LifeScale Gram Negative Kit (LSGN) with the LifeScale AST System

K211815 · Affinity Biosensors · SAN · Apr 2, 2024 · Microbiology

Device Facts

• Record ID: K211815
• Device Name: LifeScale Gram Negative Kit (LSGN) with the LifeScale AST System
• Applicant: Affinity Biosensors
• Product Code: SAN · Microbiology
• Decision Date: Apr 2, 2024
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.1650
• Device Class: Class 2
• Attributes: PCCP

Intended Use

The LifeScale AST system is a multiplexed in vitro diagnostic test that uses a microfluidic sensor and resonant frequency to calculate organism concentration and/or mass distribution for quantitative antimicrobial susceptibility testing (AST). Testing is performed directly on blood cultures signaled as positive by a continuous monitoring blood culture system and confirmed by Gram stain. The LifeScale AST system does not provide organism identification and is not indicated for use with polymicrobial samples. Interpretive results (Susceptible/Intermediate/Resistant) are provided for specific drug/organism combinations. 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 susceptibility testing of organisms present in polymicrobial samples, for testing antimicrobial agents and species not indicated for testing with the device, for epidemiologic testing and for recovery of organisms present in microbial samples. The LifeScale Gram Negative Kit (LSGN) is intended for use with the LifeScale AST system for in vitro testing of positive blood culture samples confirmed by Gram stain as containing gram-negative bacilli.

Device Story

System uses microfluidic sensor and resonant frequency technology to measure microbe count, mass, and fluid volume in positive blood culture samples; provides quantitative MIC and interpretive (S/I/R) results for specific Gram-negative bacilli/antibiotic combinations. Used in clinical laboratories; requires positive blood culture confirmed by Gram stain. System automates inoculation, incubation (3-6 hours), and readout; includes automated washing/disinfection of sipper and sensor to prevent cross-contamination. Clinicians use output alongside other findings to guide antibiotic therapy; benefits include faster AST results compared to traditional methods. Does not provide organism identification; requires subculture for polymicrobial samples or non-indicated agents.

Clinical Evidence

Clinical performance evaluated at six U.S. sites using 201 prospective positive blood cultures and 1,112 contrived samples (seeded clinical isolates). Performance measured against reference broth microdilution (BMD) per CLSI M07. Primary endpoints: Essential Agreement (EA) and Categorical Agreement (CA). Results showed acceptable performance for indicated drug/organism combinations (EA/CA generally >90%). Trending analysis identified high MIC bias for several combinations, addressed via labeling. Study confirmed system performance for on-line and off-line incubation, with specific limitations for ertapenem/E. coli off-line.

Technological Characteristics

System uses microfluidic sensor with mechanically vibrating microchannel (transduces mass to frequency). Components: LifeScale instrument, touchscreen, auto-inoculator, centrifuge, compressor/waste station, manual barcode reader. Consumables: LSGN 96-well plate with lyophilized antibiotics, cation-adjusted Mueller Hinton growth media. Connectivity: USB for peripheral communication. Software: MIC Analysis Software v2.3.58. Sterilization: Not specified. Standards: IEC 60601-1-2, IEC 61010-1, IEC 62304.

Indications for Use

Indicated for in vitro testing of positive blood culture samples confirmed by Gram stain as containing gram-negative bacilli for specific antimicrobial agents and target organisms: Ampicillin (E. coli); Aztreonam (E. coli, K. aerogenes, K. oxytoca); Cefazolin (K. pneumoniae, K. variicola); Ceftazidime (A. baumannii, A. baumannii/nosocomialis group, E. coli, K. aerogenes, K. oxytoca, K. variicola, P. aeruginosa); Ertapenem (E. coli, K. aerogenes, K. oxytoca); Trimethoprim-Sulfamethoxazole (E. coli, K. aerogenes, K. oxytoca, K. variicola).

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 Pheno system, Accelerate PhenoTest BC Kit (K192665)

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image contains two logos. On the left is the Department of Health & Human Services logo. On the right is the FDA logo, which includes the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue text.

April 2, 2024

Affinity Biosensors % Peter Trabold, Ph.D., MBA Regulatory Affairs Specialist MDC Associates. Inc. 180 Cabot Street Beverly, Massachusetts 01915

Re: K211815

Trade/Device Name: LifeScale Gram Negative Kit (LSGN) with the LifeScale AST system Regulation Number: 21 CFR 866.1650 Regulation Name: A Cellular Analysis System For Multiplexed Antimicrobial Susceptibility Testing Regulatory Class: Class II Product Code: SAN, LON Dated: October 13, 2023 Received: October 16, 2023

Dear Peter Trabold:

We have reviewed your section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (the Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

