ASTar BC G- Kit and ASTar Instrument

K221688 · Q-Linea AB · SAN · Apr 26, 2024 · Microbiology

Device Facts

• Record ID: K221688
• Device Name: ASTar BC G- Kit and ASTar Instrument
• Applicant: Q-Linea AB
• Product Code: SAN · Microbiology
• Decision Date: Apr 26, 2024
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.1650
• Device Class: Class 2

AI Performance

Output	Algorithm	Acceptance	Observed	Dev DS	Dev Readers	Test DS	Test Readers
Antimicrobial Susceptibility Testing (AST)	High-performance optical detection system with image analysis algorithms	Essential Agreement (EA) ≥ 90% compared to reference BMD	EA ranged from 89.44% to 100% across antimicrobials	—	—	Clinical study (K221688): 880 samples (256 fresh positive blood cultures, 223 contrived with clinical stock isolates, 401 contrived with challenge isolates).	—

Indications for Use

The ASTar System is intended to be used for the automated quantitative susceptibility testing for most clinically significant microorganisms. The ASTar System does not provide organism identification. The ASTAar System, comprised of the ASTar Instrument with the ASTar BC G– Kit (ASTar BC G– Consumable kit, ASTar BC G– Frozen Insert, and ASTar BC G– Kit software), utilizes high-speed, time-lapse microscopy imaging of bacteria for the in vitro, quantitative determination of antimicrobial susceptibility of on-panel gram-negative bacteria. The test is performed directly on positive blood culture samples signaled as positive by a continuous monitoring blood culture system and confirmed to contain gram- negative bacilli by Gram stain. Organism identification is required for AST result interpretation and reporting. Test results from the ASTar BC G– Kit should be interpreted 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. Sub-culturing is necessary to support further testing for: bacteria and antimicrobials not on the ASTar BC G– panel, where inconclusive results are obtained, epidemiologic testing, recovery of organisms present in microbial samples, and susceptibility testing of bacteria in polymicrobial samples.

Device Story

The ASTar System performs automated antimicrobial susceptibility testing (AST) on positive blood culture samples. The operator loads approximately 1 mL of a Gram-stained, positive blood culture sample into an ASTar Cartridge. The instrument purifies the sample, adjusts the bacterial concentration to a standardized inoculum, and transfers it to an ASTar Disc. The system uses high-speed, time-lapse microscopy to monitor bacterial growth in the presence of various antimicrobial concentrations. The instrument processes up to six samples in parallel and can incubate up to 12 discs simultaneously. Results, including Minimum Inhibitory Concentration (MIC) and qualitative susceptibility (S, I, R), are generated within approximately six hours. The system requires external organism identification to report results. Healthcare providers use these results to guide antimicrobial therapy for patients with suspected bacteremia. The system benefits patients by providing rapid, quantitative susceptibility data, potentially enabling faster optimization of antibiotic treatment.

Clinical Evidence

Clinical study evaluated 933 valid samples (256 fresh positive blood cultures, 223 contrived with clinical stock, 401 contrived with challenge isolates). Performance compared to frozen BMD (CLSI M07). Overall Essential Agreement (EA) and Category Agreement (CA) were calculated for 18 antimicrobials across various Gram-negative species. Results showed high performance across most antimicrobials, with EA and CA generally meeting acceptance criteria. 97.2% of samples produced at least partial AST results. Reproducibility studies (n=23 strains) demonstrated ≥95% reproducibility for best-case scenarios.

Technological Characteristics

The system uses high-speed, time-lapse microscopy for optical detection of bacterial growth in broth microdilution. It consists of the ASTar Instrument, ASTar Cartridge (sample prep), and ASTar Disc (incubation/imaging). It is a fully automated, networked system. Software performs image analysis to determine MICs. The system is designed for use with positive blood culture bottles (e.g., BD BACTEC, bioMérieux BACT/ALERT).

Indications for Use

Indicated for in vitro, quantitative determination of antimicrobial susceptibility of on-panel gram-negative bacteria from positive blood culture samples confirmed by Gram stain. Requires organism identification from an approved method for result interpretation. For prescription use only.

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.

Predicate Devices

• Accelerate PhenoTest BC Kit (DEN160032)

Submission Summary (Full Text)

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**FDA** U.S. FOOD & DRUG
ADMINISTRATION

# **510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY AND INSTRUMENT**

# **I Background Information:**

# **A 510(k) Number**

K221688

# **B Applicant**

Q-linea AB

# **C Proprietary and Established Names**

ASTar Instrument and ASTar BC G- Kit

# **D Regulatory Information**

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| SAN | Class II | 21 CFR 866.1650 - A Cellular Analysis System For Multiplexed Antimicrobial Susceptibility | MI - Microbiology |
| LON | Class II | 21 CFR 866.1645 - Fully automated short-term incubation cycle antimicrobial susceptibility system | MI - Microbiology |

# **II Submission/Device Overview:**

# **A Purpose for Submission:**

1. To obtain a substantial equivalence determination for use of the ASTar Instrument and ASTar BC G- Kit for testing positive blood culture samples to determine the minimum inhibitory concentration of specific antimicrobials with specific Gram-negative organisms.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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# **B Measurand:**

| Antimicrobial | Reporting Range |
| --- | --- |
| Amikacin | ≤0.5 to ≥256 μg/mL |
| Ampicillin | ≤1 to ≥128 μg/mL |
| Ampicillin/sulbactam | ≤1 to ≥128 μg/mL |
| Aztreonam | ≤0.25 to ≥128 μg/mL |
| Cefazolin | ≤0.25 to ≥32 μg/mL |
| Cefepime | ≤0.25 to ≥128 μg/mL |
| Ceftazidime | ≤0.25 to ≥128 μg/mL |
| Ceftazidime/avibactam | ≤0.125 to ≥64 μg/mL |
| Cefuroxime | ≤1 to ≥128 μg/mL |
| Ciprofloxacin | ≤0.125 to ≥16 μg/mL |
| Gentamicin | ≤0.25 to ≥64 μg/mL |
| Levofloxacin | ≤0.125 to ≥32 μg/mL |
| Meropenem | ≤0.06 to ≥128 μg/mL |
| Meropenem/vaborbactam | ≤0.25 to ≥64 μg/mL |
| Piperacillin/tazobactam | ≤0.25 to ≥512 μg/mL |
| Tigecycline | ≤0.03 to ≥32 μg/mL |
| Tobramycin | ≤0.06 to ≥64 μg/mL |
| Trimethoprim/sulfamethoxazole | ≤0.06 to ≥16 μg/mL |

# **C Type of Test:**

Quantitative antimicrobial susceptibility test (AST) system that utilizes high-speed, time-lapse microscopy imaging of organisms in positive blood culture samples to determine the minimum inhibitory concentration (MIC) of specific antimicrobial-organism combinations.

# **III Intended Use/Indications for Use:**

# **A Intended Use(s):**

The ASTar System is intended to be used for the automated quantitative susceptibility testing for most clinically significant microorganisms. The ASTar System does not provide organism identification.

# **B Indication(s) for Use:**

The ASTAar System, comprised of the ASTar Instrument with the ASTar BC G– Kit (ASTar BC G– Consumable kit, ASTar BC G– Frozen Insert, and ASTar BC G– Kit software), utilizes high-speed, time-lapse microscopy imaging of bacteria for the in vitro, quantitative determination of antimicrobial susceptibility of on-panel gram-negative bacteria. The test is performed directly on positive blood culture samples signaled as positive by a continuous monitoring blood culture system and confirmed to contain gram- negative bacilli by Gram stain. Organism identification is required for AST result interpretation and reporting.

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Test results from the ASTar BC G– Kit should be interpreted 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. Sub-culturing is necessary to support further testing for: bacteria and antimicrobials not on the ASTar BC G– panel, where inconclusive results are obtained, epidemiologic testing, recovery of organisms present in microbial samples, and susceptibility testing of bacteria in polymicrobial samples.

The ASTar BC G– Kit tests the following antimicrobial agents with the following bacterial species:

Amikacin: *Citrobacter freundii*, *Enterobacter cloacae* complex, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Pseudomonas aeruginosa*, *Serratia marcescens*

Ampicillin: *Escherichia coli*, *Proteus mirabilis*

Ampicillin-sulbactam: *Escherichia coli*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*

Aztreonam: *Citrobacter koseri*, *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Serratia marcescens*

Cefazolin: *Klebsiella pneumoniae*

Cefepime: *Citrobacter freundii*, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, *Serratia marcescens*

Ceftazidime: *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Serratia marcescens*

Ceftazidime-avibactam: *Citrobacter freundii*, *Citrobacter koseri*, *Enterobacter cloacae* complex, *Klebsiella oxytoca*, *Proteus mirabilis*, *Pseudomonas aeruginosa*, *Serratia marcescens*

Cefuroxime: *Escherichia coli*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*

Ciprofloxacin: *Citrobacter koseri*, *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, *Serratia marcescens*

Gentamicin: *Citrobacter freundii*, *Citrobacter koseri*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, *Serratia marcescens*

Levofloxacin: *Citrobacter freundii*, *Citrobacter koseri*, *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, *Serratia marcescens*

Meropenem: *Acinetobacter baumannii*, *Citrobacter freundii*, *Citrobacter koseri*, *Escherichia coli*, *Proteus mirabilis*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, *Serratia marcescens*

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Meropenem-vaborbactam: *Citrobacter freundii*, *Citrobacter koseri*, *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Serratia marcescens*

Piperacillin-tazobactam: *Citrobacter koseri*, *Escherichia coli*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Proteus vulgaris*, *Serratia marcescens*

Tigecycline: *Citrobacter freundii*, *Citrobacter koseri*, *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Serratia marcescens*

