← Product Code [MDB](/submissions/MI/subpart-c%E2%80%94microbiology-devices/MDB) · K183166

# BacT/ALERT FA Plus; BacT/ALERT PF Plus (K183166)

_bioMerieux, Inc. · MDB · Feb 11, 2019 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-c%E2%80%94microbiology-devices/MDB/K183166

## Device Facts

- **Applicant:** bioMerieux, Inc.
- **Product Code:** [MDB](/submissions/MI/subpart-c%E2%80%94microbiology-devices/MDB.md)
- **Decision Date:** Feb 11, 2019
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.2560
- **Device Class:** Class 1
- **Review Panel:** Microbiology

## Indications for Use

BacT/ALERT® FA Plus Culture Bottles are used with BacT/ALERT® Microbial Detection Systems in qualitative procedures for recovery and detection of aerobic and facultative anaerobic microorganisms (bacteria and yeast) from blood and other normally sterile body fluids. BacT/ALERT® PF Plus Culture Bottles are used with BacT/ALERT® Microbial Detection Systems in qualitative procedures for recovery and detection of aerobic and facultative anaerobic microorganisms (bacteria and yeast) from blood.

## Device Story

BacT/ALERT FA Plus and PF Plus are liquid culture media bottles for microbial recovery from blood/sterile body fluids. Inoculated bottles are incubated in BacT/ALERT 3D or VIRTUO systems. Systems use colorimetric sensors and reflected light to monitor CO2 production from microbial metabolism. Sensor color shifts from blue-green to yellow as CO2 increases, raising reflectance units. Instrument monitors reflectance every 10 minutes. Output is a positive/negative signal indicating microbial growth. Used in clinical microbiology labs; operated by technicians. Provides rapid detection of bacteremia/fungemia to guide clinical treatment.

## Clinical Evidence

No clinical data. Evidence consists of bench-only seeded analytical studies. Evaluated 39 diverse microbial species across 2,372 bottles (1,564 with blood, 808 without). Primary endpoints: percent recovery and time-to-detection (TTD). Overall detection rate 95.7% (adjusted) vs 96.0% (predicate). TTD equivalence assessed by mean difference criteria. Antimicrobial neutralization effectiveness confirmed for multiple classes.

## Technological Characteristics

Liquid culture media in glass/plastic bottles. Colorimetric CO2-sensitive sensor (blue-green to yellow). Continuous monitoring via reflected light. Compatible with BacT/ALERT 3D and VIRTUO systems. Automated detection of CO2-induced reflectance changes. No electronic components in the bottle itself.

## Regulatory Identification

A microbial growth monitor is a device intended for medical purposes that measures the concentration of bacteria suspended in a liquid medium by measuring changes in light scattering properties, optical density, electrical impedance, or by making direct bacterial counts. The device aids in the diagnosis of disease caused by pathogenic microorganisms.

## Predicate Devices

- BacT/ALERT PF Plus ([K121446](/device/K121446.md))
- BacT/ALERT FA Plus ([K121461](/device/K121461.md))

## Submission Summary (Full Text)

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>
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
K183166

B. Purpose for Submission:
To obtain a substantial equivalence determination for a formulation adjustment to the BacT/ALERT PF Plus and BacT/ALERT FA Plus liquid culture media bottles.

C. Measurand:
Aerobic and anaerobic facultative microorganisms (bacteria and yeast).

D. Type of Test:
Liquid culture medium for recovery of microorganisms (bacteria and yeast) from blood using colorimetric sensor to detect CO₂ dissolved in the culture media.

E. Applicant:
bioMérieux, Inc.

F. Proprietary and Established Names:
BacT/ALERT PF Plus, BacT/ALERT FA Plus

G. Regulatory Information:
1. Regulation section:
21 CFR 866.2560, Microbial Growth Monitor
2. Classification:
Class I
3. Product code:
MDB
4. Panel:

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83 - Microbiology

H. Intended Use:

1. Intended use(s):

BacT/ALERT FA Plus Culture Bottles are used with BacT/ALERT Microbial Detection Systems in qualitative procedures for recovery and detection of aerobic and facultative anaerobic microorganisms (bacteria and yeast) from blood and other normally sterile body fluids.

BacT/ALERT PF Plus Culture Bottles are used with BacT/ALERT Microbial Detection Systems in qualitative procedures for recovery and detection of aerobic and facultative anaerobic microorganisms (bacteria and yeast) from blood.

2. Indication(s) for use:

Same as the Intended Use.

3. Special conditions for use statement(s):

For prescription use only.

4. Special instrument requirements:

BacT/ALERT (BTA) 3D Microbial Detection System or BTA VIRTUO Microbial Detection System

I. Device Description:

The BacT/ALERT Microbial Detection System and BTA VIRTUO Microbial Detection System provide both a microbial detection system and a culture medium bottle with suitable nutritional and environmental conditions for microorganisms commonly encountered in blood taken from a patient suspected of having bacteremia/fungemia. The BacT/ALERT FA Plus and BacT/ALERT PF Plus culture media formulation was adjusted to add essential elements and to remove non-essential trace components.

For both the BacT/ALERT PF Plus and BacT/ALERT FA Plus culture bottles, an inoculated bottle is placed into the instrument where it is incubated and continuously monitored for the presence of microorganisms that will grow in the culture bottles.

The BacT/ALERT Microbial Detection System and BTA VIRTUO Microbial Detection System utilize a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO₂) that is dissolved in the culture medium. If microorganisms are present in the test sample, carbon dioxide is produced as the microorganisms metabolize the substrates in the culture medium. When growth of the

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microorganisms produces CO₂, the color of the gas-permeable sensor installed in the bottom of each culture bottle changes from blue-green to yellow. The lighter color results in an increase of reflectance units monitored by the system. Bottle reflectance is monitored and recorded by the instrument every 10 minutes.