FDA's substantial equivalence determination also included the review and clearance of your Predetermined Change Control Plan (PCCP). Under section 515C(b)(1) of the Act, a new premarket notification is not required for a change to a device cleared under section 510(k) of the Act, if such change is consistent with an

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established PCCP granted pursuant to section 515C(b)(2) of the Act. Under 21 CFR 807.81(a)(3), a new premarket notification is required if there is a major change or modification in the intended use of a device, or if there is a change or modification in a device that could significantly affect the safety or effectiveness of the device, e.g., a significant change or modification in design, material, chemical composition, energy source, or manufacturing process. Accordingly, if deviations from the established PCCP result in a major change or modification in the intended use of the device, or result in a change or modification in the device that could significantly affect the safety or effectiveness of the a new premarket notification would be required consistent with section 515C(b)(1) of the Act and 21 CFR 807.81(a)(3). Failure to submit such a premarket submission would constitute adulteration and misbranding under sections 501(f)(1)(B) and 502(o) of the Act, respectively.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (OS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatory

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assistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

# Ribhi Shawar -S

Ribhi Shawar, Ph.D. (ABMM) Branch Chief General Bacteriology and Antimicrobial Susceptibility Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known)

K211815

#### Device Name

LifeScale Gram Negative Kit (LSGN) with the LifeScale AST system

#### Indications for Use (Describe)

The LifeScale AST system is a multiplexed in vitro diagnostic test that uses and resonant frequency to calculate organism concentration and/or mass distribution for quantitative antimicrobial susceptibility testing is performed directly on blood cultures signaled as positive by a continuous monitoring blood culture system and confirmed by Gram stain. The LifeScale AST system does not provide organism identification and is not indicated for use with polymicrobial samples. Interpretive results (Susceptible/Intermediate/Resistant) are provided for specific drug/organism combinations. Results are 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 is necessary for the susceptibility testing of organisms present in polymicrobial samples, for testing antimicrobial agents and species not indicated for testing with the device, for epidemiologic testing and for recovery of organisms present in microbial samples.

The LifeScale Gram Negative Kit (LSGN) is intended for use with the LifeScale AST system for in vitro testing of positive blood culture samples confirmed by Gram staining gram-negative bacilli for the antimicrobial agents and specific target organisms identified below:

• Ampicillin: Escherichia coli

- Aztreonam: Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca
- Cefazolin: Klebsiella pneumoniae, Klebsiella variicola
- Ceftazidime: Acinetobacter baumannii/hosocomialis group, Escherichia coli, Klebsiella aerogenes,
- Klebsiella oxytoca, Klebsiella variicola, Pseudomonas aeruginosa
- · Ertapenem: Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca
- Trimethoprim-Sulfamethoxazole: Escherichia coli, Klebsiella oxytoca, Klebsiella oxytoca, Klebsiella variicola

| Type of Use (Select one or both, as applicable) | |
|-------------------------------------------------------------------------------------------------------------|-----------------------------------------------|
| <span style="text-decoration: underline;"><b> ☑ </b></span> Prescription Use (Part 21 CFR 801 Subpart D) | ☐ Over-The-Counter Use (21 CFR 801 Subpart C) |

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Affinity Biosensors LifeScale™ AST system K211815

# 510(k) Summary

The summary of the 510(k) Substantial Equivalence Determination Performance is being submitted in compliance with section 807.92(c).

# 1. Contact Details

| Sponsor: | Affinity Biosensors, LLC |
|----------|---------------------------------|
| | 222 East Canon Perdido Street |
| | Suite 101 |
| | Santa Barbara, California 93101 |
| | [phone] |

- Correspondent: MDC Associates, Inc. Peter Trabold, Regulatory Affairs Specialist 48 Dunham Ridge, Suite 4000 Beverly, MA 01915 Phone: [phone] Email: [e-mail]
# 2. Device

| Device Trade Name: | LifeScale™ Gram Negative Kit (LSGN) with the LifeScale AST system |
|--------------------------|---------------------------------------------------------------------------------|
| Regulation Name: | A cellular analysis system for multiplexed antimicrobial susceptibility testing |
| Regulation Number: | 21 CRF 866.1650 |
| Product Code: | SAN |
| Additional Product Code: | LON |
| Classification: | Class II |
| Predicate Device: | Accelerate Pheno system, Accelerate PhenoTest BC Kit (K192665) |

# 3. Device Description Summary

The LifeScale AST system is an in vitro diagnostic test designed to quantitatively assess antimicrobial susceptibility using a microfluidic sensor and resonant frequency technology. Specifically engineered for use with positive blood culture samples confirmed positive by Gram stain for Gram-negative rods, the LifeScale LSGN Panel ensures compatibility and accuracy while excluding Gram-positive or polymicrobial samples, thus maintaining specificity and reliability. During the incubation phase, the LifeScale LSGN Panel offers a standard incubation time of 3 hours, extendable up to 6 hours to accommodate varying microbial growth rates. Panels must be read within 8 hours of setup, with automatic cancellation for panels exceeding this timeframe. Panels with delayed readings can be