Tobramycin: *Citrobacter freundii*, *Citrobacter koseri*, *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Serratia marcescens*

Trimethoprim-sulfamethoxazole: *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella aerogenes*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus vulgaris*

### C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

#### General Limitations

- • The ASTar BC G- kit can only be used with the ASTar Instrument.
- • The ASTar BC G- kit has not been evaluated for specimens other than positive blood culture.
- • The performance of the ASTar BC G- kit has only been evaluated using the following blood culture bottles:
- ○ bioMérieux BACT/ALERT FA Plus Aerobic
- ○ bioMérieux BACT/ALERT FN Plus Anaerobic
- ○ bioMérieux BACT/ALERT PF Plus Peds
- ○ bioMérieux BACT/ALERT SN Standard Anaerobic
- ○ bioMérieux BACT/ALERT SA Standard Aerobic
- ○ BD BACTEC Peds Plus
- ○ BD BACTEC Lytic Anaerobic
- ○ BD BACTEC Plus Anaerobic
- ○ BD BACTEC Plus Aerobic
- ○ BD BACTEC Standard Aerobic
- ○ BD BACTEC Standard Anaerobic
- • The ASTar BC G- Kit should not be used with blood culture bottles containing charcoal.
- • Positive blood cultures should be tested immediately after a positive flag, where possible. A 16-hour sample stability claim is included in case of instrument errors or if re-testing is needed.
- • Failure to observe proper procedures for sample collection, preparation, storage, handling and/or transportation may cause incorrect results.
- • AST results should not be reported if two or more species are identified in a patient sample.
- • If an AST result is not provided by the ASTar BC G- Kit, susceptibility testing must be performed using an alternate method.
- • Subculturing of positive blood culture is necessary for organisms not claimed by the ASTar BC G- Kit and for antimicrobial agents not included on the ASTar panel.

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# Antimicrobial Susceptibility Testing (AST) Limitations

The following limitations were added to the device labeling based on performance demonstrated in the current submission:

- The ability of the ASTar system to detect resistance in the following antimicrobial/organism combinations is unknown because of an insufficient number of resistant isolates were available during the clinical study:

○ Amikacin: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens

○ Ampicillin-sulbactam: Citrobacter koseri, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris

○ Aztreonam: Citrobacter koseri, Klebsiella aerogenes, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Cefepime: Citrobacter freundii, Citrobacter koseri, Klebsiella aerogenes, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Ceftazidime: Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Ceftazidime-avibactam: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens

○ Cefuroxime: Klebsiella oxytoca, Proteus mirabilis

○ Ciprofloxacin: Citrobacter koseri, Enterobacter cloacae complex, Klebsiella aerogenes, Klebsiella oxytoca, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens

○ Gentamicin: Citrobacter freundii, Citrobacter koseri, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens

○ Levofloxacin: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Klebsiella aerogenes, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens

○ Meropenem: Citrobacter freundii, Citrobacter koseri, Escherichia coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens

○ Meropenem-vaborbactam: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Piperacillin-tazobactam: Citrobacter koseri, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Tigecycline: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Serratia marcescens

○ Tobramycin: Citrobacter freundii, Citrobacter koseri, Escherichia coli, Enterobacter cloacae complex, Proteus mirabilis, Serratia marcescens

○ Trimethoprim-sulfamethoxazole: Citrobacter koseri, Klebsiella aerogenes, Klebsiella oxytoca, Proteus vulgaris, Serratia marcescens

- Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combination(s):

○ Amikacin: Acinetobacter baumannii, Escherichia coli, Proteus vulgaris

○ Ampicillin-sulbactam: Acinetobacter baumannii

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○ Aztreonam: Escherichia coli when the ASTar MIC is 0.5 μg/mL due to one very major discrepancy, Citrobacter freundii, Pseudomonas aeruginosa

○ Cefazolin: Citrobacter koseri, Escherichia coli, Klebsiella oxytoca, Proteus mirabilis

○ Cefepime: Proteus vulgaris when the ASTar MIC is 32 μg/mL due to one major error, Enterobacter cloacae complex

○ Cefotaxime: Acinetobacter baumannii, Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Ceftazidime: Acinetobacter baumannii, Citrobacter freundii, Citrobacter koseri, Klebsiella aerogenes, Pseudomonas aeruginosa

○ Ceftazidime-avibactam: Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae

○ Ceftolozane-tazobactam: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens

○ Ceftriaxone: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens

○ Cefuroxime: Citrobacter koseri, Enterobacter cloacae complex, Klebsiella aerogenes

○ Ciprofloxacin: Citrobacter freundii

○ Ertapenem: Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens

○ Gentamicin: Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes

○ Meropenem: Escherichia coli when the ASTar MIC is either 0.5 or 1.0 μg/mL due to three very major discrepancies, Enterobacter cloacae complex, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae

○ Piperacillin-tazobactam: Escherichia coli when the ASTar MIC is 8.0 μg/mL due to one very major discrepancy, Klebsiella pneumoniae when the ASTar MIC is 8.0 μg/mL due to two very major discrepancies; Acinetobacter baumannii, Citrobacter freundii, Klebsiella aerogenes, Klebsiella oxytoca

○ Tobramycin: Klebsiella pneumoniae when the ASTar MIC is 4.0 μg/mL due to one very major discrepancy, Klebsiella aerogenes, Klebsiella oxytoca, Proteus vulgaris, Pseudomonas aeruginosa

○ Trimethoprim-sulfamethoxazole: Citrobacter freundii, Proteus mirabilis

• Perform an alternate method of testing prior to reporting results when a resistant result is obtained for the following organisms, if critical to patient care:

○ Trimethoprim-sulfamethoxazole: Klebsiella pneumoniae

# D Special Instrument Requirements:

Test with the following software or later version:

ASTar BC G- Kit Software version 1.6.4

ASTar Application Computer Image version 1.5

ASTar Instrument Computer Image version 1.7

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## IV Device/System Characteristics:

### A Device Description:

ASTar System is a fully automated system for antimicrobial susceptibility testing (AST). It consists of the ASTar Instrument in combination with dedicated application kits. The ASTar BC G- Kit contains the ASTar BC G- Consumable kit (discs and cartridges), ASTar BC G- Frozen insert, and ASTar BC G- Kit software which must be installed on the instrument to process the kit. The frozen insert is a single-use plastic container with frozen reagents and is added to the cartridge prior to sample run. The system prepares an inoculum for AST and utilizes high-speed, time-lapse microscopy imaging of pathogens in broth microdilution to determine minimum inhibitory concentration (MIC) and qualitative susceptibility results for samples. Organism identification by an alternate method is required to be entered into the ASTar Instrument for AST results to be reported.

The ASTar instrument is designed to carry out sample preparation of up to six samples in parallel using a dedicated ASTar Cartridge consumable for each sample. In the subsequent AST culturing step, the instrument transfers the prepared sample into a second dedicated consumable, referred to as the ASTar Disc. Up to 12 discs can be incubated simultaneously in the system with samples at different stages of the processing procedure. New samples can be loaded in a random-access manner when there are available slots. The operator interacts with the instrument via the touchscreen display to run test samples.

ASTar BC G- Kit is used for *in vitro* determination of AST results for commonly isolated bacteria derived from positive blood culture samples confirmed by Gram stain to be Gram-negative bacteria. To start an analysis, approximately 1 mL of a positive blood culture is pipetted into the ASTar Cartridge by the operator and loaded into the system. The instrument purifies and quantifies the bacteria, and the bacterial concentration is adjusted to the appropriate inoculum concentration. The bacterial suspensions are transferred automatically to the ASTar Disc and antimicrobial susceptibility testing is performed based on a defined protocol. The system generates an MIC and further qualitative susceptibility results (i.e., S, I, R) for the tested antimicrobials, where applicable. The ASTar System with the ASTar BC G- Kit can determine the MIC of various antimicrobials when tested against specific organisms with FDA Susceptibility Testing Interpretive Criteria (STIC) breakpoints as shown in **Table 1** below.

**Table 1:** Reportable MIC Ranges and FDA Recognized Susceptibility Test Interpretive Criteria (STIC) / 'Breakpoints' implemented in the kit software

| Antimicrobial | ASTar System Reportable Range (µg/mL) | Enterobacterales | | | *P. aeruginosa* | | | *Acinetobacter* spp. | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | S | I | R | S | I | R | S | I | R |
| Ampicillin | ≤1 to ≥128 | ≤8 | 16 | ≥32 | - | - | - | - | - | - |
| Ampicillin-sulbactam | ≤1 to ≥128 | ≤8 | 16 | ≥32 | - | - | - | - | - | - |
| Ceftazidime-avibactam | ≤0.125 to ≥ 64 | ≤8 | -^{a} | ≥16 | ≤8 | -^{a} | ≥16 | - | - | - |
| Meropenem-vaborbactam | ≤0.25 to ≥64 | ≤4 | 8 | ≥16 | - | - | - | - | - | - |
| Piperacillin-tazobactam | ≤0.25 to ≥512 | ≤8 | 16 | ≥32 | - | - | - | - | - | - |
| Cefazolin | ≤0.25 to ≥32 | ≤2 | 4 | ≥8 | - | - | - | - | - | - |

