≥ 16 |
| | <i>P. aeruginosa</i> | | | | |
## Table 4. Reportable MIC Ranges and Organism-Specific Breakpoints for Antimicrobials Included in the PhenoTest BC
4 Truncated Range for ampicillin/sulbactam: PRO (4-64)
6 Truncated Range for cefepime: PAE (2-32)
€ Truncated Range for ceftazidime: CIT, ECO, ENT, KLE, PAE (2-32)
4 Truncated Range for ceftriaxone: PRO, SMA (0.5-8)
& Truncated Range for ciprofloxacin: ENT (0.5-8)
f Truncated Range for meropenem: ENT, PAE (1-16)
§ Truncated Range for piperacillin/tazobactam: PAE (8-256)
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| Resistance Phenotype Test | ID Target | | |
|---------------------------------|-----------|---------|---------|
| | SAU | SLU | CNS |
| Cefoxitin (MRSA) | Pos/Neg | Pos/Neg | Pos/Neg |
| Erythromycin/Clindamycin (MLSB) | N/A | Pos/Neg | Pos/Neg |
Table 5. Results Applicable to Phenotypic Resistance Tests
## Performing the Assay
The sample is collected from a positive blood culture using a sterile collection device. Blood culture samples should be tested within eight hours of the positive detection as determined by the continuous monitoring blood culture system. Samples may be stored at room temperature or the blood culture bottle can remain in the blood culture instrument until the sample is withdrawn for testing. The blood culture sample is vortexed and 0.5 mL is added to the sample vial. The test must be initiated within 15 minutes of placing the blood culture sample vial. The sample vial is inserted into the sample vial receptacle on the reagent cartridge, and the test cassette and reagent cartridge are placed into the Pheno instrument.
## Test Results and Interpretation
Identification Assay. When the assay is complete, the Accelerate Pheno system displays the final results in a Patient Report or a Quality Control report. The "Identification Results" section lists all target groups tested and indicates which target group was detected. Target groups with positive and/or indeterminate results are printed at the top of the report in bold font. Identification results can be as follows: Positive (target group detected); Negative (target group not detected); and Indeterminate (result not defined, target group may or may not be present). An "invalid" result (a test for which too few cells are present in the control channel) is not specifically reported; for samples with invalid results the Patient Report will indicate "No ID results reported; too few cells for analysis. Perform alternate testing method for identification and susceptibility results."
When the PhenoTest BC kit detects an organism present in a positive blood culture for which there is no target probe in the PhenoTest BC kit (off-panel organism), the Patient Report indicates negative results for all target probes and instructs the user to perform an alternate testing method.
For samples for which two or more target species are identified, the Patient Report will recommend that culture should be performed "Recommend culture due to possibility of another organism being present."
The PhenoTest BC Kit does not provide speciation of Streptococcus spp. If the result indicates the presence of a Streptococcus species, and that additional testing should be performed to rule out Streptococcus pneumoniae. The following limitation is included in the device labeling:
Subculture is required for the identification of S. pneumoniae in cases of a positive Streptococcus spp. call.
AST Assay. Antimicrobial susceptibility results are listed on the Patient Report for a single target species. Only one AST result will be reported for any culture; susceptibility testing for additional organisms (if present) should be performed using an alternate method from subcultured sample. The following limitation is included in the device labeling:
{13}------------------------------------------------
If an AST result is not provided by the Accelerate PhenoTest BC kit, susceptibility testing must be performed using an alternative method.
The AST results included in the PhenoTest BC report will indicate the MIC, the breakpoints, and interpretive categories for the drugs for which testing was performed. Results for resistance phenotypes (cefoxitin and MLSb) are reported as positive or negative.
## Quality Control.
For the identification assay the Quality Control Report indicates an overall status of "failed" and will list the identification results and pass/fail status for each tested species based on detection (positive or negative) and correct identification of the QC strain. For the AST assay the Quality Control report lists the QC range, MIC and pass/fail status for each tested antimicrobial agent. Pass/fail status is determined by the whether the MIC result falls within the QC range (pass) or outside of the QC range (fail).