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safely stored in the offline incubator until analysis. Upon reaching sufficient growth in the positive control wells, the LifeScale AST system transitions to data acquisition and readout. Advanced sensors capture essential metrics including microbe count, mass, and fluid volume, processed through sophisticated software algorithms to generate precise AST results for each antibiotic. To maintain hygiene standards, the LifeScale AST system incorporates automated washing and disinfection protocols for the sipper and sensor, minimizing the risk of cross-contamination and organic buildup. The culmination of the testing process involves calculating and reporting AST results (MIC and interpretive results), providing clinicians with actionable insights into antibiotic efficacy. Species-level organism identification is essential for results reporting. AST results are generated based on FDA or CLSI breakpoints validated for laboratory use.

# 4. Intended Use/Indications for Use

#### Intended Use:

The LifeScale AST system is a multiplexed in vitro diagnostic test that uses a microfluidic sensor and resonant frequency to calculate organism concentration and/or mass distribution for quantitative antimicrobial susceptibility testing (AST). Testing is performed directly on blood cultures signaled as positive by a continuous monitoring blood culture system and confirmed by Gram stain. The LifeScale AST system does not provide organism identification and is not indicated for use with polymicrobial samples. Interpretive results (Susceptible/Intermediate/Resistant) are provided for specific drug/organism combinations. 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 susceptibility testing of organisms present in polymicrobial samples, for testing antimicrobial agents and indicated for testing with the device, for epidemiologic testing and for recovery of organisms present in microbial samples.

# Indications for Use:

The LifeScale Gram Negative Kit (LSGN) is intended for use with the LifeScale AST system for in vitro testing of positive blood culture samples confirmed by Gram stain as containing gram-negative bacili for the antimicrobial agents and specific target organisms identified below:

- 1. Ampicillin: Escherichia coli
- 2. Aztreonam: Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca
- 3. Cefazolin: Klebsiella pneumoniae, Klebsiella variicola
- 4. Ceftazidime: Acinetobacter baumannii, Acinetobacter baumannii/nosocomialis group, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella variicola, Pseudomonas aeruginosa
- 5. Ertapenem: Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca
- 6. Trimethoprim-Sulfamethoxazole: Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella variicola

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# 5. Substantial Equivalency

| Feature | New Device<br>Affinity Biosensors LifeScale Gram Negative Kit (LSGN) with the LifeScale AST system<br>(K211815) | Predicate Device<br>Accelerate Pheno™ System and<br>Accelerate PhenoTest BC Kit<br>(K192665) |
|----------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | The LifeScale AST system is a multiplexed <i>in vitro</i> diagnostic test that uses a microfluidic sensor and resonant frequency to calculate organism concentration and/or mass distribution for quantitative antimicrobial susceptibility testing (AST). Testing is performed directly on blood cultures signaled as positive by a continuous monitoring blood culture system and confirmed by Gram stain. The LifeScale AST system does not provide organism identification and is not indicated for use with polymicrobial samples. Interpretive results (Susceptible/Intermediate/Resistant) are provided for specific drug/organism combinations. 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 susceptibility testing of organisms present in polymicrobial samples, for testing antimicrobial agents and species not indicated for testing with the device, for epidemiologic testing and for recovery of organisms present in microbial samples. The LifeScale Gram Negative Kit (LSGN) is intended for use with the LifeScale AST system for <i>in vitro</i> testing of positive blood culture samples confirmed by Gram stain as containing gram-negative bacilli. | The Accelerate PhenoTest BC kit is a multiplexed <i>in vitro</i> diagnostic test utilizing both qualitative nucleic acid fluorescence <i>in situ</i> 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.<br><br>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: |
| Similarities | | |
| Results | Minimum Inhibitory Concentration (MIC) based Antimicrobial Susceptibility Testing direct from Positive Blood Cultures signaled as positive by a continuous monitoring blood culture system. | Minimum Inhibitory Concentration (MIC) based Antimicrobial Susceptibility Testing direct from Positive Blood Cultures signaled as positive by a continuous monitoring blood culture system. |
| Inoculation Method | Automated | Automated |
| Read Method | Automated | Automated |
| Differences | | |
| Organisms Tested | Gram-negative bacilli bacterial pathogens | Gram-positive and Gram-negative bacterial pathogens |
| Instrument | LifeScale AST system | Accelerate Pheno System |
| Antimicrobial Agents | Ampicillin<br>Aztreonam<br>Cefazolin<br>Ceftazidime<br>Ertapenem<br>Trimethoprim-Sulfamethoxazole | Amikacin<br>Ampicillin<br>Ampicillin/Sulbactam<br>Aztreonam<br>Ceftazidime<br>Ceftaroline<br>Cefepime<br>Ceftriaxone<br>Ciprofloxacin<br>Daptomycin<br>Ertapenem<br>Gentamicin<br>Linezolid<br>Meropenem<br>Piperacillin/Tazobactam<br>Tobramycin<br>Vancomycin |
| Sample Prep | Centrifugation and pipetting of sample. Direct from sample. No manual McFarland prep. | Automated. Direct from sample. No manual McFarland prep. |
| Indicated Organisms | Acinetobacter baumannii<br>Acinetobacter baumannii/nosocomialis group<br>Escherichia coli<br>Klebsiella aerogenes<br>Klebsiella oxytoca<br>Klebsiella pneumoniae<br>Klebsiella variicola<br>Pseudomonas aeruginosa | Gram-negative species:<br>Acinetobacter baumannii<br>Citrobacter spp.<br>Enterobacter spp.<br>Escherichia coli<br>Klebsiella spp.<br>Proteus spp.<br>Pseudomonas aeruginosa<br>Serratia marcescens<br><br>Additional Gram-positive bacteria and yeast are<br>also included on the Accelerate PhenoTest BC kit. |
| Time Required for Analysis of Specimen | 4 hours, on average | 7 hours |
| Technology Principles | Microfluidic Sensor, Mass Measurement | Morphokinetic Cellular Analysis |
| IVD Functions | AST only. ID results based on an alternative procedure. | ID and AST together in same system |