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| Antimicrobial | ASTar System Reportable Range (μg/mL) | Enterobacterales | | | P. aeruginosa | | | Acinetobacter spp. | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | S | I | R | S | I | R | S | I | R |
| Cefepime | ≤0.25 to ≥128 | ≤2 | 4-8 | ≥16 | ≤8 | -^{a} | ≥16 | - | - | - |
| Cefuroxime | ≤1 to ≥128 | ≤8 | -^{a} | ≥16 | - | - | - | - | - | - |
| Ceftazidime | ≤0.25 to ≥128 | ≤4 | 8 | ≥16 | ≤8 | -^{a} | ≥16 | - | - | - |
| Aztreonam | ≤0.25 to ≥128 | ≤4 | 8 | ≥16 | - | - | - | - | - | - |
| Meropenem | ≤0.06 to ≥128 | ≤1 | 2 | ≥4 | ≤2 | 4 | ≥8 | ≤2 | 4 | ≥8 |
| Gentamicin | ≤0.25 to ≥64 | ≤4 | 8 | ≥16 | ≤4 | 8 | ≥16 | - | - | - |
| Tobramycin | ≤0.06 to ≥64 | ≤4 | 8 | ≥16 | - | - | - | - | - | - |
| Amikacin | ≤0.5 to ≥256 | ≤16 | 32 | ≥64 | ≤16 | 32 | ≥64 | - | - | - |
| Tigecycline | ≤0.03 to ≥32 | ≤2 | 4 | ≥8 | - | - | - | - | - | - |
| Ciprofloxacin | ≤0.125 to ≥16 | ≤0.25 | 0.5 | ≥1 | ≤0.5 | 1 | ≥2 | - | - | - |
| Levofloxacin | ≤0.125 to ≥32 | ≤0.5 | 1 | ≥2 | ≤1 | 2 | ≥4 | - | - | - |
| Trimethoprim-sulfamethoxazole | ≤0.06 to ≥16 | ≤2 | -^{a} | ≥4 | - | - | - | - | - | - |

$^{a}$No intermediate category is defined for these drug/organism combinations.

### B Principle of Operation:

The ASTar BC G- kit, along with the ASTar Instrument, constitute a fully automated system for the in vitro determination of antimicrobial susceptibility of Gram-negative bacilli present in positive blood culture bottles (BCBs). The instrument provides inoculum preparation for AST and utilizes high-speed, time-lapse microscopy imaging of pathogens in broth microdilution to determine minimum inhibitory concentration (MIC) and qualitative susceptibility results.

The consumable kit consists of a preparation cartridge and a disk with dried antimicrobials in 1:2 dilution series. Approximately 1 mL of a positive blood culture, confirmed Gram-negative by Gram stain, is pipetted into the ASTar Cartridge by the operator and loaded into the system. All other procedures are automated. Briefly, the ASTar purifies and quantifies the bacteria from the BCB. Resin particles are removed by filtration and human-derived cells are lysed by adding lysis buffer to the sample. Bacteria are separated from the lysate by filtering and captured on a filter membrane. The pathogens present in the aliquot are resuspended in culture medium. Bacterial concentration is determined by mixing an aliquot of bacterial suspension with staining medium (fluorescent dye). The stained aliquot is added to the disc and the concentration is determined by fluorescent imaging at one wavelength. Based on the concentration, the instrument creates aliquots at a pre-defined inoculum concentration (typically 5 × 10⁵ CFU/mL). The disc is placed in an incubator carousel and each well is imaged at specified time intervals using a high-speed optical microscopy system. Bacterial growth and response to relevant concentrations of different antimicrobial drugs are measured throughout the incubation period using the optical detection system in combination with image analysis algorithms. Next, the images are analyzed for bacterial content over time, and the system generates the MIC and qualitative susceptibility results (i.e., S, I, R) for the tested antimicrobials, where applicable. The qualitative results are determined based on established breakpoints. The system allows culturing and analysis to start in the absence of bacterial ID, which is only needed for final interpretation and reporting. Results are available within approximately 6 hours.

### C Instrument Description Information:

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1. Instrument Name:

ASTar Instrument

2. Specimen Identification:

Barcodes link the cartridge and patient sample. Barcodes are located on the cartridge, disc, and frozen insert. These barcodes contain product information, lot information, and expiration date. A sample barcode is added by the operator to the cartridge on a dedicated area (only when running samples and not Quality Control strains). All barcodes on the cartridge and frozen insert are scanned when prompted. The disc barcode is scanned automatically by the instrument as the tray retracts. Materials not provided include a sterile device for safe transfer from positive blood culture (approximately 1 mL) and adhesive sample barcode label. Pathogen identification results from a separate method are entered into the ASTar Instrument to generate AST results.

3. Specimen Sampling and Handling:

New samples can be loaded in a random-access manner if there are available slots for cartridges and discs. After placing the sample barcode in the dedicated area, approximately 1 mL of positive blood culture sample is added into the sample input of the cartridge. The end-user should check that the minimum fill-level is reached. Sample processing can be initiated without entering organism ID information, which is entered during or after processing of the sample to determine AST results.

4. Calibration:

The ASTar System requires no calibration by the end-user. Adjustments and settings of mechanical optical components are carried out as part of the instrument production process and by field service engineers to address any issues with the instrument.

5. Quality Control:

Quality control (QC) for AST testing of ASTar BC G– Consumable kit is performed by running pure cultures of characterized QC strains. An overnight culture of a QC strain is suspended in media (Cation-Adjusted Mueller-Hinton Broth, CAMHB) and added to the sample inlet of a cartridge (with a frozen insert added). For guidance, the bacterial suspension should be at 0.3–2.0 McFarland as measured in a densitometer. Cartridge and disc are loaded in the instrument and run. The instrument automatically determines the pathogen concentration, prepares the inoculum, and loads the inoculum into appropriate culture chambers in the disc. A QC test can be run at any time a slot in the instrument is available. Pass/fail status depends on whether the actual MIC result falls within the expected QC range (pass) or outside of the expected QC range (fail) (Table 2).

Table 2. QC Strains Tested with the ASTar System

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| Antimicrobial Agent | QC Strain | ASTar BC G–Reportable range^{a} (μg/mL) | CLSI QC Organism range^{a} (μg/mL) |
| --- | --- | --- | --- |
| Ampicillin | *E. coli* ATCC 25922 *K. pneumoniae* ATCC 700603^{b} | ≤0.5 - ≥128 | 2-8 >128^{c} |
| Ampicillin-sulbactam | *E. coli* ATCC 25922 *K. pneumoniae* ATCC 700603 | ≤1 - ≥128 | 2-8 8-32 |
| Ceftazidime-avibactam | *P. aeruginosa* ATCC 27853 *K. pneumoniae* ATCC 700603 | ≤0.06 - ≥64 | 0.5-4 0.25-2 |
| Meropenem-vaborbactam | *P. aeruginosa* ATCC 27853 *K. pneumoniae* ATCC BAA 2814 | ≤0.06 - ≥64 | 0.125-1 0.125-0.5 |
| Piperacillin-tazobactam | *P. aeruginosa* ATCC 27853 *K. pneumoniae* ATCC 700603 | ≤0.125 - ≥512 ≤0.25 - ≥512 | 1-8 8-32 |
| Cefazolin | *E. coli* ATCC 25922 | ≤0.125 - ≥32 | 1-4 |
| Cefepime | *P. aeruginosa* ATCC 27853 | ≤0.125 - ≥128 | 0.5-4 |
| Cefuroxime | *E. coli* ATCC 25922 | ≤0.5 - ≥128 | 2-8 |
| Ceftazidime | *P. aeruginosa* ATCC 27853 *K. pneumoniae* ATCC 700603^{b} | ≤0.125 - >128 ≤0.25 - ≥128 | 1-4 16-64 |
| Aztreonam | *P. aeruginosa* ATCC 27853 | ≤0.125 - ≥128 | 2-8 |
| Meropenem | *P. aeruginosa* ATCC 27853 | ≤0.03 - >128 | 0.125-1 |
| Gentamicin | *P. aeruginosa* ATCC 27853 | ≤0.25 - ≥64 | 0.5-2 |
| Tobramycin | *E. coli* ATCC 25922 | ≤0.06 - ≥64 | 0.25-1 |
| Amikacin | *E. coli* ATCC 25922 | ≤0.125 - ≥256 | 0.5-4 |
| Tigecycline | *E. coli* ATCC 25922 | ≤0.008 - ≥32 | 0.03-0.25 |
| Ciprofloxacin | *P. aeruginosa* ATCC 27853 | ≤0.06 - ≥16 | 0.125-1 |
| Levofloxacin | *P. aeruginosa* ATCC 27853 | ≤0.125 - ≥32 | 0.5-4 |
| Trimethoprim-sulfamethoxazole | *E. coli* ATCC 25922 | ≤0.03 - ≥16 | ≤0.5 |

$^{a}$All concentrations are in μg/mL.

$^{b}$Tested to confirm the integrity of the QC strain for testing with the beta-lactam/beta-lactam-inhibitor combination antimicrobial.

$^{c}$ASTar MIC will report ≥128 μg/mL as an acceptable result.

