ePlex Blood Culture Identification Panel - Gram Positive (BCID-GP) Panel

K181663 · Genmark Diagnostics, Incorporated · PAM · Dec 20, 2018 · Microbiology

Device Facts

• Record ID: K181663
• Device Name: ePlex Blood Culture Identification Panel - Gram Positive (BCID-GP) Panel
• Applicant: Genmark Diagnostics, Incorporated
• Product Code: PAM · Microbiology
• Decision Date: Dec 20, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3365
• Device Class: Class 2
• Attributes: Real-World Evidence, Pediatric

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K181663 · Dec 20, 2018	ePlex Blood Culture Identification Panel - Gram Positive (BCID-GP) Panel	Genmark Diagnostics, Incorporated	Retrospective clinical samples from routine clinical practice	Retrospective clinical samples were used to supplement the prospective clinical study to ensure sufficient positive samples for low-prevalence targets, supporting the clinical performance evaluation of the ePlex BCID-GP Panel.	Retrospective clinical samples; Clinical performance study; Diagnostic accuracy

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
ePlex BCID-GP Panel Clinical Performance Study; Multi-center clinical study (prospective and retrospective collection); Study Period: June 2014 through July 2016 (prospective); January through February 2018 (prospective)	Individuals exhibiting signs and/or symptoms of bloodstream infection; Sample Size: 586 retrospective samples; Number of Sites: 7	Standard laboratory procedures (traditional and automated culture, MALDI-TOF IVD, microbiological/biochemical techniques, and sequencing)	Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for organism identification and resistance markers

Indications for Use

The GenMark ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-positive bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GP Panel is capable of detecting a wide variety of gram-negative bacteria (Pan Gram-Negative assay) and several Candida species (Pan Candida assay). The ePlex BCID-GP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-positive organism. The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GP Panel: Bacillus cereus group, Bacillus subtilis group, Corynebacterium, Cutibacterium acnes (Propionibacterium acnes), Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus, Streptococcus agalactiae (GBS), Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes (GAS), mecA, mecC, vanA and vanB. The ePlex BCID-GP Panel contains assays for the detection of genetic determinants associated with resistance to methicillin (mecA and mecC) and vancomycin (vanA and vanB) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. The ePlex BCID-GP Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include a broad Pan Gram-Negative assay as well as a Pan Candida assay, which is designed to detect four of the most prevalent Candida species: Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GP Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-GP Panel and for susceptibility testing, differentiation of mixed growth and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of blood stream infection.

Device Story

Device uses multiplex nucleic acid hybridization to identify gram-positive bacteria, resistance markers (mecA, mecC, vanA, vanB), and pan-targets (Gram-negative, Candida) in positive blood culture samples; operates on GenMark ePlex instrument; utilizes automated microfluidic extraction, PCR/RT-PCR amplification, and electrochemical detection via gold electrode array; provides qualitative results to clinicians; aids in rapid diagnosis of bloodstream infections; results must be interpreted alongside Gram stain and clinical findings; requires sub-culturing for definitive susceptibility testing.

Clinical Evidence

Prospective multicenter study (N=711) and retrospective study (N=586) plus contrived samples (N=565). Performance compared to standard culture, MALDI-TOF, and sequencing. Sensitivity/PPA and specificity/NPA reported for all targets; overall high agreement with comparator methods.

Technological Characteristics

Competitive nucleic acid hybridization sandwich assay; gold electrode array; ferrocene-labeled signal probes; alternating current voltammetry (ACV) detection; automated microfluidic cartridge-based extraction and PCR/RT-PCR; requires GenMark ePlex instrument; storage 2–8 °C.

Indications for Use

Indicated for patients of all ages exhibiting signs/symptoms of bloodstream infection. Used as an aid in diagnosis of bacteremia/fungemia in positive blood culture samples containing gram-positive organisms. Results must be interpreted with Gram stain and other clinical/laboratory findings.

Regulatory Classification

Identification

A multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures is a qualitative in vitro device intended to simultaneously detect and identify microorganism nucleic acids from blood cultures that test positive by Gram stain or other microbiological stains. The device detects specific nucleic acid sequences for microorganism identification as well as for antimicrobial resistance. This device aids in the diagnosis of bloodstream infections when used in conjunction with other clinical and laboratory findings. However, the device does not replace traditional methods for culture and susceptibility testing.

Special Controls

In combination with the general controls of the FD&C Act, the Verigene® Gram Positive Blood Culture Nucleic Acid Test is subject to the following special controls: The special controls for the BC-GP Assay are contained in the guideline document entitled "Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures."

*Classification.* Class II (special controls). The special control for this device is FDA's guideline document entitled “Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures.” For availability of the guideline document, see § 866.1(e).

Predicate Devices

• FilmArray Blood Culture Identification Panel (K130914)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

A. 510(k) Number:

K181663

B. Purpose for Submission:

To obtain clearance for the ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel

C. Measurand:

Bacillus cereus group, Bacillus subtilis group, Corynebacterium, Cutibacterium acnes (P. acnes), Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus, Streptococcus agalactiae (GBS), Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes (GAS), mecA, mecC, vanA and vanB.