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The differences noted above do not impact the safety and effectiveness of the device.

# 6. Performance Characteristics

#### Comparison Study

A comparison study was conducted to evaluate the performance of the LifeScale™ Gram Negative Kit (LSGN) in testing prospective clinical blood cultures confirmed positive by Gram Stain for Gramnegative bacilli. This study encompassed testing both prospective blood cultures (PBCs) and blood cultures contrived using isolates chosen to generate data required to fulfill intended use claims. All LifeScale AST sample results were compared to the reference Broth Microdilution Method (BMD). Each sample submitted for BMD testing was assigned a unique Trial ID, and LifeScale results were kept blinded to prevent bias. Performance was evaluated by comparing quantitative (MIC) and qualitative (S-I-R) AST results generated by the LifeScale LSGN kit with those of the reference BMD.

Prospective PBCs consistent with the inclusion criteria were enrolled and tested at 6 US Clinical sites. For testing of prospective samples, organism ID was performed using an FDA-cleared direct from positive blood culture ID system. Testing was performed on the LifeScale AST system in accordance with the manufacturer's instructions for use. PBC bottles were sub-cultured onto Tryptic Soy Agar supplemented with 5% Sheep Blood panels (BAP) and MacConkey Agar panels (MAC) incubated for 18-24 hours and examined for purity and colony morphology. If more than one colony type was observed, each organism was isolated for purity. All organisms isolated were identified using matrix-assisted laser desorption/ionization (MALDI). Polymicrobial samples were withdrawn from the study. ID results generated using a direct from Blood Culture ID system and/or MALDI were entered into the LifeScale AST system, and a final MIC/SIR result was generated using the final LifeScale AST system software. If there was a discordant organism identification between MALDI and a Direct from Blood Culture system, MALDI ID was considered the organism's final identification.

Contrived samples were prepared from frozen isolates supplied by Affinity Biosensors, or they were prepared from contemporary isolates collected by the laboratory and agreed upon by Affinity for study inclusion. Blood cultures with the required amount of blood were spiked with isolated organisms and incubated in the blood culture system. When flagged as positive blood culture was tested on the LifeScale AST system in accordance with manufacturer's instructions for use. The PBC was sub-cultured to confirm purity. If a mixed (contaminated) culture was observed, a fresh contrived sample was prepared and tested.

Indicated CDC Challenge and Challenge isolates from other reference laboratories were prepared from frozen isolates supplied by Affinity Biosensors to one trial site. Blood cultures with the required amount of blood were spiked with isolated organisms and incubated in the blood culture system. When flagged as positive, the positive blood culture was tested on the LifeScale AST system in accordance with manufacturer's instructions for use. The PBC was sub-cultured to confirm purity. If a mixed (contaminated) culture was observed, a new sample was prepared and tested.

All LifeScale LSGN testing was performed within 12 hours of the blood culture bottle being flagged as positive. LifeScale LSGN panels were read upon system confirmation of growth. Positive growth was determined automatically by the LifeScale AST system as part of the reading process. If the panel was

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incubated offline, it was placed on the LifeScale system to be read. To generate the final AST report, the organism ID was entered into LifeScale AST system. The LifeScale AST system software generated the final AST results (MIC and S/I/R).