### V Substantial Equivalence Information:

#### A Predicate Device Name(s):

Accelerate Pheno system, Accelerate Phenotest BC Kit

#### B Predicate 510(k) Number(s):

DEN160032

#### C Comparison with Predicate(s):

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| Device & Predicate Device(s): | **K221688 (Device)** | **DEN160032 (Predicate)** |
| --- | --- | --- |
| Device Trade Name | ASTar BC G– Kit | Accelerate PhenoTest BC Kit |
| **General Device Characteristic Similarities** | | |
| Intended Use/Indications For Use | The ASTar System is intended to be used for the automated quantitative susceptibility testing for most clinically significant microorganisms. The ASTar System does not provide organism identification. The ASTAar System, comprised of the ASTar Instrument with the ASTar BC G– Kit (ASTar BC G– Consumable kit, ASTar BC G– Frozen Insert, and ASTar BC G– Kit software), utilizes high-speed, time-lapse microscopy imaging of bacteria for the *in vitro*, quantitative determination of antimicrobial susceptibility of on-panel gram-negative bacteria. The test is performed directly on positive blood culture samples signaled as positive by a continuous monitoring blood culture system and confirmed to contain gram- negative bacilli by Gram stain. Organism identification is required for AST result interpretation and reporting. Test results from the ASTar BC G– Kit should be interpreted 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. Sub-culturing is necessary to support further testing for: bacteria and antimicrobials not on the ASTar BC G– panel, where inconclusive results are obtained, epidemiologic testing, recovery of organisms present in microbial samples, and susceptibility testing of bacteria in polymicrobial samples. | 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. |
| Blood Culture Bottle Types | BD BACTEC: Standard/10 Aerobic, Anaerobic; Lytic/10 Anaerobic; PEDS PLUS, Plus Aerobic, Anaerobic; | Same |

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| Device & Predicate Device(s): | **K221688 (Device)** | **DEN160032 (Predicate)** |
| --- | --- | --- |
| | BioMeriuex BacT/ALERT: Standard Aerobic, Anaerobic; Plus Aerobic, Anaerobic; PF Plus | |
| Technology | High-speed, time-lapse microscopy imaging | Similar |
| Sample Type | Positive Blood Culture | Same |
| Sample Prep | Automated direct from sample | Same |
| **General Device Characteristic Differences** | | |
| IVD Function | Provides AST results only. ID is required but provided by alternative method | Provides both ID and AST |
| Instrument Platform | ASTar Instrument | Accelerate Pheno System |
| Blood Culture Bottle Type (Versa TREK:REDOX 1 and 2 Tested) | No | Yes |
| Antimicrobial Panel | Amikacin, Ampicillin, Ampicillin-sulbactam, Aztreonam Cefazolin, Cefepime, Ceftazidime, Ceftazidime- avibactam, Cefuroxime, Ciprofloxacin, Gentamicin, Meropenem, Meropenem- vaborbactam, Levofloxacin, Piperacillin-tazobactam, Tobramycin, Tigecycline, Trimethoprim- sulfamethoxazole | Amikacin, Ampicillin, Ampicillin-sulbactam, Aztreonam, Ceftazidime, Ceftaroline, Cefepime, Ceftriaxone, Ciprofloxacin, Daptomycin, Erythromycin, Ertapenem, Gentamicin, Linezolid, Meropenem, Piperacillin-tazobactam, Tobramycin, Vancomycin |
| Types Organisms Tested | Gram-negative bacteria | Gram-positive and Gram-negative bacteria |
| Sample per Instrument | 12 | 1 |
| Time to AST Results | Approximately 6 hours | Approximately 7 hours |

## VI Standards/Guidance Documents Referenced:

- • IEC 60601-1-2 Edition 4.0 2014-02, Medical electrical equipment -Part 1-2: General requirements for basic safety and essential performance
- • IEC 61010-1 Edition 3.1 2017-01, Safety requirements for electrical equipment for measurement, control, and laboratory use
- • IEC 62304 Edition 1.1 2015-06 Consolidated Version, Medical device software - Software life cycle processes
- • IEC 62366-1 Edition 1.1 2020-06 Consolidated Version, Medical devices - Part 1: Application of usability engineering to medical devices
- • ISO 14971 Third Edition 2019-12 Medical devices - Application of risk management to medical devices
- • ISO 15223-1 Fourth edition 2021-07 Medical devices - Symbols to be used with information to be supplied by the manufacturer - Part 1: General requirements
- • ISO 20417 First edition 2021-04 Corrected version 2021-12 Medical devices- Information to be supplied by the manufacturer

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• CLSI M100, 33rd Ed., 2021 Performance Standards for Antimicrobial Susceptibility Testing
• FDA Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems; Guidance for Industry and FDA (Issued August 28, 2009)

# VII Performance Characteristics (if/when applicable):

# A Analytical Performance:

# 1. Precision/Reproducibility:

A reproducibility study for the ASTar System (ASTar BC G– Kit run on ASTar Instrument) with contrived positive blood culture bottles (BCBs) included the evaluation of 23 bacterial strains. Triplicate samples from each contrived blood culture were tested at three sites on at least two days. Reproducibility was determined from the total number (and percent) of results that were within one dilution (+/- one doubling dilution) of the modal MIC result divided by the total number of results. All samples were tested within 16 hours of bottle positivity. Both best-case (assumes that off-scale results are within one dilution of the mode) and worst-case (assumes that off-scale results are more than one dilution of the mode) performance was determined for each antimicrobial. Antimicrobials with inter-site worst-case reproducibility of <89% or an insufficient number of results generated in the initial study were further evaluated in a supplemental study in which testing was performed with three instruments. The supplemental reproducibility study was conducted in-house with three individual instruments to increase the number of valid results. Testing was performed similar to the initial reproducibility study protocol.

Data from the Reproducibility Study are summarized in Table 3 and show the performance for each drug with claimed species. In total, thirty-three (33) samples were re-run. Twenty (20) samples that did not complete a run were re-run under the same sample ID within 16 hours of positive BCB. Thirteen (13) samples were re-run with new inoculation with new sample ID due to ASTar instrument error, and 4 samples were withdrawn as these were duplicates. QC was run on the Q-linea ASTar system each day of reproducibility testing. Inter-site and intra-site reproducibility were determined to be acceptable.

Table 3. Reproducibility of ASTar System (ASTar BC G– Kit)

| Antibiotic | Original reproducibility study results (3 sites) | | Supplemental Study Data (single site) | |
| --- | --- | --- | --- | --- |
| | Best-case^{a} | Worst-case^{b} | Best case^{a} | Worst case^{b} |
| Amikacin | 144/144 (100%) | 139/144 (96.5%) | 53/53 (100%) | 53/53 (100%) |
| Ampicillin^{c} | 118/126 (93.7%) | 118/126 (93.7%) | 36/36 (100%) | 36/36 (100%) |
| Ampicillin-sulbactam | 162/162 (100%) | 162/162 (100%) | 36/36 (100%) | 34/36 (94.4%) |
| Aztreonam | 108/108 (100%) | 99/108 (91.7%) | 18/18 (100%)^{d} | 18/18 (100%)^{d} |
| Cefazolin | 126/126 (100%)^{e} | 126/126 (100%)^{e} | 36/36 (100%)^{e} | 36/36 (100%)^{e} |
| Cefepime | 107/108 (99.1%) | 107/108 (99.1%) | 35/35 (100%) | 35/35 (100%) |
| Ceftazidime | 89/90 (98.9%) | 82/90 (91.1%) | 18/18 (100%) | 13/18 (72.2%) |
| Ceftazidime-avibactam | 89/89 (100%) | 78/89 (88.8%) | 18/18 (100%) | 18/18 (100%) |
| Cefuroxime | 125/125 (100%) | 125/125 (100%) | 36/36 (100%) | 36/36 (100%) |
| Ciprofloxacin | 144/144 (100%) | 144/144 (100%) | 53/53 (100%) | 53/53 (100%) |

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| Antibiotic | Original reproducibility study results (3 sites) | | Supplemental Study Data (single site) | |
| --- | --- | --- | --- | --- |
| | Best-case^{a} | Worst-case^{b} | Best case^{a} | Worst case^{b} |
| Gentamicin | 108/108 (100%) | 108/108 (100%) | 53/53 (100%) | 53/53 (100%) |
| Levofloxacin | 180/180 (100%) | 170/180 (94.4%) | 71/71 (100%) | 71/71 (100%) |
| Meropenem | 36/36 (100%)^{f} | 36/36 (100%)^{f} | 36/36 (100%) | 36/36 (100%) |
| Meropenem-vaborbactam | 90/90 (100%) | 90/90 (100%) | 53/53 (100%) | 53/53 (100%) |
| Piperacillin-tazobactam | 233/233 (100%) | 233/233 (100%) | 53/53 (100%) | 53/53 (100%) |
| Tigecycline | 284/288 (98.6%) | 284/288 (98.6%) | 89/89 (100%) | 89/89 (100%) |
| Tobramycin | 263/270 (97.4%) | 263/270 (97.4%) | 87/89 (97.8%) | 87/89 (97.8%) |
| Trimethoprim-sulfamethoxazole | 180/180 (100%) | 171/180 (95%) | 0/0 (N/A)^{g} | 0/0 (N/A)^{g} |

$^{a}$Best case scenario calculation for reproducibility assuming the off-scale result is within one well from the mode.

$^{b}$Worst case scenario calculation for reproducibility assuming the off-scale result is greater than one well from the mode.

$^{c}$All antimicrobials show a reproducibility of ≥ 95% for best-case scenario calculations, except Ampicillin (93.7%). Supplemental testing with additional samples showed performance with ampicillin ≥95%.

$^{d}$Aztreonam testing with other indicated species (not on panel) for drug: P. aeruginosa.

$^{e}$Cefazolin testing with other indicated species (not on panel) for drug: E. coli and P. mirabilis.

$^{f}$Meropenem reproducibility with all indicated species (not on panel) was 144/144 (100%) for best and worst case scenarios.

$^{g}$No on-scale species tested for Trimethoprim-sulfamethoxazole.

## 2. Linearity:

Not Applicable

## 3. Analytical Specificity/Interference:

Analytical Specificity—Not Applicable

### Interference Study

The aim of this study was to demonstrate the ability of ASTar System to report accurate AST results in the presence of endogenous and exogenous substances in blood samples. Three (3) bacterial isolates from the following bacterial species were included in the analytical study: E. coli, A. baumannii, and P. aeruginosa. The isolates were selected to provide a variety of bacterial characteristics to demonstrate the robustness of the test and to include species that may be affected by the presence of the potential interferents. Organisms were inoculated into BCBs with and without interfering substances and cultured until positive. A purity check was performed on each positive BCB to verify that only monomicrobial samples were included in the study. Positive BCB (BD BACTEC Plus Aerobic/F Culture Vials Plastic) samples with and without the interferents were run on the ASTar System. All conditions were tested in triplicate. The MIC values obtained from the interferent samples were compared to the mode MIC results obtained from the samples without the potential interfering substances. If a MIC value was within ±1 doubling dilution from the initial value (without potential interferent), then the sample passed. At least one QC sample was run each day of testing on each instrument used that day. All QC isolates were run within each week on all instruments used that week. Table 4 lists the potentially interfering endogenous and exogenous substances tested with the ASTar BC G- Kit.