## Gram Stain Correlation
All identification results provided by the PhenoTest BC kit are intended to be interpreted in conjunction with results obtained from Gram stain of the positive bottle. Gram reaction (gram-positive or gram-negative) and cellular morphology (gram-positive cocci in clusters, pairs or chains, gramnegative rods) should be considered in the correlation with PhenoTest BC results. Gram stain results showing gram-negative cocco-bacilli may indicate the presence of Acinetobacter baumannii; however the cellular morphology is inconsistent or difficult to interpret for members of this genus. If the Gram stain results differ from the expected Gram stain morphology for the organism(s) identified by the PhenoTest BC kit, results should be confirmed with an alternate method. In addition, for any sample for which PhenoTest BC has indicated a monomicrobial result and for which the Gram stain of the positive blood culture shows multiple morphologies, results should be confirmed with an alternate method. All Patient Reports include the following footnote:
Identification results that are discordant with Gram stain should be confirmed with an alternate method.
## Monomicrobial Call
The PhenoTest BC kit will report a monomicrobial call for samples for which a target organism is identified and for which there is no additional evidence of an additional organism (on- or off-panel). Results of all monomicrobial calls should be correlated with results from Gram stain of the positive blood culture bottle. Patient results for samples that are considered monomicrobial will include the following footnote:
Monomicrobial: sample positive for only one pathogen
## J. Standard/Guidance Document Referenced:
- CLSI EP-05-A3, Evaluation of Precision Performance of Quantitative Measurement, Approved ● Guideline, 2004
- CLSI M100-S26, Performance Standards for Antimicrobial Susceptibility Testing, 2015 ●
- CLSI M02-A12, Performance Standards for Antimicrobial Disk Susceptibility Testing, 2015 ●
- CLSI EP07-A2, Interference Testing in Clinical Chemistry, Approved Guideline, 2005 ●
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- CLSI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, Approved Guideline, 2012
- . CLSIM07-A10. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard, 2015
- . EN 62304-2006, Medical Device Software - Software Life-Cycle Processes, 2006
## K. Test Principle:
The Accelerate Pheno system uses an automated sample preparation and surface immobilization method to enable microscopy-based. single-cell analysis for identification (ID) and antimicrobial susceptibility testing (AST). Identification is accomplished via fluorescence in situ hybridization (FISH). Antimicrobial susceptibility testing uses microscopic observation of individual, live, growing bacterial cells in near real time (approximately every 10 minutes) in the presence of antimicrobial agents.
The Accelerate Pheno system employs automated sample and reagent pipetting, temperature controlled incubation, digital microscopy, image acquisition and analysis in an integrated and fully automated system.
## L. Performance Characteristics:
## 1. Analytical performance:
#### a. Precision/Reproducibility:
Reproducibility studies of the Accelerate PhenoTest BC Kit for positive blood culture organism identification (ID) and antimicrobial susceptibility testing (AST) included evaluation of four replicates of the same positive blood culture sample on the same day at three testing sites for a total of 12 tests per blood culture sample. (Four replicates were initially tested to assure at least three valid results for each sample.) Samples were tested within eight hours of positivity. Primary probe targets utilized for reproducibility testing of the ID assay included representative species of each major organism group (S. aureus, S. pneumoniae, E. faecium, E. coli, A. baumannii and C. albicans). Additional species were tested in order to obtain the expected number of AST results (K. pneumoniae, P. aeruginosa, Citrobacter spp., Enterobacter spp., coagulase negative Staphylococcus, Proteus spp., and S. marcescens, and E. faecalis). A minimum of 90 data points were evaluated for each probe target.
The reproducibility of the organism identification assay was assessed on a per probe basis across all study sites and within each study site. A minimum of nine ID results (three per site) was evaluated for each sample. The ID results were classified as "correct" or "not correct" by comparing the observed ID to the known ID for each test strain.
Isolates that provided at least three ID results per site but which failed to produce at least three AST results per site were retested. All ID results were included in the analysis of probe performance. In order not to confound ID probe performance with overrepresentation of a single species, weighted percentages were calculated.
For the identification assay, 11 of the 12 probe targets showed reproducibility > 95%. The Enterobacter probe (ENT) initially showed a reproducibility of 87.5% due to false negative results. Root cause analysis resulted in a post study imaging processing change; the original data was reevaluated with regression analysis. The resulting reproducibility was 93.2%
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## (Table 6).