Reference testing was performed on all enrolled samples in triplicate. Testing was done in accordance with the reference protocol and was performed at one clinical sites shipped isolates on transport media from the PBC purity panel following verification of pure culture. Samples contrived from laboratory stock underwent organism identification using MALDI prior to the reference site for testing. Reference testing was performed in triplicate. The procedure for Broth Microdilution reference testing follows CLSI guidance (CLSI M07).

The performance of the LifeScale LSGN kit was compared to the FDA-recognized reference BMD method for determining quantitative (MIC) AST results direct from Gram-negative blood cultures. Acceptable clinical performance was assessed across the following parameters for each antimicrobial agent on the LSGN panel; Essential Agreement (EA), Category Agreement (CA), Essential Agreement of evaluable results (Evaluable EA), Very Major Discrepancy (VMJ), Major Discrepancy (MAJ), Minor Discrepancy (MIN), Growth Failure Rate. Assessment of categorical agreement (Susceptible/Intermediate/Resistant, S/I/R) was conducted utilizing FDA breakpoints (Antimicrobial Susceptibility Test Interpretive Criteria/STIC) and CLSI M100 guidelines, if applicable.

# Exclusion Data

# Summary of LifeScale LSGN Tests Initiated and Failed to Report a Result

The provided table summarizes tests initiated on the LifeScale AST system and the reasons for exclusion or incomplete results during clinical, and quality control (QC) phases. Out of 5885 tests initiated:

Plate Failures (0.19%): This category includes issues such as being unable to verify positive controls, sensor clogs detected, and the system being unable to calculate MIC.

Growth Failures (0.54%): These failures occurred due to issues related to growth during testing.

LifeScale Failures (1.19%): This category involves failures directly attributable to the LifeScale system, including software and hardware failures.

Other Reasons (1.31%): This includes a variety of reasons such as operator error, incubation time exceeding 8 hours, user cancellation, and protocol errors.

The total percentage of tests excluded or incomplete is 3.23%, with clinical tests accounting for 3.23%, analytical tests for 4.43%, and QC tests for 0.86%.

Upon initiation of any test on the LifeScale system, results were available 96.77% (5695/5885) of the time.

| Reason for Exclusion/Incomplete Test | Clinical | Analytical | QC | Overall |
|--------------------------------------|----------------|------------------|-----------------|------------------|
| Plate Failures* | [3/682] 0.44% | [8/3452] 0.23% | [0/1751] 0.00% | [11/5885] 0.19% |
| Growth Failures | [6/682] 0.88% | [26/3452] 0.75% | [0/1751] 0.00% | [32/5885] 0.54% |
| LifeScale Failures** | [4/682] 0.59% | [51/3452] 1.48% | [15/1751] 0.86% | [70/5885] 1.19% |
| Other Reasons*** | [9/682] 1.32% | [68/3452] 1.97% | [0/1751] 0.00% | [77/5885] 1.31% |
| Total Excluded/Incomplete Tests | [22/682] 3.23% | [153/3452] 4.43% | [15/1751] 0.86% | [190/5885] 3.23% |

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#### Table 1. Summary of LifeScale AST tests initiated and failed to report a result

*Plate Failures include: unable to verify positive controls, sensor clog detected, system unable to calculate MIC **LifeScale Failures include: LifeScale system software and hardware failures

***Other Reasons include: operator error, incubation time greater than 8 hours, user canceled, protocol error

#### Clinical Performance Data

Overall AST performance for the LifeScale LSGN kit was evaluated with 6 antimicrobials, and an overview of the overall performance is presented in Table 2, below. All antimicrobial/organism combinations met the overall FDA acceptance criteria of >90% Essential (EA) and Categorical agreement (CA) rates except for the following: Aztreonam/K. pneumoniae, P. aeruginosa, Cefazolin/E. coli, Ceftazidime/Acinetobacter spp. (other than A. baumannii), K. pneumoniae, Ertapenem/K. pneumoniae, and Trimethoprim-Sulfamethoxazole/K. pneumoniae. For Ceftazidime, major errors occurred in P. aeruginosa isolates with a MIC value of 16 µg/mL, affecting 5 out of 61 susceptible isolates (8.2%). Adjusted for a lack of an intermediate breakpoint, this equated to 2 major errors (3.3%). Regarding Ertapenem, very major errors were observed in Klebsiella oxytoca isolates with MIC values of 0.5 µg/mL, where 2 out of 10 resistant isolates (20%) were affected. Limitations are included in the product labeling.