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Table 4. Potential Endogenous Interferents and Concentrations Tested for the ASTar BC G- Kit.

| Potential Interferent | Concentration Tested |
| --- | --- |
| *Endogenous Substances* | |
| Conjugated bilirubin | 400 mg/L |
| Gamma-globulin | 50 g/L (plasma concentration) |
| RBCs (Hemoglobin/Hematocrit) | 20 g/dL |
| WBC | 12,000 WBCs/μL |
| Platelets | 400,000 PLTs/μL |
| *Exogenous Substances* | |
| Intralipid | 20 g/L |
| Sodium polyanethole sulfonate (SPS)^{a} | 0.1% w/v (in bottle with blood) |
| Heparin | 3000 Units/L |

$^{a}$Organisms were inoculated with SPS to match levels in BCBs. SPS in exogenous substances study tested at a higher concentration.

All samples after spiking into blood turned positive as expected (within five days) and could be loaded onto the ASTar instruments as intended (within 16 hours of positivity). However, 10 samples failed during instrument run but could be re-run within 16 hours. In addition, six samples were re-run with new BCB inoculations because four samples failed an agar plate purity check, and two samples failed in the instrument but a re-run on the same day was not possible. One platelet interferent sample (E. coli QM324) gave no results after data re-analysis, due to a failed quality control of a positive growth control in the AST disc (indicating a defective consumable). This sample was not re-run since this information was available at the time of analysis, resulting in 112 samples included in the study. In total, 74 QC isolate samples were run. One (1) QC sample had to be re-run due to one failed MIC, which passed on the second run. In addition, five re-runs were required due to instrument related errors. Remaining QC samples yielded passed QC results for all antimicrobials.

All eight evaluated interfering substances had a >95% pass rate after comparison to samples without interferent (Table 5). No interference could be detected for any of the evaluated substances. The study results suggest that none of the tested interferents reduce quantitative AST performance of positive blood cultures run on the ASTar System.

Table 5. Performance with Potential Exogenous and Endogenous Interferents

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| Potential Interferent | Number of MIC values ±1 from mode MIC values of control sample/ Total number of evaluated MIC values | Pass rate |
| --- | --- | --- |
| Conjugated bilirubin | 117/117 | 100% |
| Gamma-globulin | 117/117 | 100% |
| Intralipid | 117/117 | 100% |
| SPS | 117/117 | 100% |
| Heparin | 117/117 | 100% |
| RBCs (Hemoglobin/Ht) | 116/117 | 99.1% |
| WBCs | 117/117 | 100% |
| Platelets | 95/95 | 100% |

### Interfering Antibiotics Study

The aim of this study was to demonstrate if the presence of antibiotics in a positive blood culture sample can affect the ASTar System's capability to report AST results. Nine (9) bacterial isolates from the following bacterial species were included in the analytical study: E. coli (4), K. pneumoniae (4) and P. aeruginosa (1). Each potentially interfering antibiotic (i.e., Cefotaxime, Ciprofloxacin and Meropenem) was tested using three bacterial isolates from the nine (9) organism panel that were resistant to that antibiotic. In addition to the resistant isolates, a tenth (10th) isolate (K. pneumoniae) that was sensitive to all three antibiotics was used in the study to confirm bioactivity of the antibiotic solutions. The MIC values obtained from the interferent samples were compared to the mode MIC values obtained from the control samples (no antibiotic) of the same isolate/BCB-combination. If the MIC value was within ±1 from the control value, then that MIC value passed. Results were pooled within each category (interfering antibiotic/BCB-combination) and the pass rate (%) when compared to control samples (without potential interferent) was determined. Mode MIC-values were determined for most control samples. However, in some cases the median MIC values were used as the control value instead.

Antibiotic test concentrations were selected to represent the highest expected drug concentration under therapeutic treatment (peak level) (Table 6). Positive BCB samples with and without the antibiotics were run on the ASTar System. Two different bottle types from two main suppliers were used—one containing resins (BD BACTEC Plus Aerobic/F Culture Vials Plastic) and the other lacking resins (bioMérieux BACT/ALERT SA Standard Aerobic). Each resistant isolate/antibiotic/BCB-combination was tested in triplicate. A purity check was performed on each positive BCB to verify that only monomicrobial samples were included in the study. The positive blood culture samples were then run on the ASTar System with kit consumables. At least one QC sample was run on each day of testing on every instrument used that day. All QC isolates were run within each week on all instruments used that week.

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Table 6. Performance with Potentially Interfering Antibiotics

| Antibiotic | Antibiotic class | Test concentration |
| --- | --- | --- |
| Cefotaxime | Cephalosporin | 52.8 mg/dL |
| Ciprofloxacin | Fluoroquinolone | 1.20 mg/dL |
| Meropenem | Carbapenem | 33.90 mg/dL |

The study initially included 108 samples to be run on the ASTar System. All turned positive as expected (within five days) and could be loaded onto the study instruments as intended. Although 10 samples failed during instrument run, these were re-run on the same day. In addition, two samples had to be re-run with new BCB inoculations because one sample failed an agar plate purity check and one sample failed in the instrument and a re-run on the same day was not possible. In total, 17 QC isolates were run. One re-run was required due to instrument related errors. The remaining QC isolates completed their ASTar runs, and all showed passed QC results for all antimicrobials.

Test results for each evaluated potentially interfering antibiotic are shown with pass rate (%) as compared to the control sample mode/median MIC values for the ASTar BC G- Kit (Table 7).

Table 7. Results of Potential Interferent (Antibiotic Study)

| Interferent | BCB type^{a} | Number of MIC values ±1 from mode value of control sample / Total number of evaluated MIC values | Pass Rate^{b} |
| --- | --- | --- | --- |
| Cefotaxime | BACTEC | 191/194 | 98.5% |
| | BACT/ALERT | 192/192 | 100% |
| Ciprofloxacin | BACTEC | 194/194 | 100% |
| | BACT/ALERT | 189/195 | 96.9% |
| Meropenem | BACTEC | 158/159 | 99.4% |
| | BACT/ALERT | 152/158 | 96.2% |

$^{a}$BACTEC bottles contained resins whereas the BACT/ALERT bottles did not contain resins.

$^{b}$Pass rates <90% was observed for some combinations of interferent/bottle type/antimicrobial. For these combinations the results are specified per antibiotic/bottle type (numbers within parenthesis show the ratio of passed replicates/total) as follows—Cefotaxime/BACTEC: Ceftolozane-tazobactam 88.9% (8/9),

Trimethoprim-sulfamethoxazole 77.8% (7/9); Ciprofloxacin/BacT/ALERT: Amikacin 77.8% (7/9), Ampicillin-sulbactam 88.9% (8/9), Cefotaxime 88.9% (8/9), Tobramycin 77.8% (7/9); Meropenem / BACTEC:

Trimethoprim-sulfamethoxazole 83.3% (5/6); Meropenem/BacT/ALERT: Meropenem-vaborbactam 50% (3/6), Trimethoprim-sulfamethoxazole 50% (3/6).

The combined total of potentially interfering antibiotics/BCB-combinations evaluated passed the acceptance criteria of >95% as compared to samples without interfering antibiotics.

# 4. Assay Reportable Range:

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Not Applicable

# 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

**Quality Control Testing.** Quality control samples were run each day that testing was conducted. CLSI recommended QC strains for each antimicrobial were tested a sufficient number of times (i.e., at least 20 times/site) at each testing site using the ASTar System, including the reference site using the broth microdilution reference method. QC organisms were tested on a rotating basis. QC expected ranges and results for the ASTar System are summarized in **Table 8**. For all antimicrobials, greater than 95% of results were within the expected range, which is acceptable.