J. Substantial Equivalence Information:

1. Predicate device name(s):
BacT/ALERT PF Plus,
BacT/ALERT FA Plus

2. Predicate 510(k) number(s):
K121446,
K121461

3. Comparison with predicate:

3

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|  Similarities  |   |   |
| --- | --- | --- |
|  Item | Device: BacT/ALERT PF Plus, BacT/ALERT FA Plus (K183166) | Predicate: BacT/ALERT PF Plus, BacT/ALERT FA Plus (K121446, K121461)  |
|  Intended Use | BacT/ALERT FA Plus Culture Bottles are used with BacT/ALERT Microbial Detection Systems in qualitative procedures for recovery and detection of aerobic and facultative anaerobic microorganisms (bacteria and yeast) from blood and other normally sterile body fluids.
BacT/ALERT PF Plus Culture Bottles are used with BacT/ALERT Microbial Detection Systems in qualitative procedures for recovery and detection of aerobic and facultative anaerobic microorganisms (bacteria and yeast) from blood. | Same  |
|  Specimen Type | Human blood, Sterile body fluids - BacT/ALERT FA Plus only | Same  |
|  Detection Technology | Continuous monitoring; utilizes a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO2) that is dissolved in the culture medium, produced by the growth of aerobic and facultative anaerobic bacteria and yeast. | Same  |
|  Differences  |   |   |
| --- | --- | --- |
|  Item | Device: BacT/ALERT PF Plus and BacT/ALERT FA Plus (K183166) | Predicate: BacT/ALERT PF Plus (K121446) and BacT/ALERT FA Plus (K121461)  |
|  Media Formulation | The adjusted BacT/ALERT FA Plus and adjusted BacT/ALERT PF Plus culture media formulations differ from the previous (original) culture media. The formulation was adjusted to add essential elements and to remove non-essential trace components.  |   |

# K. Standard/Guidance Document Referenced (if applicable):

Not Applicable

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L. Test Principle:

The BacT/ALERT Microbial Detection System and BTA VIRTUO Microbial Detection Systems utilize a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO₂) dissolved in the culture medium. If microorganisms are present in the test sample, carbon dioxide is produced as the microorganisms metabolize the substrates in the culture medium. When growth of the microorganisms produces CO₂, the color of the gas-permeable sensor installed in the bottom of each culture bottle changes from blue-green to yellow. The lighter color results in an increase of reflectance units monitored by the system. Bottle reflectance is monitored by the system. Bottle reflectance is monitored and recorded by the instrument every 10 minutes.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

Antimicrobial neutralization

An internal study was conducted to demonstrate that the adjusted media formulation does not impact the rate of recovery (percent) of microorganisms in the presence of antimicrobials. At least one antimicrobial agent from each class claimed in the FA Plus culture bottle package insert was evaluated in this study. Single agents and combinations of antimicrobials, in clinically relevant concentrations, were added to FA Plus bottles with the susceptible aerobic and facultative anaerobic microorganisms listed in Table 1 and Table 2. The targeted inoculum for each microorganism was 10-100 CFU/bottle. The actual inoculum ranged from 15 CFU/bottle to 109 CFU/bottle. All organisms were added to FA Plus bottles that were supplemented with phosphate buffered saline (PBS) to simulate a challenging environment with limited nutrients and high antimicrobial bioavailability. In addition, a subset of organisms were added to bottles supplemented with blood. Adjusted BTA FA Plus bottles were tested on both the BTA 3D and BTA VIRTUO Microbial Detection Systems. The effectiveness of the antimicrobials was confirmed by parallel testing using a non-neutralizing medium as a control.

Antimicrobial neutralization was evaluated with at least three lots of adjusted FA Plus culture bottles for each tested microorganism/antimicrobial. For each microorganism/antimicrobial, at least six bottles were inoculated and tested on the BTA 3D or the BTA VIRTUO. Testing of all antimicrobials was evaluated at 100% Peak Serum Level (PSL), unless otherwise noted. Antimicrobials were considered effectively neutralized by the adjusted FA Plus bottle based on 100% recovery of the microorganisms tested at the indicated percent of PSL tested.

The study demonstrated that antimicrobials from the following categories were neutralized by the adjusted FA Plus media, resulting in a positive bottle signal (as expected) within five days of inoculation: penicillins, glycylcyclines, polyenes, macrolides, triazoles, aminoglycosides, fluoroquinolones, lincosamides, glycopeptides, and oxazolidinones.

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Antimicrobial neutralization was also achieved for ceftaroline.

Antimicrobial neutralization was not achieved for S. aureus with the following single and combinations of antimicrobials: Cefoxitin, Piperacillin/Tazobactam, Vancomycin + Piperacillin/Tazobactam, and Daptomycin + Piperacillin /Tazobactam. In addition, antimicrobial neutralization was not achieved for C. albicans grown in the presence of Caspofungin, an echinocandin. Based on the C. albicans/Caspofungin result, follow up testing was conducted with five additional echinocandin/yeast pairs. All additional echinocandin/yeast pairs demonstrated 100% microorganism recovery.

Less than complete neutralization was observed for cefazolin. Cefazolin was neutralized at 50% PSL for E. coli in PBS.

Antimicrobial neutralization study results are summarized in Table 1 and Table 2 for single and combinations of antimicrobial agents, respectively. Table 3 illustrates results of follow up testing with additional echinocandin/yeast pairs.

Table 1. Antimicrobial Agents and Paired Microorganisms

|  Antimicrobial Agent | 100% PSL (ug/mL)^{1} | Antimicrobial Class/Subclass | Organism (Species) | MIC (ug/mL)^{2} | Diluent | % Recovery (n/N)  |
| --- | --- | --- | --- | --- | --- | --- |
|  Amphotericin B | 2.5 | Polyene | Candida albicans | 0.25^{3}-1.0^{4} | PBS | 100% (6/6)  |
|  Caspofungin | 9.9 | Echinocandin | Candida albicans | 0.1^{4} | PBS | 77.8% (14/18)^{5}  |
|  Fluconazole | 14 | Triazole | Candida albicans | 0.25-1 | PBS | 100% (6/6)  |
|  Amikacin | 30 | Aminoglycoside | Escherichia coli | 0.5-4 | PBS | 100% (6/6)  |
|  Amikacin | 30 | Aminoglycoside | Escherichia coli | 0.5-4 | Human blood^{6} | 100% (6/6)  |
|  Amikacin | 30 | Aminoglycoside | Pseudomonas aeruginosa | 1-4 | PBS | 100% (6/6)  |
|  Ampicillin | 47 | Penicillin | Enterococcus faecalis | 0.5-2 | PBS | 100% (6/6)  |
|  Ampicillin/ Sulbactam | 47, 28^{7} | β-lactam, β-lactamase inhibitor | Escherichia coli | 2/1-8/4 | PBS | 100% (6/6)  |
|  Azithromycin | 3.6 | Marcolide | Streptococcus pneumoniae | 0.06-0.25 | PBS | 100% (6/6)  |
|  Cefazolin | 94^{8} | Cephems/1^{st} generation Cephalosporin | Escherichia coli | 1-4 | PBS | 100% (6/6)  |
|  Cefoxitin | 110 | Cephems/2^{nd} generation Cephalosporin | Staphylococcus aureus | 1-4 | PBS | 88.9% (16/18)^{9}  |