| Total | | | | No. Eval | | | | | | #MIN | #MAJ | #VMJ |
|-------------------------------------------------------------------------------------------------------------------------|--------|--------|----------|----------|----------|--------|--------|-------|-------|------------|-----------|-----------|
| Evaluated | No. EA | EA% | Eval Tot | EA | Eval EA% | No. CA | CA% | No. R | No. S | (MIN%) | (MAJ%) | (VMJ%) |
| Ampicillin - E. coli [Breakpoints (µg/mL): 8.0 (S), 32.0 (R)] | | | | | | | | | | | | |
| 137 | 137 | 100.0% | 23 | 23 | 100.0% | 137 | 100.0% | 87 | 50 | 0 (0.00%) | 0 (0.00%) | 0 (0.00%) |
| Aztreonam - E. coli, K. aerogenes, K. oxytoca [Breakpoints (µg/mL): 4.0 (S), 16.0 (R)] | | | | | | | | | | | | |
| 301 | 295 | 98.0% | 45 | 39 | 86.7% | 295 | 98.0% | 91 | 207 | 5 (1.66%) | 1 (0.48%) | 0 (0.00%) |
| Cefazolin - K. pneumoniae, K. variicola [Breakpoints (µg/mL): 2.0 (S), 8.0 (R)] | | | | | | | | | | | | |
| 143 | 140 | 97.9% | 69 | 66 | 95.7% | 132 | 92.3% | 77 | 62 | 11 (7.69%) | 0 (0.00%) | 0 (0.00%) |
| Ceftazidime - E. coli, K. aerogenes, K. oxytoca, K. variicola [Breakpoints (µg/mL): 4.0 (S), 16.0 (R)] | | | | | | | | | | | | |
| 340 | 332 | 97.6% | 34 | 26 | 76.5% | 334 | 98.2% | 101 | 238 | 6 (1.76%) | 0 (0.00%) | 0 (0.00%) |
| Ceftazidime - P. aeruginosa [Breakpoints (µg/mL): 8.0 (S), 16.0 (R)] | | | | | | | | | | | | |
| 116 | 107 | 92.2% | 85 | 76 | 89.4% | 109 | 94.0% | 55 | 61 | 0 (0.00%) | 5 (8.20%) | 2 (3.64%) |
| Ceftazidime - A. baumannii, A. baumannii/nosocomialis group [Breakpoints (µg/mL): 8.0 (S), 32.0 (R)] | | | | | | | | | | | | |
| 73 | 72 | 98.6% | 25 | 24 | 96.0% | 73 | 100.0% | 56 | 15 | 0 (0.00%) | 0 (0.00%) | 0 (0.00%) |
| Ertapenem - E. coli, K. aerogenes, K. oxytoca [Breakpoints (µg/mL): 0.5 (S), 2.0 (R)] | | | | | | | | | | | | |
| 226 | 212 | 93.8% | 28 | 14 | 50.0% | 216 | 95.6% | 43 | 182 | 7 (3.1%) | 1 (0.55%) | 2 (4.65%) |
| Trimethoprim-Sulfamethoxazole - E. coli, K. aerogenes, K. oxytoca, K. variicola [Breakpoints (µg/mL): 2.0 (S), 4.0 (R)] | | | | | | | | | | | | |
| 340 | 337 | 99.1% | 16 | 13 | 81.3% | 337 | 99.1% | 91 | 249 | 0 (0.00%) | 3 (1.20%) | 0 (0.00%) |

Table 2. LifeScale LSGN kit performance: Interpretation of MIC results are based on FDA Susceptibility Test Interpretative Criteria (STIC) and the 33ª edition of the CLSI M100, as recognized by FDA.

#### Ampicillin (AMP)

A total of 137 samples were evaluated with Ampicillin including 91 clinical (prospective and seeded) (66.4%) and 46 challenge (33.6%) samples. The combined results from clinical and challenge testing demonstrated an EA of 100% and a CA of 100% with no VMJs (0%) and no MAJs (0%)

#### Species-level performance

A total of 137 E. coli samples were included in the performance analysis including 91 clinical (66.4%) and 46 challenge (33.6%) samples. The combined results from clinical and challenge testing demonstrated an EA of 100% and a CA of 100% with no VMJs (0%) and no MAJs (0%).

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# Aztreonam (AZT)

A total of 301 samples were evaluated with Aztreonam including 235 clinical (prospective and seeded) (78.1%) and 66 challenge (21.9%) samples. The combined results from clinical and challenge testing from all claimed species demonstrated an EA of 98.0% with no VMJs (0%) and one MAJs (0.48%)

# Species-level performance

A total of 137 E. coli samples were evaluated with Aztreonam. The combined results from clinical and challenge testing demonstrated an EA of 98.5% and a CA of 97.8% with no VMJs (0%) and one MAJ (1.23%).