| Probea | No. Detected/<br>No. Valid | No. Detected/<br>No.Valid | No. Detected/<br>No.Valid | Total<br>Identified | Percent<br>Identified | Weighted<br>Percent<br>Identified |
|--------|----------------------------|---------------------------|---------------------------|---------------------|-----------------------|-----------------------------------|
| | Site 34 | Site 35 | Site 36 | | | |
| ABA | 62/62 | 63/63 | 60/64 | 185/189 | 97.9% | 98.7% |
| CITb | 17/17 | 30/32 | 30/32 | 77/81 | 95.1% | 94.4% |
| ECO | 55/55 | 51/52 | 55/55 | 161/162 | 99.4% | 99.2% |
| EFM | 51/53 | 55/56 | 49/54 | 155/163 | 95.1% | 97.0% |
| EFSb | 8/8 | 8/8 | 8/8 | 24/24 | 100.0% | 100.0% |
| ENTb | 19/24 | 21/23 | 20/24 | 60/71 | 84.5%b | 87.5%c |
| KLE | 46/48 | 46/48 | 44/48 | 136/144 | 94.4% | 96.3% |
| PAE | 43/43 | 48/48 | 48/48 | 139/139 | 100.0% | 100.0% |
| SAU | 131/135 | 138/140 | 136/137 | 405/412 | 98.3% | 98.0% |
| SMAb | 8/8 | 8/8 | 8/8 | 24/24 | 100.0% | 100.0% |
| STR | 38/39 | 38/38 | 35/35 | 111/112 | 99.1% | 99.2% |
Table 6. Summary of Reproducibility of Identification Assay Results:
4 See Table 1 for abbreviation definitions
b Isolates of Citrobacter spp., E. faecalis, Enterobacter spp. and S. marcescens were evaluated in order to obtain sufficient results for AST Testing; total number of data points for each species <90.
6 An Image processing change was implemented post study to correct a known issue. Regression analysis of the ENT probe produced a percent identified of 93.0% and a weighted percent identified score of 93.2%.
> For AST, reproducibility for all antimicrobials was assessed by evaluating approximately 10 organisms with on-scale MIC values (a value within the assay's reportable range) for each antimicrobial reported by the assay. For Daptomycin fewer than ten isolates were tested due to the lack of isolates with on-scale MICs for these antimicrobials; however, more than four replicates per isolate were tested to achieve 90 data points for those antimicrobials. AST reproducibility was determined from the total number of results that fell within 1 dilution (+/- one doubling dilution) of the mode results divided by the total number of results. On-scale AST performance for each antimicrobial agent was evaluated between sites and within each site. Both Best Case (assumes that off-scale results are within one dilution of the mode) and Worse Case (assumes that off-scale results are more than one dilution of the mode) performance was determined for each drug when the mode across all sites was on-scale for each drug. Worst case performance was not determined for the resistance phenotypes as this result is positive/negative with no off-scale results. Only those samples that met the required number of total results per site were used in the final calculations.
> For the AST assay testing the reproducibility of 17 of the 18 antimicrobials and resistance phenotypes were acceptable with best case reproducibility of > 95%. However, erythromycin demonstrated best and worst case reproducibility of 93.6% which was considered acceptable (Table 7).
{16}------------------------------------------------
Indeterminate, false positive and invalid results were obtained during the course of the reproducibility study. See Tables 35 and 36 for summaries of the indeterminate results. See Tables 37 and 38 for a summary of the false positive and invalid results, respectively.
| Antimicrobial | Organisms<br>Tested | No.<br>isolates | Best Case<br>No. Within 1 ± Dil/<br>Total Tests (%) | Worst Case<br>No. Within 1 ± Dil/<br>Total Tests (%) |
|-----------------------|----------------------|-----------------|-----------------------------------------------------|------------------------------------------------------|
| Amikacin | A. baumannii | 6 | 128/128 (100.0) | 118/128 (92.2) |
| | P. aeruginosa | 4 | | |
| | S. marcescens | 1 | | |
| Ampicillin | E. faecium | 8 | 93/93 (100.0) | 88/93 (94.6) |