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|  Cefoxitin | 110 | Cephems/2ndgenerationCephalosporin | Escherichia coli | 2-8 | PBS | 100%(6/6)  |
| --- | --- | --- | --- | --- | --- | --- |
|  Ceftaroline | 21 | Cephems/4thgenerationCephalosporin | Escherichia coli | 0.03-0.12 | PBS | 100%(6/6)  |
|  Ceftaroline | 2110 | Cephems/4thgenerationCephalosporin | Staphylococcus aureus(MRSA) | 111 | PBS | 100%(6/6)  |
|  Ciprofloxacin | 4.6 | Fluoroquinolone | Pseudomonas aeruginosa | 0.25-1 | PBS | 100%(6/6)  |
|  Clindamycin | 10 | Lincosamide | Staphylococcus aureus | 0.06-0.25 | PBS | 100%(6/6)  |
|  Linezolid | 20 | Oxazolidinone | Enterococcus faecalis | 1-4 | PBS | 100%(6/6)  |
|  Tigecycline | 0.63 | Glycylcyclines | Staphylococcus aureus | 0.03-0.25 | PBS | 100%(6/6)  |
|  Vancomycin | 50 | Glycopeptide | Staphylococcus aureus | 0.5-2 | PBS | 100%(6/6)  |
|  Vancomycin | 50 | Glycopeptide | Staphylococcus aureus | 0.5-2 | Human blood6 | 100%(6/6)  |
|  Piperacillin | 400 | Penicillin | Pseudomonas aeruginosa | 1-8 | PBS | 100%(6/6)  |
|  Piperacillin | 400 | Penicillin | Pseudomonas aeruginosa | 1-8 | Human blood6 | 100%(6/6)  |
|  Piperacillin/Tazobactam | 190, 1912 | β-lactam, β-lactamase inhibitor | Staphylococcus aureus | 0.25/4-2/4 | PBS | 75%(9/12)13  |
|  Trimethoprim/Sulfamethoxazole | 11, 12614 | Sulfonamides | Escherichia coli | 0.5/9.5 | PBS | 100%(6/6)  |

Table 1 footnotes:
1. Peak serum level as defined in The Sanford Guide to Antimicrobial Therapy 2007, unless indicated otherwise
2. MIC as determined by the respective CLSI Reference Methods: (i) Reference method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Third Informational Supplement; CLSI document M27-S3; Wayne, Pa; Clinical and Laboratory Standards Institute;2011. (ii) CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Supplement. Wayne, PA. Clinical and Laboratory Standards Institute; CLSI M100-S-24; Wayne, Pa; Clinical and Laboratory Standards Institute; 2014
3. Pai, Manjunath P. 2009 Antifungal Combination against Simulated Candida albicans Endocardial Vegetations. Antimicrobial Agents and Chemotherapy, 53: p2626-2631
4. Li XC, MR Jacob SI Khan, MK Ashfaq, S Babu, AK Agarwal, HN El Sohly, SP Manly, and AM Clark. 2008. Potent In Vitro Antifungal Activities of Naturally Occurring Acetylenic Acids. Antimicrobial Agents and Chemotherapy, 52: p2442-2448
5. Discordant resolution was conducted by supplementating with either human blood (10 mL) or horse blood (3 mL + 7 mL PBS). Percent recovery was 94.4% (17/18) for human blood and 100% (18/18) for horse blood
6. Supplemented with  $10~\mathrm{mL}$  human blood

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7.  $50\%$  PSL.  $100\%$  PSL of Ampicillin alone is  $47~\mathrm{ug / mL}$  per Sanford 2007. When combined with Sulbactam, the combined PSL is  $109 - 150~\mathrm{ug / mL}$  (2:1 AM:SB). Therefore at  $50\%$  PSL, the range is  $54.5 - 75~\mathrm{ug / mL}$ . The total amount tested was  $75~\mathrm{ug / mL}$  with a 2:1 ratio
8.  $50\%$  PSL
9. S. aureus testing yielded  $66.7\%$  (4/6) recovery for original results and  $100\%$  (12/12) recovery during retesting
10. PSL as defined in The Sanford Guide to Antimicrobial Neutralization 2015. Developed as a new therapy in 2007
11. Ceftaroline with S. aureus 29213: MIC = 0.12-0.5 ug/mL. M100-S24. With MRSA; MIC = 1. Sweeney et al. Diagnostic Microbiology and Infectious Disease. 89 (2017) p.83-85.5
12. The Sanford Guide to Antimicrobial Therapy 2007 states the PSL for Pip-Taz is 209. The established performance of the predicate device was based on the following ratio: 10 Pip : 1 Taz. Thus,  $100\%$  PSL was 190/19 (190 Pip to 19 Taz for a total of 209). For the triple combinations not previously tested during Validation of the predicate device, the PSL of 209 was used for Pipercillin per The Sanford Guide to Antimicrobial Neutralization 2007
13. Original testing conducted at 36 CFU/bottle. Re-testing with S. aureus at 200-300 CFU/bottle yielded  $100\%$  (6/6) percent recovery
14.  $120\%$  PSL

Table 2. Combinations of Antimicrobial Agents and Paired Microorganisms

|  Antimicrobial Agents | 100% PSL (ug/mL)1 | Antimicrobial Class/Subclass | Organism (Species) | MIC (ug/mL)2 | % Recovery (n/N)  |
| --- | --- | --- | --- | --- | --- |
|  Vancomycin + Piperacillin/Tazobactam | 50, 209, 19 | Glycopeptide, β-lactam, β-lactamase inhibitor combination | Staphylococcus aureus | 0.5-2 (Vanc)/ 0.25/4-2/4 (PIP-TAZ) | 66.7% (8/12)3  |
|  Daptomycin + Piperacillin/Tazobactam | 99, 209, 19 | Lipopeptide, β-lactam, β-lactamase inhibitor combination | Staphylococcus aureus | 0.12-1 (Dapto)/ 0.25/4-2/4 (PIP-TAZ) | 66.7% (8/12)3  |
|  Gentamycin + Piperacillin/Tazobactam | 10, 20, 19 | Aminoglycoside, β-lactam, β-lactamase inhibitor combination | Pseudomonas aeruginosa | 0.12-1 (Dapto)/ 0.25/4-2/4 (PIP-TAZ) | 100% (6/6)  |
|  Fluconazole + Amphotericin B | 14, 3.5 | Polyene, Azole combination | Candida albicans | 0.25-1/0.5-2.04 | 100% (6/6)  |