Table 8. QC Expected Ranges and Results for the Quantitative ASTar System

| Antimicrobial | QC Organism | Expected Range (μg/mL) | No. in Range (%) | |
| --- | --- | --- | --- | --- |
| | | | ASTar | Reference |
| Ampicillin | *E. coli* ATCC 25922 | 2-8 | 149/149 (100) | 108/108 (100) |
| | *K. pneumoniae* ATCC 700603^{a} | >128^{b} | 144/144 (100) | 117/117 (100) |
| Ampicillin/sulbactam | *E. coli* ATCC 25922 | 2-8 | 148/150 (98.7) | 116/117 (99.1) |
| | *K. pneumoniae* ATCC 700603 | 8-32 | 146/146 (100) | 114/116 (98.3) |
| Ceftazidime/avibactam | *P. aeruginosa* ATCC 27853 | 0.5-4 | 162/162 (100) | 113/114 (99.1) |
| | *K. pneumoniae* ATCC 700603 | 0.25-2 | 146/146 (100) | 115/115 (100) |
| Meropenem/vaborbactam | *P. aeruginosa* ATCC 27853 | 0.125-1 | 160/161 (99.4) | 115/115 (100) |
| | *K. pneumoniae* ATCC BAA 2814 | 0.125-0.5 | 147/147 (100) | 118/118 (100) |
| Piperacillin/tazobactam | *P. aeruginosa* ATCC 27853 | 1-8 | 162/162 (100) | 115/116 (99.1) |
| | *K. pneumoniae* ATCC 700603 | 8-32 | 146/146 (100) | 113/113 (100) |
| Cefazolin | *E. coli* ATCC 25922 | 1-4 | 150/150 (100) | 116/117 (99.1) |
| Cefepime | *P. aeruginosa* ATCC 27853 | 0.5-4 | 161/162 (99.4) | 118/119 (99.2) |
| Cefuroxime | *E. coli* ATCC 25922 | 2-8 | 150/150 (100) | 115/116 (99.1) |
| Ceftazidime | *P. aeruginosa* ATCC 27853 | 1-4 | 161/161 (100) | 109/110 (99.1) |
| | *K. pneumoniae* ATCC 700603^{a} | 16-64 | 146/146 (100) | 115/115 (100) |
| Aztreonam | *P. aeruginosa* ATCC 27853 | 2-8 | 161/162 (99.4) | 118/118 (100) |
| Meropenem | *P. aeruginosa* ATCC 27853 | 0.125-1 | 145/145 (100) | 117/118 (99.2) |
| Gentamicin | *P. aeruginosa* ATCC 27853 | 0.5-2 | 159/159 (100) | 116/116 (100) |
| Tobramycin | *E. coli* ATCC 25922 | 0.25-1 | 149/150 (99.3) | 113/116 (97.4) |
| Amikacin | *E. coli* ATCC 25922 | 0.5-4 | 149/150 (99.3) | 114/115 (99.1) |
| Tigecycline | *E. coli* ATCC 25922 | 0.03-0.25 | 149/150 (99.3) | 114/115 (99.1) |
| Ciprofloxacin | *P. aeruginosa* ATCC 27853 | 0.125-1 | 160/162 (98.8) | 111/112 (99.1) |
| Levofloxacin | *P. aeruginosa* ATCC 27853 | 0.5-4 | 162/162 (100) | 114/115 (99.1) |
| Trimethoprim/ sulfamethoxazole | *E. coli* ATCC 25922 | ≤0.5 | 149/149 (100) | 102/103 (99.0) |

$^{a}$ Tested to confirm the integrity of the QC strain for testing with the beta-lactam/beta-lactam-inhibitor combination antimicrobial.

$^{b}$ Highest concentration of Ampicillin on the ASTar BC G− panel is ≥128 μg/mL. ASTar MIC results of ≥128 μg/mL were considered acceptable.

### Microbial Suspension Accuracy Study

The objective of this study was to provide performance data supporting the ASTar System accuracy of bacterial concentration measurement and adjustment and to determine the system's capability to abort AST assay if a set low inoculum is detected. *E. coli*, *P. aeruginosa*, *E. cloacae complex* and *K. aerogenes* were included in this analytical study to represent a variety of species found in positive BCB, including two isolates that have been found to give high bacterial counts in positive BCBs. A dilution series (10-fold dilutions) consisting of six concentrations was prepared for each bacterial isolate. Each bacterial isolate

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dilution was tested in triplicate for a total of 72 samples. Up to 12 samples were run per day on the ASTar System.

Seeded blood culture bottles using the four organisms were cultured until positive. Bottles seeded with blood in the absence of bacteria were also cultured in parallel. A viable count was performed on each positive BCB to determine the starting input concentration for the test system. Also, a purity check was performed on each positive BCB to verify that only monomicrobial samples were included in the study. Material from each positive BCB was then diluted in material from the BCBs without bacteria (negative) in five steps of ten-fold dilutions. A wide range of BCB input concentrations were prepared ($4.0 \times 10^3 - 4.3 \times 10^9$ CFU/mL). The undiluted sample and the dilution series were then loaded on the ASTar System with kit consumables. Samples were run in the ASTar System until the “*Eject*” function of the cartridge was available for each sample, i.e., when the AST wells were filled. The inoculum was extracted directly from cartridge and further diluted. Viable count for the bacterial input concentration of each sample (BCB viable count) and concentration-adjusted sample were reported. Viable count was performed on the concentration-adjusted sample within 15 minutes, except in cases where the instrument aborted the sample before the inoculum was prepared (i.e., when the bacterial count was already determined by the ASTar System to be out of range). Additionally, any samples which were automatically aborted by the instrument were recorded and the reason for run failure noted.

When run on the ASTar System, the concentration-adjusted samples of *E. coli*, *E. cloacae complex*, *P. aeruginosa* and *K. aerogenes* were required to be in a range of $2 - 8 \times 10^5$ CFU/mL by viable count in order to pass the acceptance criteria. A viable count was performed by plating three agar plates per sample and then calculating a mean CFU/mL. The viable count value was then used to assess the pass/fail criteria. The total pass rate was calculated using viable count data from all concentration-adjusted samples. If the mean CFU/mL was within acceptable range, the sample passed. On each study day where the ASTar System was run, at least one QC isolate was run on each operating instrument. In total, 11 QC isolates were run during the study. All QC samples yielded acceptable QC results.

Seventy-two (72) samples were included in the study (**Table 9**). Purity check was performed on all samples. Thirty-two (32) out of the 72 samples completed concentration adjustment by the ASTar System, and a subsequent viable count performed. Of these samples, the pass rate was 96.9% (31/32). Twenty-four (24) samples were within the ASTar System requirements input range of $5 \times 10^7 - 5 \times 10^9$ CFU/mL. 95.8% (23/24) of those samples completed the concentration adjustment step, and all these adjustments were within an acceptable range. Twelve (12) samples were in a concentration range between $5 \times 10^6$ and $< 4.99 \times 10^7$ CFU/mL. Of these, twelve samples, 75.0% (9/12) completed concentration adjustment with 88.9% (8/9) within an acceptable output range. Thirty-six (36) samples had a BCB concentration of $< 5 \times 10^6$ CFU/mL, where none of the samples completed the concentration adjustment step.

**Table 9.** BCB Viable Count Input Concentration Range and Distribution of Samples and Completed Concentration Adjustment by the ASTar System

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| BCB Input Viable Count (CFU/mL) | Quantity | Completed Concentration Adjustment | ASTar Output Viable Count within Acceptance Range |
| --- | --- | --- | --- |
| ≥ 5 x 10^{9} | 0 | 0% (0/0) | N/A (0/0) |
| 5 x 10^{8} to < 4.99 x 10^{9} | 12 | 100% (12/12) | 100% (12/12) |
| 5 x 10^{7} to < 4.99 x 10^{8} | 12 | 91.7% (11/12) | 100% (11/11) |
| 5 x 10^{6} to < 4.99 x 10^{7} | 12 | 75.0% (9/12) | 88.9% (8/9)^{a} |
| < 5 x 10^{6} | 36 | 0% (0/36) | N/A (0/0) |

$^{a}$The viable count for *K. aerogenes* QM409 sample was outside the acceptable range. This was likely due to an operator mistake, resulting in the wrong dilution plated for one of the three replicates used to determine viable count.

Out of the 72 tested samples in this study, 40 samples did not complete the sample concentration adjustment step on the ASTar System and were aborted by the instrument. Of those 40 samples not completing sample concentration adjustment, 39/40 (97.5%) had BCB input concentrations determined to be below the ASTar System requirements of $5 \times 10^7 - 5 \times 10^9$ CFU/mL. Only one (1) sample (*P. aeruginosa* QM276) was within the system requirements and did not complete concentration adjustment. No samples in the study were above the $5 \times 10^9$ CFU/mL limit in system requirements. Samples that passed the concentration adjustment step had a BCB input concentration range between $2.4 \times 10^7 - 4.3 \times 10^9$ CFU/mL. The ASTar System was able to perform a concentration determination and adjustment for the majority of samples where the BCB input concentration was near the ASTar System requirements input range.

### Sample Stability Study

The objective of this study was to provide data to support sample stability when samples come from blood culture bottles are stored either in blood culture cabinet or at room temperature. Nine (9) isolates from the following organisms were included in the study: *E. coli*, *K. oxytoca*, *K. pneumoniae*, *P. aeruginosa*, *P. mirabilis*, *E. cloacae*, *S. marcescens*, *C. koseri*, and *A. baumannii*. The isolates represent the product panel and were selected to favor resistance phenotypes and to include as many on-scale MIC values as possible. At least one QC sample was run each day of testing. The nine different isolates were run after three time/incubation conditions:

- 16-24 hours after positivity

The MIC values after 16-24 hours of incubation time (RT and 35 °C) were compared to the modal MIC values obtained from the samples run within one hour after positivity (**Table 10**). Mode MIC-values could be determined for most samples (<1 hour after positivity). In cases where a mode value could not be determined, the median was used. Results from all antimicrobials were pooled within each time/incubation condition. MIC values (within ±1 of the modal MIC value of samples loaded <1 hour) were evaluated for all time/incubation conditions. Overall, the number of MIC values of sample conditions tested within ±1 dilution to the mode value in the <1 hour sample were 100% (441/441) for 16-24 hours at RT and 99.6% (459/461) for 16-24 hours at 35 °C. Pass rates are shown below in **Table 10** at 16-18 hours and >18-24 hours at room temperature and at 35°C.