A total of 109 K. oxytoca samples were evaluated with Aztreonam. The combined results from clinical and challenge testing demonstrated an EA of 99.1% and a CA of 98.2% with no VMJs (0%) and no MAJs (0%).

A total of 55 K. aerogenes samples were evaluated with Aztreonam. The combined results from clinical and challenge testing demonstrated an EA of 94.5% and a CA of 98.2% with no VMJs (0%) and no MAJs (0%).

# Cefazolin (FAZ)

A total of 143 samples were evaluated with Cefazolin including 114 clinical (prospective and seeded) (79.7%) and 29 challenge (20.3%) samples. The combined results from clinical and challenge testing from all claimed species demonstrated an EA of 97.9% and a CA of 92.3% with no VMJs (0%) and no MAJ (0%).

# Species-level performance

A total of 105 K. pneumoniae samples were evaluated with Cefazolin. The combined results from clinical and challenge testing demonstrated an EA of 93.3% with no VMJs (0%) and no MAJs (0%).

A total of 38 K. variicola samples were evaluated with Cefazolin. The combined results from clinical and challenge testing demonstrated an EA of 97.4% and a CA of 89.5% with no VMJs (0%) and no MAJs (0%). EA evaluable is 96.3%.

# Ceftazidime (TAZ)

A total of 529 samples were evaluated with Ceftazidime including 378 clinical (prospective and seeded) (71.5%) and 151 challenge (28.5%) samples. The combined results from clinical and challenge testing from all claimed species demonstrated an EA of 97.2% with two VMJs (0.94%) and five MAJs (1.59%).

# Species-level performance

A total of 73 A. baumannii/nosocomialis group) samples were evaluated with Ceftazidime. The combined results from clinical and challenge testing demonstrated an EA of 98.6% and a CA of 100% with no VMJs (0%) and no MAJs (0%).

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A total of 137 E. coli samples were evaluated with Ceftazidime. The combined results from clinical and challenge testing demonstrated an EA of 97.8% and a CA of 97.1% with no VMJs (0%) and no MAJs (0%).

A total of 55 K. aerogenes samples were evaluated with Ceftazidime. The combined results from clinical and challenge testing demonstrated an EA of 92.7% and a CA of 100% with no VMJs (0%) and no MAJs (0%).

A total of 109 K. oxytoca samples were evaluated with Ceftazidime. The combined results from clinical and challenge testing demonstrated an EA of 99.1% and a CA of 98.2% with no VMJs (0%) and no MAJs (0%).

A total of 39 K. variicola samples were evaluated with Ceftazidime. The combined results from clinical and challenge testing demonstrated an EA of 100% and a CA of 100% with no VMJs (0%) and no MAJs (0%).

A total of 116 P. aeruginosa samples were evaluated with Ceftazidime. The combined results from clinical and challenge testing demonstrated an EA of 94.0% with two VMJs (3.64%) and five MAJ (8.20%). Due to lack of an intermediate breakpoint, one VMJ was in Essential agreement bringing adjusted VMJ rate to 1.82%.

Perform an alternative method of testing prior to reporting results for:

P. aeruginosa at MIC value of 16 µg/mL due to the occurrence of major errors (5 / 61 susceptible isolates, (8.2%) adjusted to 2 major errors (3.3%) due to a lack of an intermediate breakpoint).

# Ertapenem (ETP)

A total of 224 samples were evaluated with Ertapenem including 176 clinical (prospective and seeded) (78.6%) and 48 challenge (21.4%) samples. The combined results from clinical and challenge testing from all claimed species demonstrated an EA of 93.8% and a CA of 95.5% with two VMJs (4.76%) and two MAJs (0.55%).

# Species-level performance

A total of 66E. coli samples were evaluated with Ertapenem. The combined results from clinical and challenge testing demonstrated an EA of 95.5% and a CA of 93.9% with no VMJs (0%) and one MAJs (2.33%).

A total of 51 K. aerogenes samples were evaluated with Ertapenem. The combined results from clinical and challenge testing demonstrated an EA of 94.1% and a CA of 96.1% with no VMJs (0%) and no MAJs (0%).

A total of 137 K. oxytoca samples were evaluated with Ertapenem. The combined results from clinical and challenge testing demonstrated an EA of 92.5% and a CA of 96.3% with two VMJs (20.0%) and no MAJs (0%). Both VMJs occurred at MIC values of 0.5 µg/mL.

Perform an alternative method of testing prior to reporting results for:

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Ertapenem: K. oxytoca at MIC values of 0.5 µg/mL due to the occurrence of very major errors (2 /10 resistant isolates, 20%).

# Trimethoprim-sulfamethoxazole (SXT)

A total of 340 samples were evaluated with Trimethoprim-Sulfamethoxazole including 264 clinical (prospective and seeded) (77.6%) and 76 challenge (22.4%) samples. The combined results from clinical and challenge testing from all claimed species demonstrated an EA of 99.1% and a CA of 99.1% with no VMJs (0%) and three MAJs (1.20%).