Table 2 footnotes:
1. Peak serum level as defined in The Sanford Guide to Antimicrobial Therapy 2007 unless indicated otherwise
2. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Supplement. Wayne, PA. Clinical and Laboratory Standards Institute; CLSI M100-S-24; Wayne, Pa; Clinical and Laboratory Standards Institute; 2014
3. Original testing conducted at 36 CFU/bottle. Re-testing with S. aureus at 200-300 CFU/bottle yielded  $100\%$  (6/6) percent recovery

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4. CLSI. Reference method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Third Informational Supplement; CLSI document M27-S3; Wayne, Pa; Clinical and Laboratory Standards Institute; 2011

Table 3. Results of follow up antimicrobial neutralization testing conducted with yeasts and Echinocandins

|  Echinocandin | 100% PSL (ug/mL)1 | Organism (yeast) | MIC (ug/mL)2 | % Recovery (n/N)  |
| --- | --- | --- | --- | --- |
|  Caspofungin | 9.9 | Candida krusei | 0.12-1.0 | 100% (39/39)  |
|   |  9.9 | Candida parapsilosis | 0.25-1.0 | 100% (40/40)  |
|  Micafungin | 16.4 | Candida albicans | 0.0153 | 100% (40/40)  |
|   |  16.4 | Candida krusei | 0.12-0.5 | 100% (40/40)  |
|   |  16.4 | Candida parapsilosis | 0.5-2 | 100% (40/40)  |

Table 3 footnotes:
1. Peak serum level as defined in The Sanford Guide to Antimicrobial Therapy 2007 unless indicated otherwise
2. CLSI. Reference method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Third Informational Supplement; CLSI document M27-S3; Wayne, Pa; Clinical and Laboratory Standards Institute; 2011
3. Pai, Manjunath P. 2009 Antifungal Combination against Simulated Candida albicans Endocardial Vegetations. Antimicrobial Agents and Chemotherapy, 53: p2626-2631

# Delayed Entry

An internal study was conducted to evaluate the effect of a delay, from the time the adjusted FA Plus culture bottle was inoculated, to the time the bottle was placed on the instrument. This seeded study was conducted using three lots of adjusted FA Plus culture bottles. Three replicate suspensions of each of the following 11 species were evaluated: Staphylococcus aureus, Candida albicans, Candida krusei, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Streptococcus pneumoniae, Enterococcus faecium, Haemophilus influenzae, and Neisseria meningitidis. Target concentrations of  $\leq 100$  CFU per bottle (actual inoculum levels from 16 CFU/bottle to 97 CFU/bottle) were prepared in 0, 4, and  $10\mathrm{mL}$  human blood. Testing in the absence of blood was performed to simulate the worst case scenario for normally sterile body fluids. Four and ten mL blood represent the recommended fill volume for pediatric and adult bottles, respectively. Haemophilus influenzae and Neisseria meningitidis were inoculated into bottles supplemented with 1, 4, 10 or 4 and  $10\mathrm{mL}$  blood, respectively. Haemophilus influenzae and Neisseria meningitidis were only inoculated with blood added due to growth requirements. Sixty negative controls (i.e., bottles containing blood and media alone without organism) were evaluated on both the BTA 3D and BTA VIRTUO instruments. All samples were held at the specified temperatures and times prior to loading into the BTA 3D or BTA VIRTUO instruments. Percent recovery reflects time to positive flag by the instrument.

The study demonstrated that all delayed entry conditions showed  $\geq 100\%$  percent recovery of

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inoculated organisms, except for delayed entry at  $2 - 8^{\circ}\mathrm{C}$  for 48 hours. Holding bottles at  $2 - 8^{\circ}\mathrm{C}$  for 48 hours is not a claim in the adjusted FA Plus package insert. These results are acceptable. Study results are illustrated in Table 4, below.

Table 4. Delayed Entry Summary

|  Sample Input | Instrument | Hold Temperature (℃) | Hold Time (hours) | % Recovery | TTD from Sample Inoculation (Hold Time + Instrument TTD in hours)  |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |  Mean | Range  |
|  Inoculated Test Bottles | BTA 3D | Control | No delay | 100% (96/96) | 16.0 | 10.8-32.6  |
|   |   |  2-8 | 48 | 95.8% (92/96) | 64.1 | 58.3-75.6  |
|   |   |  20-25 | 24 | 100% (96/96) | 34.1 | 28.1-47.5  |
|   |   |  20-25 | 36 | 100% (96/96) | 43.8 | 37.9-59.8  |
|   |   |  35-37 | 8 | 100% (96/96) | 17.1 | 12.6-30.1  |
|   |  BTA VIRTUO | Control | No delay | 100% (96/96) | 13.8 | 8.6-26.5  |
|   |   |  2-8 | 48 | 97.9% (94/96) | 62.2 | 50.8-75.8  |
|   |   |  20-25 | 24 | 100% (96/96) | 32.3 | 25.2-48.2  |
|   |   |  20-25 | 36 | 100% (96/96) | 42.2 | 37.2-59.5  |
|  35-37 |   | 8 | 100% (96/96) | 15.5 | 11.0-25.9  |   |
|  Negative Controls (Blood only) | BTA 3D | All conditions | 3.4% (2/58)* | NA  |   |   |
|   |  BTA VIRTUO |   |   |   |   | 0.0% (0/60)  |

*Two bottles were removed from analysis due to contamination, leaving 58 bottles for percent recovery calculations. Two of the remaining 58 bottles yielded false positive results.

# a. Precision:

# Within-laboratory Precision

Within-laboratory precision was evaluated by conducting an in-house seeded study over 19 days using multiple systems (BTA 3D and BTA VIRTUO), varied blood contents (0, 4, and  $10~\mathrm{mL}$ ), adjusted FA Plus bottles lots, and operators. The combined data from a total of 717

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bottles was used to evaluate the percent recovery for the species listed in Table 5a. Three of the species were tested with the addition of antimicrobial agents: Escherichia coli – Amikacin (30 μg/mL), Pseudomonas aeruginosa – Pipercillin (400 μg/mL), and Staphylococcus aureus – Vancomycin (50 μg/mL). All species were seeded into adjusted FA Plus bottles at a target inoculum of ≤100 CFU/bottle (actual inoculum from 16 CFU/bottle to 109 CFU/bottle). Percent recovery reflects positive flag by the instrument and sub-culture consistent with the appropriate seeded organism. Mean TTD reflects the average time to detection for all evaluated microorganisms, combined. The study demonstrated that percent recovery was 100% for yeast and bacteria with and without blood supplementation (Table 5a). Percent recovery was not affected by adjusted FA Plus bottle lot.