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Table 10. Pass Rate of Samples Loaded onto the ASTar System During Different Timeframes after BCB positivityᵃ

| Antimicrobial agent | Room Temperature | | 35 °C | |
| --- | --- | --- | --- | --- |
| | 16-18 hoursᵇ,ᶜ | >18-24 hoursᵇ | 16-18 hoursᵇ | >18-24 hoursᵇ |
| Amikacin | 1/1 (100%) | 24/24 (100%) | 3/3 (100%) | 23/23 (100%) |
| Ampicillin | -- | 5/5 (100%) | 3/3 (100%) | 3/3 (100%) |
| Ampicillin-sulbactam | -- | 17/17 (100%) | 3/3 (100%) | 15/15 (100%) |
| Aztreonam | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Cefazolin | -- | 14/14 (100%) | 3/3 (100%) | 12/12 (100%) |
| Cefepime | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Ceftazidime | 1/1 (100%) | 24/24 (100%) | 3/3 (100%) | 23/23 (100%) |
| Ceftazidime-avibactam | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Cefuroxime | -- | 17/17 (100%) | 3/3 (100%) | 14/14 (100%) |
| Ciprofloxacin | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Gentamicin | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Levofloxacin | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Meropenem | 1/1 (100%) | 24/24 (100%) | 3/3 (100%) | 23/23 (100%) |
| Meropenem-vaborbactam | -- | 19/19 (100%) | 3/3 (100%) | 17/17 (100%) |
| Piperacillin-tazobactam | -- | 22/22 (100%) | 3/3 (100%) | 19/20 (95.0%) |
| Tigecycline | -- | 17/17 (100%) | -- | 16/17 (94.2%) |
| Tobramycin | 1/1 (100%) | 21/21 (100%) | 3/3 (100%) | 20/20 (100%) |
| Trimethoprim-sulfamethoxazole | -- | 19/19 (100%) | 3/3 (100%) | 17/17 (100%) |
| Total | 11/11 (100%) | 430/430 (100%) | 63/63 (100%) | 396/398 (99.5%) |

ᵃ Bottle type was the BD BACTEC Plus Aerobic medium in plastic culture vials.

ᵇ Stability of samples loaded to the ASTar System within different timeframes after BCB positivity for each time/incubation condition. The pass rate represents the number of MIC values ±1 from mode MIC values in initial sample (<1 hour)/total MIC values at that condition. Data also includes indicated species, but not claimed, due to panel alteration after the study was completed.

ᶜ Greater number of data points collected across the panel of antimicrobials under more challenging conditions related to time (>18 hrs) and temperature (35°C).

In total, 103 positive blood culture samples were loaded onto the ASTar System. Eight (8) samples failed during instrument run and did not generate MIC results; however, these were re-run within the respective timeframe using the same positive BCB. Twelve (12) samples had to be re-run with a new BCB inoculation due to failed purity check. Two (2) samples were accidentally taken out of cabinet before turning positive and had to be re-run with new BCB inoculations. Two samples (E. cloacae, 16-24 hours at 35 °C, and P. mirabilis, 16-24 hours at RT) gave no results due to a failed quality check of a positive growth control in the ASTar disc (indicating a defect consumable). One (1) additional sample (S. marcescens, 16-24 hours at RT) yielded no results due to a delay in transfer of ASTar culture images during the run. These last three samples were not re-run at the time of sample testing. In total 64 QC isolate samples were run during the study. Two (2) QC samples failed QC results in the initial run (E. coli ATCC 25922 run with Cefazolin), but were successfully re-run. Three (3) QC samples were re-run successfully after instrument related errors, and one (1) QC sample was not re-run within the week it failed. Remaining QC samples yielded acceptable results for all antimicrobials.

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Samples stored for up to 16 hours after positivity at either room temperature or in 35 °C in a blood culture cabinet produced similar results to samples loaded into the ASTar instrument within 1-hour of positivity. Language will be added to labeling to indicate positive blood cultures should be “tested immediately after a positive flag,” as well as language indicating that testing within 16 hours of positivity may be performed in case of instrument errors or if re-testing is needed.

6. Detection Limit:

Not Applicable

7. Assay Cut-Off:

Not Applicable

8. Accuracy (Instrument):

Not Applicable

9. Carry-Over:

The objective was to provide supporting data of the ASTar Systems’ ability to run multiple samples in parallel without carry-over or cross contamination between samples. Two (2) isolates of E. coli—one highly resistant and one susceptible—were included in this analytical study. MIC values for the two E. coli isolates differed for several antimicrobials. BCBs (BD BACTEC Plus Aerobic/F Culture Vials Plastic) were seeded separately with each isolate and spiked into healthy human donor blood and incubated until positive. Positive BCB samples were then run on the ASTar System within 16 hours of positive BCB ring. Up to 12 samples were run per day on the ASTar System. The operators loaded and started a run of 6 samples with either alternating susceptible or resistant isolates or a full run of the isolate in each ASTar instrument. At least one QC sample was run each day of testing on every ASTar Instrument used that day. The MIC values of isolates tested in parallel were within ± 1 dilution compared to the mode MIC values for each respective strain. When evaluating the susceptible isolate samples, no carry-over or cross contamination was observed as evidenced by 99.7% total pass rate (307/308). In conclusion, the ASTar System was capable of running multiple samples in parallel without carry-over or cross contamination between samples.

For re-runs, two (2) samples required re-runs due to sample failures and were re-loaded within 16 hours. Six (6) samples required re-runs on another day with new BCB inoculations due to a single system failure. During re-run, a full alternated isolate run with six samples was loaded to ASTar, which resulted in three additional datapoints for a total of 27 samples. In total, 11 QC isolate samples were run on ASTar instruments during the study. Two (2) out of 11 QC samples were additional re-runs due to sample and system failures on ASTar; all antimicrobials had a passed QC result on each instrument.

# B Comparison Studies:

1. Method Comparison with Predicate Device:

The purpose of the clinical study was to demonstrate the clinical performance of the ASTar BC G- Kit with the ASTar Instrument in providing quantitative AST results direct from

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positive blood culture containing Gram-negative bacteria. Results were compared to reference frozen Broth Microdilution (BMD) results performed according to CLSI M07. Positive blood cultures included fresh, left-over samples from patients with suspected bacteremia, along with positive blood cultures contrived with clinical stock isolates from the clinical sites or challenge isolates. Clinical stock isolates and challenge isolates were enrolled to supplement fresh positive blood cultures due to low prevalence of certain species and antimicrobial resistance expected during prospective collection. Isolates were sub-cultured on appropriate media (Tryptic Soy Agar with 5% sheep blood), spiked into blood culture bottles at a concentration of 10³ to 10⁴ CFU per bottle containing fresh human donor blood, and incubated on the appropriate blood culture system until ring positivity.

Clinical performance testing on the ASTar System was initially performed at four sites (3 U.S. test sites and 1 internal site). For instances in which testing was required to supplement existing data from the original study and support specific claims, testing was performed on ASTar System instruments at three testing sites (2 US and one internal). Testing with the ASTar BC G– Kit on the ASTar Instrument was performed within 16 hours of blood culture positivity during which time the blood culture bottle was either kept on the automated blood culture instrument or stored at room temperature until testing. Organism identification was obtained from an FDA cleared bacterial identification method and/or FDA cleared MALDI-TOF method for input into the ASTar Instrument. An aliquot from contrived positive blood cultures was sub-cultured onto Tryptic Soy Agar with 5% Sheep Blood or other appropriate media plate and incubated for 18-24 hours. Isolation plates were checked for purity and colony morphology, which was used to verify isolate identification via MALDI. If more than one colony type was observed, each colony type was isolated for purity assessment. If direct-from-blood culture ID system (first bacterial ID method) results were not available or if the results from the specified ID method did not provide a specific species on the ASTar panel, results from MALDI were performed on subcultured isolates and used for input into the ASTar System. During the study, MALDI was performed on all isolates for confirmation. MALDI from the subcultured organism was used for final organism identification. If more than one organism was identified by the first bacterial ID method, the ASTar test was aborted or the pathogen ID not entered in the ASTar Instrument, and the sample was excluded from the final performance data according to the exclusion criteria. Samples with multiple organisms were tracked and reported as mixed or polymicrobial.

A total of 1,068 samples were enrolled in the study that included both Fresh PBC (positive blood cultures) and contrived PBC with either clinical stock or challenge isolates. 188 samples were excluded due to off-panel organisms, contamination of contrived samples (either due to the blood used for contriving or other sources), non-viable stock isolates, and protocol deviations. In total 880 samples were included in the performance analysis consisting of 256 fresh, positive blood culture samples (29.1%), 223 contrived samples with clinical stock isolates (25.3%), and 401 contrived blood culture samples with challenge isolates (45.6%). In the choice of clinical stock isolates, on-panel organisms (i.e., Gram-negative bacteria species included in the ASTar BC G- panel) were selected from isolate banks at the external clinical sites to supplement species and antimicrobial resistance requirements. These isolates were originally sourced from clinical specimens from patients admitted at the clinical site. Pure isolates were cultured and contrived in blood cultures using healthy human donor blood and enrolled and tested after positivity. The set of challenge isolates were provided for testing at both the internal clinical site and selected external clinical sites. These challenge isolates included CDC AR Bank isolates obtained in the

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United States as well as isolates obtained from clinical samples at hospitals throughout Europe. Challenge isolates were used to supplement Fresh PBC and contrived stock isolates for species inclusion, specific antimicrobial resistance, and on-scale MIC results closer to the breakpoints. In addition, challenge isolates were shipped to the central reference site for BMD testing and characterization. Subsequently, selected on-panel challenge isolates were cultured and contrived in blood cultures using healthy human donor blood, and enrolled and tested after ring positivity. ASTar BC G- Kit testing was compared to frozen BMD run in triplicate according to CLSI to establish a reference Mode MIC for each antimicrobial evaluated. If a Mode MIC could not be established with the first set of three replicates a second set of three frozen replicates was tested. If a Mode MIC could not be established with the second set of plates, the Median from all six plates was used. A total of 933 valid samples were analyzed on the ASTar during the clinical study and 97.2% (907/933) of samples produced at least a partial AST result. Of the 26 samples that failed to produce an AST result, 96% (25/26) were resolved upon retesting.