| Aztreonam | C. freundii | 3 | 348/350 (99.4) | 348/350 (99.4) |
| | K. pneumoniae | 10 | | |
| | E. coli | 11 | | |
| | P. aeruginosa | 6 | | |
| | S. marcescens | 1 | | |
| Ceftazidime | C. freundii | 3 | 324/331 (100.0) | 317/331 (95.8) |
| | K. pneumoniae | 7 | | |
| | E. coli | 11 | | |
| | P. aeruginosa | 7 | | |
| | S. marcescens | 1 | | |
| Ceftaroline | S. aureus | 11 | 151/151 (100.0) | 146/151 (96.7) |
| Ciprofloxacin | C. freundii | 1 | 136/139 (100.0) | 133/139 (95.7) |
| | K. pneumoniae | 2 | | |
| | E. coli | 2 | | |
| | P. aeruginosa | 5 | | |
| | S. marcescens | 1 | | |
| Ceftriaxone | E. aerogenes | 1 | 151/151 (100.0) | 140/151 (92.7) |
| | C. freundii | 1 | | |
| | K. pneumoniae | 2 | | |
| | E. coli | 8 | | |
| | S. marcescens | 2 | | |
| Daptomycin | S. aureus | 3 | 92/92 (100.0) | 82/92 (89.1) |
| | S. haemolyticus | 1 | | |
| | E. faecalis | 1 | | |
| Erythromycin | S. aureus | 10 | 117/125 (93.6) | 117/125 (93.6) |
| Ertapenem | C. freundii | 3 | 356/356 (100.0) | 354/356 (99.4) |
| | K. pneumoniae | 11 | | |
| | E. coli | 12 | | |
| | S. marcescens | 2 | | |
| | E. aerogenes | 3 | | |
| Cefepime | C. freundii | 1 | 158/162 (97.5) | 152/162 (93.8) |
| | K. pneumoniae | 1 | | |
| | E. coli | 8 | | |
| | P. aeruginosa | 2 | | |
| | S. marcescens | 1 | | |
| | E. aerogenes | 1 | | |
| Antimicrobial | Organisms<br>Tested | No.<br>isolates | Best Case<br>No. Within 1 ± Dil/<br>Total Tests (%) | Worst Case<br>No. Within 1 ± Dil/<br>Total Tests (%) |
| Gentamycin | <i>K. pneumoniae</i> | 6 | 233/234 (99.6) | 218/234 (93.2) |
| | <i>E. coli</i> | 5 | | |
| | <i>P. aeruginosa</i> | 6 | | |
| | <i>S. marcescens</i> | 2 | | |
| | <i>E. aerogenes</i> | 1 | | |
| Linezolid | <i>E. faecium</i> | 10 | 350/355 (98.6) | 350/355 (98.6) |
| | <i>E. faecalis</i> | 2 | | |
| | <i>S. aureus</i> | 17 | | |
| Meropenem | <i>P. aeruginosa</i> | 7 | 105/106 (99.1) | 104/106 (98.1) |
| | <i>S. marcescens</i> | 2 | | |
| Amp/Sulbactam | <i>K. pneumoniae</i> | 6 | 157/157 (100.0) | 152/157 (96.8) |
| | <i>E. coli</i> | 8 | | |
| Tobramycin | <i>K. pneumoniae</i> | 2 | 102/104 (98.1) | 101/104 (97.1) |
| | <i>E. coli</i> | 2 | | |
| | <i>P. aeruginosa</i> | 3 | | |
| | <i>S. marcescens</i> | 2 | | |
| | <i>C. freundii</i> | 2 | | |
| Pip/Tazo | <i>K. pneumoniae</i> | 4 | 215/222 (96.9) | 205/222 (92.3) |
| | <i>E. coli</i> | 4 | | |
| | <i>P. aeruginosa</i> | 7 | | |
| | <i>S. marcescens</i> | 1 | | |
| | <i>E. aerogenes</i> | 1 | | |
| Vancomycin | <i>E. faecium</i> | 1 | 268/282 (95.0) | 232/282 (82.3) |
| | <i>S. aureus</i> | 7 | | |
| Resistance Phenotypes | | | | |
| Cefoxitin | <i>S. aureus</i> | 21 | 206/206 (100.0) | N/Ab |
| MLSb | <i>S. aureus</i> a | 21 | 217/222 (97.8) | N/Ab |
Table 7. Summary of Reproducibility of AST Assay Results for all Antimicrobials and Resistance Phenotypes across All Sites
{17}------------------------------------------------
ª Reproducibility was performed with 21 isolates of S. aureus that were MLSb positive; however in the clinical study, major and minor errors occurred that could not be resolved; there is no claim for testing S. aureus for MLSb and results will be suppressed.
b Resistance phenotype, no worst case evaluation.
- b. Linearity/assay reportable range: Not Applicable
- Traceability, Stability, Expected values (controls, calibrators, or methods): C. Three internal assay controls are included in each Accelerate PhenoTest BC kit and require no action by the user:
Universal Bacterial Probe (or Universal Eukaryotic Probe for Yeast). The universal bacterial probe binds to rRNA in all bacterial cells to signal that bacterial cells are present in the flowcells and to differentiate bacteria from debris. The universal eukaryotic probe binds to rRNA in all eukaryotic cells, including yeast, to signal that yeast cells are present in the flowcells and to differentiate yeast from debris. Colocalization of universal and target probe is required for identification of a target organism.