Table 5a. Precision Study - Percent Recovery Results (All data combined)

|  Species
(Antimicrobial Agent) | Adjusted FA Plus Bottle |   | % Recovery  |
| --- | --- | --- | --- |
|   |  # Bottles Tested | # Bottles Positive  |   |
|  Candida albicans | 129 | 129 | 100%  |
|  Escherichia coli | 105 | 105 | 100%  |
|  Klebsiella pneumoniae | 36 | 36 | 100%  |
|  Pseudomonas aeruginosa | 105 | 105 | 100%  |
|  Staphylococcus aureus | 72 | 72 | 100%  |
|  Streptococcus pneumoniae | 90 | 90 | 100%  |
|  Escherichia coli
(Amikacin - 30 μg/mL) | 60 | 60 | 100%  |
|  Pseudomonas aeruginosa
(Pipercillin - 400 μg/mL) | 60 | 60 | 100%  |
|  Staphylococcus aureus
(Vancomycin -50 μg/mL) | 60 | 60 | 100%  |

Mean TTD did not vary by bottle lot or blood volume. Mean TTD results for the adjusted FA Plus bottle with and without blood addition are illustrated in Table 5b and Table 5c, respectively.

Table 5b. Time to Detection - Lot-to-Lot Difference by Blood Volume

|  Adjusted FA Plus Bottle Lot | Blood Volume (mL) | Mean TTD (Hours)  |   |
| --- | --- | --- | --- |
|   |   |  Inoculum: 10-100 CFU/bottle (95% CI) | Inoculum: 101-199 CFU/bottle (95% CI)  |
|  1 | 4 | 14.7
(12.3, 17.1) | 10.4^{a}  |
|  1 | 10 | 15.0
(13.0, 16.9) | 20.7
(15.3, 26.0)  |
|  2 | 4 | 14.8
(12.2, 17.3) | 10.3^{a}  |

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Table 5c. Time to Detection - Lot-to-Lot Difference for Media Only (No Blood)

|  Adjusted FA Plus Bottle Lot | Blood Volume (mL) | Mean TTD (Hours)  |   |
| --- | --- | --- | --- |
|   |   |  Inoculum: 10-100 CFU/bottle (95% CI) | Inoculum: 101-199 CFU/bottle (95% CI)  |
|  1 | 0 | 18.8 (17.5, 19.9) | 13.7 (12.0, 15.3)  |
|  2 | 0 | 18.9 (17.8, 19.9) | 13.7 (11.9, 15.5)  |
|  3 | 0 | 18.6 (17.5, 19.6) | 14.1 (12.0, 16.1)  |

a. Linearity/assay reportable range:

Not applicable

b. Traceability, Stability, Expected values (controls, calibrators, or methods):

# External QC Testing

During the analytical studies, daily external quality control was conducted on the BTA 3D system and included positive and negative control bottles. Control organisms were seeded into bottles at target inoculum levels of 10-100 CFU/bottle, with actual inoculums ranging from 36 CFU/bottle to 91 CFU/bottle. Organism concentrations for bottle seeding were confirmed by plate counting procedures. One obligate aerobe (P. aeruginosa) and two facultative organisms (E. coli and S. aureus) were evaluated. On each testing day, at least two quality control organisms were evaluated. In addition, uninoculated negative control bottles were tested daily.  $100\%$  of positive controls generated the expected positive signal by BTA 3D and  $100\%$  of negative controls were reported as negative.

# Stability of the adjusted BTA FA Plus bottle (shelf-life)

Studies were conducted to determine the adjusted FA Plus bottle shelf-life. Three lots of adjusted FA Plus bottles produced for product validation and verification were tested until they were 13 months old. At the beginning of the study, a set of adjusted FA Plus bottles were stored under normal conditions (15-30°C) while another set of bottles were thermally stressed before final storage under normal conditions. At each time point, growth performance (% recovery and TTD), antimicrobial neutralization,

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and physical parameters were evaluated on the BTA 3D system. In addition, FA Plus bottles were evaluated to determine if they possessed sufficient vacuum to direct draw 10 ml blood. These studies demonstrated that stability can be achieved for the claimed shelf-life and that direct blood draw was maintained over the claimed shelf-life.

c. Detection limit:

Limit of Detection (LoD)

An in-house LoD study was conducted with seeded samples to evaluate/confirm the LoD of the microorganisms in the adjusted FA Plus bottle based on the established LoD of the original bottle. All microorganisms were inoculated into adjusted FA Plus Bottles at specific organism concentrations based on the LoD of the predicate FA Plus bottle and different bottles were incubated on the BTA 3D and BTA VIRTUO Systems. Due to inherent inaccuracy in preparing suspensions of microorganisms at a low density, inoculum levels were deemed acceptable if they were within a factor of 2 (±2-fold) of the previously established LoD for each detection system. The following 11 species were tested on the BTA 3D System: Candida albicans, Klebsiella (Enterococcus) aerogenes, Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, and Streptococcus pneumoniae. Of the species evaluated on the BTA 3D System, all were tested on the BTA Virtuo System, except for Klebsiella (Enterococcus) aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, and Salmonella enterica. Three production lots of adjusted FA Plus Bottles were tested. A minimum of 60 bottles were tested for each species on each detection system. LoD was determined in the absence of blood for all organisms except H. influenzae, which was supplemented with 4 mL human blood due to growth requirements. At least 95% detection was achieved at the established LoD for all evaluated species, indicating that the LoD was the same in the adjusted and predicate FA Plus bottles.

Percent Recovery (Detection) Study

An in-house seeded study was conducted to demonstrate that changes to the media formulation do not negatively impact percent recovery (detection). Percent recovery was evaluated in a head-to-head study of 2372 adjusted and predicate bottles, of which 1564 were supplemented with human blood (4 or 10 mL) and 808 without blood. A set of 39 diverse microorganisms commonly found in blood were evaluated using three lots of the adjusted FA Plus bottle (a total of five different lots were tested) and two lots of the predicate. The species tested are listed in Table 6d. All organisms were seeded into adjusted FA Plus bottles at a target inoculum range of 10-100 CFU/bottle (actual inoculum ranged from 3 CFU/bottle to 137 CFU/bottle). Seeded bottles were incubated on the BTA 3D and BTA VIRTUO Systems.

The adjusted FA Plus bottle was considered equivalent to the predicate based on the Newcombe's Hybrid Score CI of the percent recovery difference of the adjusted

13

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bottle compared to the predicate bottle.