Performance was determined generally based on criteria outlined in the Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems including essential agreement (EA), categorical agreement (CA), and categorical errors (minor, major and very major errors). EA was calculated as the percentage of ASTar MIC results that were within plus or minus one serial two-fold dilution of the reference result. CA was calculated as the percentage of ASTar interpretive results (S/I/R) that were identical to the interpretive results of the reference result. EA of evaluable results (on-scale ASTar and reference results or results in which an off-scale result was at least two doubling dilutions from the on-scale result) were also calculated. Performance was considered acceptable if the EA and CA were ≥90%, major error rate was ≤3%, and very major error rate was ≤2%. For antimicrobials that lack an intermediate interpretive criterion, further analysis of the category errors was performed, and adjustments were made by considering the MIC values that were one doubling dilution from the reference MIC value.

A high-level summary of the ASTar BC G- Kit AST System performance is described below for each antimicrobial and indicated species. Complete details and results including EA, CA and error rate analyses are summarized in Table 11.

**Ampicillin.** A total of 236 Enterobacterales isolates (202 *E. coli*, 34 *P. mirabilis*) were evaluated with ampicillin. The combined results from clinical and challenge testing demonstrated an EA of 97.5% and CA of 97.9%. There were 3 minor, 2 major, and 0 very major errors. Overall, performance was acceptable.

**Amikacin.** A total of 49 *A. baumannii* isolates were evaluated with amikacin. The combined results from clinical and challenge testing demonstrated an EA of 89.8% and CA of 85.7%, which was not acceptable. There were 6 minor, 1 major (1/36 = 2.8%), and 0 very major errors. Due to the unacceptable performance for *A. baumannii*, this drug/organism combination is not indicated for use with the ASTar System. The following limitation is included in the device labeling to restrict reporting of *A. baumannii* due to unacceptable performance:

Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combination(s):

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- Amikacin: Acinetobacter baumannii

A total of 64 P. aeruginosa isolates were evaluated with amikacin. The combined results from clinical and challenge testing demonstrated an EA of 92.2% and CA of 96.9%. There were 2 minor, 0 major, and 0 very major errors. Overall, performance was acceptable.

A limitation statement is included in the device labeling to address the lack of testing with resistant Pseudomonas aeruginosa isolates.

A total of 735 Enterobacterales isolates (indicated species: 200 E. coli, 65 E. cloacae complex, 27 C. freundii, 48 K. aerogenes, 50 K. oxytoca, 142 K. pneumoniae, 89 P. mirabilis, 33 P. vulgaris, 39 S. marcescens; non-indicated species: 42 C. koseri) were evaluated with amikacin. The combined results from clinical and challenge testing demonstrated an EA of 86.7% and CA of 99.1%. There were 7 minor, 0 major, and 0 very major errors. When evaluating results by individual species, E. coli had an EA of 68% and CA of 99% with 2 minor, 0 major, and 0 very major errors. The performance was not acceptable. P. vulgaris had an EA of 81.8% with 0 minor, 0 major, and 0 very major errors. The performance was not acceptable. Due to the unacceptable performance for E. coli and P. vulgaris, these drug/organism combinations are not indicated for use with the ASTar System. The following limitation is included in the device labeling to restrict reporting of E. coli and P. vulgaris due to unacceptable performance:

Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combination(s):

- Amikacin: Escherichia coli, Proteus vulgaris

A limitation statement is included in the device labeling to address the lack of testing with resistant Citrobacter koseri, Citrobacter freundii, Enterobacter cloacae complex, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, and Serratia marcescens isolates.

Ampicillin-Sulbactam. A total of 49 A. baumannii isolates were evaluated with ampicillin-sulbactam. The combined results from clinical and challenge testing demonstrated an EA of 89.8% and CA of 71.4%. The EA of evaluable for A. baumannii was 88.6%, which was not acceptable. In addition, there were 13 minor, 1 major (1/28=3.6%), and 0 very major errors, which was not acceptable. Due to the unacceptable performance for A. baumannii, this drug/organism combination is not indicated for use with the ASTar System. The following limitation is included in the device labeling to restrict reporting of A. baumannii due to unacceptable performance:

Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combination(s):

- Ampicillin-Sulbactam: Acinetobacter baumannii

A total of 487 Enterobacterales isolates (indicated species: 201 E. coli, 50 K. oxytoca, 140 K. pneumoniae, 34 P. mirabilis, 20 P. vulgaris; non-indicated species: 42 C. koseri) were evaluated with ampicillin-sulbactam. The combined results from clinical and challenge testing demonstrated an EA of 97.3% and CA of 90.1%. There were 47 minor, 1 major, and 0

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very major errors. When evaluating results by individual species, K. oxytoca had a CA <90%, which was considered acceptable since all of the categorical errors were minor and the EA of evaluable results was good (>95%). P. vulgaris had an EA of 85% and CA of 95%. The EA of the evaluable was 85%, which was low. However, analysis of performance of P. vulgaris using a truncated reporting range (≤2 μg/mL - ≥128 μg/mL) improved the EA to 90%. This performance was acceptable.

A limitation statement is included in the device labeling to address the lack of testing with resistant Citrobacter koseri, Klebsiella oxytoca, Proteus mirabilis, and Proteus vulgaris isolates.

Aztreonam. A total of 679 Enterobacterales isolates (201 E. coli, 42 C. freundii, 42 C. koseri, 65 E. cloacae complex, 48 K. aerogenes, 49 K. oxytoca, 141 K. pneumoniae, 34 P. mirabilis, 18 P. vulgaris, 39 S. marcescens) were evaluated with aztreonam. The combined results from clinical and challenge testing demonstrated an EA of 96.2% and CA of 96.3%. There were 20 minor, 0 major, and 5 very major errors. When evaluating results by individual species, C. freundii had a CA <88.1% and EA of Evaluable of <69.2% with 4 minor, 0 major, and 1 (1/13=7.7%) very major errors. The performance was unacceptable. For K. oxytoca, an EA of 85.7% was observed with a CA of 98% with 0 minor and 0 major errors. One 1(1/4=25%) very major error was observed, which was considered a random error due to the limited number of resistant isolates tested. Analysis of performance of K. oxytoca using a truncated reporting range (≤0.25 μg/mL- ≥128 μg/mL) improved the EA to 93.9%. This performance was acceptable. E. coli showed 2 very major errors (2/52=3.8%)—one of which was at ASTar MIC value of 0.5 μg/mL (representing 8.2%) of results from this drug/organism combination. Restricting the reporting at an MIC value of 0.5 μg/mL mitigated one very major error and was acceptable. For P. vulgaris, 1 very major error was reported, which was considered a random error due to the limited number of resistant isolates tested. Due to the unacceptable performance for C. freundii, this drug/organism combination is not indicated for use with the ASTar System. The following limitation is included in the device labeling to address one of the E. coli very major errors and restrict reporting of C. freundii due to unacceptable performance:

Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combination(s):

- Aztreonam: Escherichia coli when the ASTar MIC is 0.5 μg/mL due to 1 very major error; Citrobacter freundii

A limitation statement is included in the device labeling to address the lack of testing with resistant Citrobacter koseri, Klebsiella aerogenes, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, and Serratia marcescens isolates.

A total of 64 P. aeruginosa isolates were evaluated with aztreonam. The combined results from clinical and challenge testing demonstrated an EA of 87.5% and CA of 76.6%. There were 11 minor, 0 major, and 4 very major (4/22 = 18.2%) errors. Due to the unacceptable performance for P. aeruginosa, this drug/organism combination is not indicated for use with the ASTar System. The following limitation is included in the device labeling to restrict reporting of P. aeruginosa due to unacceptable performance:

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Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combination(s):

- Aztreonam: Pseudomonas aeruginosa

Cefazolin. A total of 351 Enterobacterales isolates (indicated species: 49 E. coli, 50 K. oxytoca, 140 K. pneumoniae, 90 P. mirabilis; non-indicated species: 22 C. koseri) were evaluated with cefazolin. The combined results from clinical and challenge testing demonstrated an EA of 89.2% and CA of 76.1%. There were 72 minor, 11 major, and 1 very major errors. When evaluating results by individual species, C. koseri had an EA of 63.6% and CA of 45.4%, which was unacceptable. E. coli had an EA of 85.7% and CA of 81.6% with 4 minor, 5 (5/26=19.2%) major, and 0 very major errors, which was not acceptable. K. oxytoca had an EA of 80% and CA of 72% with 12 minor, 2 major (2/10=20%), and 0 very major errors, which was not acceptable. For K. pneumoniae, the CA was <90%, which was considered acceptable since most of the categorical errors were minor and the EA of evaluable results was good (93.5%). P. mirabilis had an EA of 91.1% and CA of 64.4% with 30 minor, 2 major (2/4=100%), and 0 very major errors with an EA of evaluable of 90.6%. However, the 2 (2/4=50%) major errors yielded an unacceptable major error rate. Due to the unacceptable performance for C. koseri, E. coli, K. oxytoca, and P. mirabilis, these drug/organism combinations are not indicated for use with the ASTar System. The following limitation statement is included in the device labeling to restrict reporting of C. koseri, E. coli, K. oxytoca, and P. mirabilis due to unacceptable performance:

Perform an alternative method of testing prior to reporting results for the following antibiotic/organism combinations:

- Cefazolin: Citrobacter koseri, Escherichia coli, Klebsiella oxytoca, Proteus mirabilis

Cefepime. A total of 738 Enterobacterales isolates (indicated species: 41 C. freundii, 64 E. cloacae complex, 202 E. coli, 49 K. aerogenes, 50 K. oxytoca, 141 K. pneumoniae, 90 P. mirabilis, 20 P. vulgaris, 39 S. marcescens; non-indicated: 42 C. koseri) were evaluated with cefepime. The combined results from clinical and challenge testing demonstrated an EA of 95.1% and CA of 95.5%. There were 31 minor, 2 majo…