# Species-level performance

A total of 138 E. coli samples were evaluated with Trimethoprim-Sulfamethoxazole. The combined results from clinical and challenge testing demonstrated an EA of 99.3% with no VMJs (0%) and one MAJ (1.32%).

A total of 55 K. aerogenes samples were evaluated with Trimethoprim-Sulfamethoxazole. The combined results from clinical and challenge testing demonstrated an EA of 98.2% and a CA of 96.4% with no VMJs (0%) and two MAJs (3.92%). Due to a lack of an intermediate breakpoint, the MAJ rate was adjusted to 1.96% (1 MAJ).

A total of 108 K. oxytoca samples were evaluated with Trimethoprim-Sulfamethoxazole. The combined results from clinical and challenge testing demonstrated an EA of 100% and a CA of 100% with no VMJs (0%) and no MAJs (0%).

A total of 39 K. variicola samples were evaluated with Trimethoprim-Sulfamethoxazole. The combined results from clinical and challenge testing demonstrated an EA of 97.4% and a CA of 100% with no VMJs (0%) and no MAJs (0%).

# Trending

In the clinical study or in the Inoculum Density analytical study, the majority of drug/organism combinations tested with the LifeScale LSGN kit showed MIC values equal to or at least one doubling dilution higher than the reference method. Use caution when reporting drug resistance for any antimicrobial. The following drug/organism combinations showed high trending:

- Ampicillin- E. coli
- Aztreonam – E. coli, K. aerogenes, K. oxytoca
- Cefazolin — K. pneumoniae
- Ceftazidime – E. coli, K. aerogenes, K. variicola, A. baumannii, P. aeruginosa
- Ertapenem — E. coli, K. aerogenes, K. oxytoca
- Trimethoprim/sulfamethoxazole – E. coli, K. aerogenes, K. variicola

# Quality Control Performance Data

Strains recommended by the FDA and CLSI were tested for each antimicrobial agent evaluated using the LifeScale LSGN kit and the CLSI Reference Broth Microdilution Method. The quality control (QC) strains tested were E. coli ATCC 25922, P. aeruginosa ATCC 27853, and K. pneumoniae ATCC 700603. The QC testing results indicate that this device can reliably yield acceptable QC results for >95% of tests.

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The measured MIC range for QC data from the six sites are shown in Table 3 through Table 8, along with data from BMD data, QC ranges are shaded grey.

All Trial Site data, 1(LS) to 6(LS), refer to QC data obtained on the LifeScale LSGN kit (LS). Ampicillin

| | | | Trial Site | | | | | | |
|-----------------------|-----------------------|-----------|-------------------|-------------------|-----------------|-----------------|-----------------|-----------------|-----------------|
| QC Organism | QC Range | MIC | BMD | 1 (LS) | 2 (LS) | 3 (LS) | 4 (LS) | 5 (LS) | 6 (LS) |
| E. coli<br>ATCC 25922 | 2 µg/mL to<br>8 µg/mL | ≤ 2 µg/mL | 9 | 38 | 15 | 24 | 35 | 13 | 22 |
| | | = 4 µg/mL | 102 | 80 | 14 | 20 | 13 | 17 | 4 |
| | | = 8 µg/mL | 1 | 3 | 0 | 0 | 1 | 0 | 0 |
| Total | | | 112/112<br>(100%) | 121/121<br>(100%) | 29/29<br>(100%) | 44/44<br>(100%) | 49/49<br>(100%) | 30/30<br>(100%) | 26/26<br>(100%) |

Table 3. LifeScale LSGN MIC QC distribution for Ampicillin.

N.B. Ampicillin: Does not include the full CLSI expected range.

#### Aztreonam

| | | | Trial Site | | | | | | |
|------------------------------------|-----------------------|------------|----------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|
| QC Organism | QC Range | MIC | BMD | 1 (LS) | 2 (LS) | 3 (LS) | 4 (LS) | 5 (LS) | 6 (LS) |
| <i>P. aeruginosa</i><br>ATCC 27853 | 2 µg/mL to<br>8 µg/mL | = 2 µg/mL | 2 | 80 | 24 | 51 | 57 | 24 | 20 |
| | | = 4 µg/mL | 85 | 0 | 0 | 1 | 3 | 1 | 4 |
| | | = 8 µg/mL | 23 | 0 | 0 | 0 | 1 | 0 | 0 |
| | | = 16 µg/mL | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Total | | | 110/111(99.1%) | 81/81<br>(100%) | 26/26<br>(100%) | 55/55<br>(100%) | 65/65<br>(100…