The overall detection rate was  $96.0\%$  for the predicate bottle and  $95.7\%$  for the adjusted bottle, with an overall difference in percent recovery of  $0.3\%$ . Results are shown in shown in Table 6a, stratified by inoculum level/blood content, and Table 6b, stratified by Detection System. Percent recovery results by adjusted bottle lot are illustrated in Table 6c.

Table 6a. Percent Recovery Difference Stratified by Inoculum Level and Blood Content

|  Actual Inoculum Level (CFU/bottle) | Blood Content | Percent Recovery |   | Diff. % Recovery (Predicate - Adjusted) (95% CI)  |
| --- | --- | --- | --- | --- |
|   |   |  Predicate Bottle | Adjusted Bottle  |   |
|  1-9 | No Blood | 50.0 (12/24) | 50.0 (12/24) | 0.0 (-26.3, 26.3)  |
|   |  Blood | 100.0 (48/48) | 100.0 (48/48) | 0.0 (-7.4, 7.4)  |
|  10-100 | No Blood | 91.9 (601/654) | 91.4 (598/654) | 0.5 (-2.6, 3.5)  |
|   |  Blood | 98.2 (1289/1312) | 99.3 (1303/1312) | -1.1 (-2.0, -0.2)  |
|  >100 | No Blood | 94.6 (123/130) | 80.8 (105/130)a | 13.8 (5.9, 21.9)  |
|   |  Blood | 100.0 (204/204) | 99.5 (203/204) | 0.5 (-1.4, 2.7)  |
|  Overall | No Blood | 91.1 (736/808) | 88.5 (715/808) | 2.6 (-0.4, 5.7)  |
|   |  Blood | 98.5 (1541/1564) | 99.4 (1554/1564) | -0.8 (-1.7, -0.1)  |
|  Overall | Overall | 96.0 (2277/2372) | 95.7 (2269/2372) | 0.3 (-0.8, 1.5)  |

a 18 of 25 unexpected negative results were for Abiotropha. defectiva, which upon investigation was found to be incapable of growth in the adjusted formulation without blood supplementation. Detection of Abiotropha defectiva in the absence of blood is no longer a claim for the FA Plus bottle

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Table 6b. Percent Recovery Difference Stratified by Microbial Detection System

|  System | Percent Recovery |   | Diff. Percent Recovery (Predicate – Adjusted) (95% CI)  |
| --- | --- | --- | --- |
|   |  Predicate Bottle | Adjusted Bottle  |   |
|  BTA 3D | 95.8 (1142/1192)a | 95.0 (1133/1193) | 0.8 (-0.9, 2.5)  |
|  BTA VIRTUO | 96.2 (1135/1180) | 96.4 (1136/1179)a | -0.2 (-1.7, 1.4)  |

a One bottle removed due to contamination

Table 6c. Percent Recovery for the Adjusted Bottle Stratified by Lot

|  Lot # | Number of Bottles Tested | Percent Recovery  |
| --- | --- | --- |
|  1 | 727 | 95.9% (697/727)  |
|  2 | 728 | 95.5% (695/728)  |
|  3 | 725 | 95.9% (695/725)  |
|  4 | 96 | 94.8% (91/96)  |
|  5 | 96 | 94.8% (91/96)  |

The study demonstrated that the adjusted bottle performed equivalently when compared to the predicate bottle for all evaluated incolulum levels and blood volumes, except for an inoculum of  $>100$  CFU/bottle in the absence of blood. For this condition, the detection rate was  $94.6\%$  for the predicate bottle and  $80.8\%$  for the adjusted bottle, with a difference in percent recovery of  $13.8\%$ . It was noted that for this condition, all adjusted bottles  $(n = 18)$  inoculated with A. defectiva yielded negative results. Excluding A. defectiva from the analysis, Newcombe's Hybrid Score CI of the percent recovery difference between the adjusted and predicate FA Plus bottle met the acceptance criteria for equivalence. A note has been added to the adjusted FA Plus Package Insert indicating that detection of A. defectiva necessitates blood.

In the adjusted bottle, percent recovery of bottles inoculated with  $H.$  parainfluenzae (with blood supplementation) was  $58\%$  and percent recovery for  $C.$  hominis (without blood supplementation) was  $50\%$ . These results are consistent with what has previously been observed for these organisms/conditions and is reported in the predicate bottle Package Insert. The following notes have been maintained in the adjusted FA Plus bottle Package Insert.

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-  $H$  parainfluenzae is not always detected in the presence of blood
- C. hominis is not always detected in the absence of blood

In the adjusted bottle, without blood supplementation, less than  $100\%$  recovery was observed for the fastidious organisms N. gonorrhoea, N. menigitidis, C. jeikeium, H. influenzae, and H. parainfluenzae. These results are consistent with results for these organisms grown in the predicate bottle without blood supplementation and denoted in the predicate Package Insert. A limitation that recovery of fastidious organisms requires blood supplementation has been maintained in the adjusted FA Plus Package Insert.

The adjusted bottle was found to be equivalent to the predicate when read on either the BTA 3D or BTA VIRTUO Systems. Percent recovery was not affected by the adjusted bottle lot.

Table 6d. Percent Recovery by Organism and Blood Content

|  Microorganism* | Strains Tested | Blood Content** | % Recovery |   | Diff. in % Recovery (Pred - Adj)* | 95% CI (Lower, Upper)  |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  Predicate | Adjusted  |   |   |
|  Abiotrophadefectiva | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 56.0 (20/36) | 0.0 (0/36) | 56.0 | (34.5, 71.7)  |
|  AcinetobacterBaumannii | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Aggregatibacteractinomycetem Comitans | 1 | Blood | 100.0 (32/32) | 100.0 (32/32) | 0.0 | (-13.3, 13.3)  |
|   |   |  No Blood | 97.0 (31/32) | 94.0 (30/32) | 3.0 | (-12.6, 19.4)  |
|  CampylobacterJejuni | 2 | Blood | 100.0 (24/24) | 100.0 (23/23)c | 0.0 | (-17.2, 17.8)  |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  CandidaAlbicans | 11 | Blood | 100.0 (135/135) | 100.0 (135/135) | 0.0 | (-3.4, 3.4)  |
|   |   |  No Blood | 100.0 (66/66) | 100.0 (66/66) | 0.0 | (-6.9, 6.9)  |
|  CandidaGlabrata | 11 | Blood | 100.0 (131/131)c | 100.0 (132/132) | 0.0 | (-3.6, -3.5)  |
|   |   |  No Blood | 100.0 (66/66) | 100.0 (66/66) | 0.0 | (-6.9, 6.9)  |