{18}------------------------------------------------
General Nucleic Acid Stain. A cell permeant nucleic acid fluorescent dye that binds to double-stranded DNA (dsDNA) is run in a separate flowcell channel as a DNA staining control. Data from this control is used to quantify the total number of microbial cells in a sample. Based on this result the remainder of the sample is diluted in order to load the desired number of cells per microscopic field of view for susceptibility testing.
Growth Control Channel. The growth control channel is used as a positive control for AST. It measures the number of healthy clones as well as the growth density and growth rate of healthy cells that are not exposed to any antibiotics. The channel consists of the sample and Mueller-Hinton Agar as the growth media for the cells as recommended by CLSI for susceptibility testing.
External Controls. Organisms used for QC testing of the PhenoTest BC kit are included in four panels (Table 8); multiple isolates are tested in a single run by loading the members of a single panel into specified wells in the reagent cartridge. OC organism panels can be rotated to ensure that each ID and AST channel is tested on a regular basis.
| Panel Name | Organisms | Strain |
|-------------------|----------------|--------------|
| EPEES Panel (AST) | E. coli | ATCC 25922 |
| | E. coli | ATCC 35218 |
| | P. aeruginosa | ATCC 27853 |
| | E. faecalis | ATCC 29212 |
| | S. aureus | ATCC 29213 |
| SES Panel (AST) | S. aureus | ATCC 43300 |
| | S. aureus | ATCC BAA-977 |
| CASKS Panel (ID) | S. lugdunensis | ATCC 700328 |
| | K. pneumoniae | ATCC 700603 |
| | A. baumannii | ATCC 19606 |
| | C. freundii | ATCC 6879 |
| | C. glabrata | ATCC 2001 |
| | S. agalactiae | ATCC 12403 |
| SPECS Panel (ID) | E. faecium | ATCC 19434 |
| | S. marcescens | ATCC 43862 |
| | E. aerogenes | ATCC 13048 |
| | P. vulgaris | ATCC 6380 |
| | C. albicans | ATCC 96268 |
| | S. pneumoniae | ATCC 49619 |
Table 8. Quality Control Organism Panels
QC Reportable Range. The dilutions used for evaluation of QC strains are different than the reportable ranges used for patient isolates (Table 9). These ranges were added for the purpose of generating on-scale QC results; these ranges are not the same as those used for reporting patient samples from the PhenoTest BC kit.
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| QC Strain | Drug | QC Reportable Range (µg/mL) |
|-----------------------------|-------------------------|-----------------------------|
| E. coli<br>ATCC 25922 | Ceftriaxone | 0.015 - 0.25 |
| E. coli<br>ATCC 25922 | Ceftazidime | 0.03 - 1 |
| E. coli<br>ATCC 25922 | Amikacin | 0.5 - 8 |
| E. coli<br>ATCC 25922 | Aztreonam | 1 - 16 |
| E. coli<br>ATCC 25922 | Cefepime | 0.25 - 8 |
| P. aeruginosa<br>ATCC 27853 | Ciprofloxacin | 0.12 - 2 |
| P. aeruginosa<br>ATCC 27853 | Ertapenem | 1 - 16 |
| P. aeruginosa<br>ATCC 27853 | Gentamicin | 0.25 - 4 |
| P. aeruginosa<br>ATCC 27853 | Meropenem | 0.25 - 2 |
| P. aeruginosa<br>ATCC 27853 | Tobramycin | 0.25 - 2 |
| E. coli<br>ATCC 35218 | Ampicillin/Sulbactam | 4 - 64 |
| E. coli<br>ATCC 35218 | Piperacillin/Tazobactam | 0.25 - 4 |
| E. coli<br>ATCC 35218 | Ampicillin | 0.25 - 4 |
| E. faecalis<br>ATCC 29212 | Daptomycin | 0.5 - 8 |
| E. faecalis<br>ATCC 29212 | Linezolid | 0.5 - 8 |
| E. faecalis<br>ATCC 29212 | Vancomycin | 0.5 - 8 |
| S. aureus<br>ATCC 29213 | Cefoxitin | Negative* |
| S. aureus<br>ATCC 29213 | Ceftaroline | 0.25 - 1 |
| S. aureus<br>ATCC 29213 | Erythromyc…