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|  Candida Krusei | 10 | Blood | 100.0 (120/120) | 100.0 (120/120) | 0.0 | (-3.9, 3.9)  |
| --- | --- | --- | --- | --- | --- | --- |
|   |  | No Blood | 100.0 (60/60) | 100.0 (60/60) | 0.0 | (-7.5, 7.5)  |
|  Candida parapsilosis | 10 | Blood | 100.0 (120/120) | 100.0 (120/120) | 0.0 | (-3.9, 3.9)  |
|   |  | No Blood | 100.0 (60/60) | 100.0 (60/60) | 0.0 | (-7.5, 7.5)  |
|  Candida Tropicalis | 9 | Blood | 100.0 (108/108) | 100.0 (108/108) | 0.0 | (-4.3, 4.3)  |
|   |  | No Blood | 100.0 (54/54) | 100.0 (54/54) | 0.0 | (-8.3, 8.3)  |
|  Cardiobacterium Hominis | 1 | Blood | 100.0 (32/32) | 100.0 (32/32) | 0.0 | (-13.3, 13.3)  |
|   |  | No Blood | 100% (16/16) | 50% (8/16)^{s} | 50.0 | (15.7, 74.5)  |
|  Corynebacterium Jeikeium | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 0.0 (0/12) | 0.0 (0/12) | 0.0 | (-30.1, 30.1)  |
|  Cryptococcus neoformans | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Eikenella Corrodens | 1 | Blood | 100.0 (32/32) | 100.0 (32/32) | 0.0 | (-13.3, 13.3)  |
|   |  | No Blood | - | - | - | NA  |
|  Klebsiella (Enterobacter) Aerogenes | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Enterobacter Cloacae | 2 | Blood | 100.0 (30/30) | 100.0 (30/30) | 0.0 | (-14.1, 14.1)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Enterococcus Faecalis | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Enterococcus Faecium | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Escherichia Coli | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |

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|  Haemophilus Influenzae | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
| --- | --- | --- | --- | --- | --- | --- |
|   |  | No Blood | 0.0 (0/12) | 0.0 (0/12) | 0.0 | (-30.1, 30.1)  |
|  Haemophilus parainfluenzae | 2 | Blood | 67.0 (16/24) | 58.0 (14/24) | 8.3 | (-20.6, 35.6)  |
|   |  | No Blood | 0.0 (0/12) | 0.0 (0/12) | 0.0 | (-30.1, 30.1)  |
|  Klebsiella Oxytoca | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Klebsiella pneumoniae | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Listeria monocytogenes | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Micrococcus Luteus | 1 | Blood | 100.0 (32/32) | 100.0 (32/32) | 0.0 | (-13.3, 13.3)  |
|   |  | No Blood | 100.0 (16/16) | 100.0 (16/16) | 0.0 | (-24.1, 24.1)  |
|  Neisseria gonorrhoeae | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 83.0 (10/12) | 92.0 (11/12) | -8.3 | (-41.7, 26.4)  |
|  Neisseria Menigitidis | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 8.0 (1/12) | 33.0 (4/12) | -25.0 | (-57.2, 13.8)  |
|  Proteus Mirabilis | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Proteus Vulgaris | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Pseudomonas Aeruginosa | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Salmonella Enterica | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |  | No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |

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|  Serratia marcescens | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Shigella Flexneri | 4 | Blood | 75.0 (45/60)d | 100.0 (60/60) | -25.0 | (-38.1, -12.6)  |
|   |   |  No Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|  Staphylococcus Aureus | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Staphylococcus epidermidis | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Stenotrophomonas maltophilia | 4 | Blood | 100.0 (48/48) | 100.0 (48/48) | 0.0 | (-9.2, 9.2)  |
|   |   |  No Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|  Streptococcus Agalactiae | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |
|  Streptococcus Mitis | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 75.0 (18/24) | 92.0 (22/24) | -16.7 | (-39.8, 8.1)  |
|  Streptococcus pneumoniae | 5 | Blood | 100.0 (60/60) | 100.0 (60/60) | 0.0 | (-7.5, 7.5)  |
|   |   |  No Blood | 100.0 (30/30) | 100.0 (30/30) | 0.0 | (-14.1, 14.1)  |
|  Streptococcus Pyogenes | 2 | Blood | 100.0 (24/24) | 100.0 (24/24) | 0.0 | (-17.2, 17.2)  |
|   |   |  No Blood | 100.0 (12/12) | 100.0 (12/12) | 0.0 | (-30.1, 30.1)  |

*Inoculum concentration 10-100 CFU/bottle. Actual concentration ranged from 3 to 137 CFU/bottle
**Results pooled for 4 and  $10\mathrm{mL}$  blood supplementation
a All false negative results  $(n = 8)$  were obtained on the BTA 3D instrument
b The following statement has been added to the FA Plus Package Insert: Recovery of A. defectiva in FA Plus bottles, tested on either instrument, necessitates blood
One bottle was removed from analysis due to contamination
d All unexpected negative results  $(n = 15)$  occurred for a single strain, S. flexneri 33948, in the predicate bottle
Positive values indicate decreased recovery rate in the adjusted bottle compared to the predicate, whereas negative values indicate increased recovery rate in the adjusted bottle compared to the predicate

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20

# Time-To-Detection (TTD) Study

An in-house seeded study was conducted to demonstrate that changes to the media formulation do not negatively impact Time-To-Detection (TTD). TTD (in hours), was evaluated in a head-to-head study of 2372 adjusted and predicate bottles, of which 1564 were supplemented with blood (4 or 10 mL) and 808 without blood. A set of 39 diverse species commonly found in blood were evaluated using three lots of the adjusted FA Plus bottle (five lots in total were tested) and two lots of the predicate. The species tested are the same as those listed in Table 6d, above. All organisms were seeded into adjusted FA Plus bottles at a target inoculum of 10-100 CFU/bottle. The actual inoculum ranged from 3 CFU/bottle to 137 CFU/bottle. Seeded bottles were read on the BTA 3D and BTA VIRTUO Systems.

To demonstrate equivalence, the mean difference in TTD between the adjusted and predicate bottle was calculated. For all microorganisms, the following criteria were used to assess equivalence: (i) for organisms with TTDs of < 10 hours in the predicate bottle, the mean difference in TTD should be ≤ 1 hour; (ii) for organisms with TTDs >10 hours in the predicate bottle, the mean difference should be within 10% of the predicate bottle TTD.

The overall TTD was 25.7 hours for the predicate bottle and 21.8 hours for the adjusted bottle, with an overall mean difference in TTD of 3.9 hours (Table 7a). Results are shown in Table 7a, stratified by inoculum level/blood content and Table 7b, stratified by Detection System. TTD by adjusted bottle lot is shown in Table 7c.

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Table 7a. Mean Time-to-Detection Difference by Inoculum Level and Blood Content

|  Actual Inoculum Level (CFU/Bottle) | Blood Content | # Bottles Evaluated (Predicate, Adjusted)a | Mean TTD (in Hours) |   | Mean TTD Diff. (Predicate-Adjusted) (in Hours) (95%CI) | % Diff in TTDb  |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  Predicate Bottle | Adjusted Bottle  |   |   |
|  1-9 | No Blood | 12, 12 | 18.2 | 18.3 | -0.1 (-1.6, 1.3) | -0.7%  |
|   |  Blood | 48, 48 | 16.1 | 16.2 | -0.2 (-0.9, 0.6) | -1.0%  |
|  10-100 | No Blood | 601, 598 | 28.3 | 23.2 | 5.1 (3.3, 6.8) | 18.0%  |
|   |  Blood | 1289, 1303 | 26.3 | 22.1 | 4.3 (3.2, 5.3) | 16.2%  |
|  >100 | No Blood | 123, 105 | 24.4 | 21.8 | 2.6 (-1.5, 6.6) | 10.5%  |
|   |  Blood | 204, 203 | 17.3 | 17.1 | 0.3 (-1.2, 1.7) | 3.0%  |
|  Overall | No Blood | 736, 715 | 27.5 | 22.9 | 4.6 (3.0, 6.1) | 16.6%  |
|   |  Blood | 1541, 1554 | 24.8 | 21.3 | 3.6 (2.7, 4.5) | 14.2%  |
|  Overall | Overall | 2277, 2269 | 25.7 | 21.8 | 3.9 (3.1, 4.7) | 15.1%  |

${}^{a}$  Bottles where growth was not detected do not have TTD data, and were removed from analysis
b Positive values denote faster TTD in the adjusted bottle, while negative values denote slower TTD in the adjusted bottle

Table 7b. Mean Time-to-Detection Difference by Detection System

|  System | # Bottles Evaluated (Predicate, Adjusted)a | Mean TTD (in Hours) |   | Mean TTD Diff. (Predicate-Adjusted) (in Hours) (95%CI) | % Diff in TTDb  |
| --- | --- | --- | --- | --- | --- |
|   |   |  Predicate Bottle | Adjusted Bottle  |   |   |
|  BTA 3D | 1142, 1133 | 27.5 | 23.1 | 4.4 (3.2, 5.6) | 15.9%  |
|  BTA VIRTUO | 1135, 1136 | 23.8 | 20.4 | 3.4 (2.4, 4.5) | 14.3%  |

${}^{a}$  Bottles where growth was not detected do not have TTD data, and were removed from analysis
b Positive values denote faster TTD in the adjusted bottle, while negative values denote slower TTD in the adjusted bottle

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Table 7c. Mean Time-to-Detection Difference of the Adjusted Bottle Stratified by Lot

|  Lot # | Number of Bottles Tested | # Bottles Evaluated^{a} | Mean TTD (in Hours) (95% CI)  |
| --- | --- | --- | --- |
|  1 | 727 | 697 | 20.6 (19.8, 21.3)  |
|  2 | 728 | 695 | 20.6 (19.8, 21.3)  |
|  3 | 725 | 695 | 20.8 (19.9, 21.6)  |
|  4 | 96 | 91 | 34.6 (32.7, 36.5)  |
|  5 | 96 | 91 | 34.8 (32.7, 36.9)  |

a Bottles where growth was not detected do not have TTD data, and were removed from analysis

The study demonstrated that the adjusted bottle performed equivalently when compared to the predicate bottle for all evaluated incolulum levels and blood volumes. The adjusted bottle was found to be equivalent to the predicate when read on either the BTA 3D or BTA VIRTUO Systems. TTD was not affected by the adjusted bottle lot.

When stratified by species, it was noted that the TTD for 38 of the 39 species evaluated in the adjusted FA Plus bottle, in the presence of blood, met the criteria for equivalency to the predicate bottle. In the presence of 4 mL blood, H. influenzae and H. parainfluenzae had a mean TTD delay of 1.7 and 32.1 hours, respectively, in the adjusted bottle. A note in the adjusted FA Plus Package Insert states that H. parainfluenzae is not always detected in the presence of blood, and was therefore found to be equivalent. In the presence of blood (4 and 10 mL), faster TTD was achieved for C. glabrata, with a mean TTD improvement of >30 hours. Of note, the following species had a faster mean TTD when supplemented with 10 mL blood in the adjusted bottle: A. baumannii (5 hours), C. albicans (2.4 hours), K. aerogenes (2.8 hours), E. cloacae (5.7 hours), M. luteus (5.9 hours), S. enterica (4.3 hours), S. flexneri (10.9 hours).

The TTD for 32 out of 34 species evaluated in the adjusted bottle, in the absence of blood, met the criteria for equivalency to the predicate bottle. In the absence of blood, S. aureus and S. pneumoniae had a mean TTD delay of 1.7 hours and 5 hours, respectively, when tested in the adjusted bottle. Of note, C. albicans and C glabrata had a faster mean TTD of 3.6 hours and 36.6 hours, respectively, when tested in the adjusted bottle.

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d. Analytical specificity:
Not Applicable

Assay cut-off:

e. Prozone/Hook Effect:
Not Applicable

2. Comparison studies:
a. Method comparison with predicate device:
Not applicable
b. Matrix comparison:
Not Applicable

3. Clinical studies:
Not Applicable; seeded analytical studies to compare the adjusted FA Plus bottle to the predicate FA Plus bottle.
a. Clinical Sensitivity:
b. Clinical specificity:
See section M3a. above.
c. Other clinical supportive data (when a. and b. are not applicable):
Not Applicable

4. Clinical cut-off:
Not Applicable

5. Expected values/Reference range:
Not Applicable. Only analytical studies were conducted.

N. Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

23

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O. Conclusion:

The submitted information in this premarket notification is complete and supports substantial equivalence decision.

24

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**Source:** [https://fda.innolitics.com/submissions/MI/subpart-c%E2%80%94microbiology-devices/MDB/K183166](https://fda.innolitics.com/submissions/MI/subpart-c%E2%80%94microbiology-devices/MDB/K183166)

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