D. Type of Test:

A multiplexed nucleic acid-based test intended for use with the GenMark's ePlex instrument for the qualitative in vitro detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID-GP assay is performed directly on positive blood culture samples that demonstrate the presence of organisms as determined by Gram stain.

E. Applicant:

GenMark Diagnostics, Incorporated

F. Proprietary and Established Names:

ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel

G. Regulatory Information:

1. Regulation section:

21 CFR 866.3365 - Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures

2. Classification:

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Class II

3. Product codes:

PAM, PEN, PEO

4. Panel:

83 (Microbiology)

# H. Intended Use:

1. Intended use(s):

The GenMark ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-positive bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GP Panel is capable of detecting a wide variety of gram-negative bacteria (Pan Gram-Negative assay) and several Candida species (Pan Candida assay). The ePlex BCID-GP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-positive organism.

The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GP Panel: Bacillus cereus group, Bacillus subtilis group, Corynebacterium, Cutibacterium acnes (Propionibacterium acnes), Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus, Streptococcus agalactiae (GBS), Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes (GAS), mecA, mecC, vanA and vanB.

The ePlex BCID-GP Panel contains assays for the detection of genetic determinants associated with resistance to methicillin (mecA and mecC) and vancomycin (vanA and vanB) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease.

The ePlex BCID-GP Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include a broad Pan Gram-Negative assay as well as a Pan Candida assay, which is designed to detect four of the most prevalent Candida species: Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis.

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The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GP Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-GP Panel and for susceptibility testing, differentiation of mixed growth and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of blood stream infection.

# 2. Indication(s) for use:

Same as Intended Use

# 3. Special conditions for use statement(s):

- For prescription use only
- For in vitro diagnostic use only

# Limitations:

- For prescription use only.
- This test is a qualitative test and does not provide a quantitative value.
- This product should not be used with blood culture media that contains charcoal.
- False results were observed for some targets using the BacT Alert FN Plus bottle type (see the Sample Matrix Equivalency (Bottle Evaluation) section of the package insert for additional details) and with a specific lot of BD BACTEC™ Plus Anaerobic bottles.
- Bacterial and fungal nucleic acids may be present in blood culture, independent of bacterial or fungal viability. Detection of an assay target does not guarantee that the corresponding bacteria or fungi are infectious or are the causative agents for clinical symptoms.
- There is a risk of false negative results due to the presence of sequence variants in the bacterial or fungal targets of the test.
- For some strains within the *Corynebacterium*, *Streptococcus* and Pan *Candida* results, 100% detection was not observed at concentrations expected at bottle positivity. See the Analytical Reactivity (Inclusivity) section for additional details.
- A result of "No Targets Detected" on the ePlex BCID-GP Panel does not preclude the possibility of bacterial or fungal infection. A specimen with a result of No Targets Detected may contain an organism not targeted by the ePlex BCID-GP Panel.
- Antimicrobial resistance can occur via multiple mechanisms. A "Not Detected" result for

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the BCID-GP antimicrobial resistance gene assays does not indicate antimicrobial susceptibility. Subculturing and standard susceptibility testing of isolates is required to determine antimicrobial susceptibility.

- In mixed cultures, the ePlex BCID-GP Panel may not identify all organisms in the specimen, depending upon the concentration of each target present.
- The results of the ePlex BCID-GP Panel should not be used as the sole basis for diagnosis, treatment or other patient management decisions.
- Bacillus badius was shown to cross-react with the Bacillus subtilis group assay.
- Burkholderia cepacia was shown to cross-react with the Corynebacterium assay at levels ≥1x10⁷ CFU/mL.
- An unspecified Rhodococcus strain (ATCC 49988) was shown to cross-react with the Micrococcus assay at levels ≥1x10⁷ CFU/mL.
- The genus level and group assays included as a part of the BCID-GP Panel are designed to detect a broad range of species but will not necessarily detect all species within a genus or group. For species detected by these assays please refer to the analytical and in silico inclusivity sections of this package insert.
- For genus level assays it is possible that an unspecified target may be masked in the case of a co-infection. For example, in the event that an unspecified Staphylococcus species is present in the same sample as a Staphylococcus epidermidis, there is no ability to determine that the unspecified Staphylococcus species is present.

# 4. Special instrument requirements:

For use with the GenMark ePlex instrument

# I. Device Description:

The ePlex BCID-GP Panel is based on the principles of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR), and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization.

Nucleic acid extraction from biological specimens occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to specific

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target sequence / region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets.

A summary of the ePlex BCID-GP Panel nucleic acid targets is presented in Table 1 below.

Table 1: Targets Detected by the ePlex BCID-GP Panel

| Bacterial Targets | |
| --- | --- |
| Bacillus cereus group | Micrococcus |
| Bacillus subtilis group | Staphylococcus |
| Corynebacterium | Staphylococcus aureus |
| Cutibacterium acnes (Propionibacterium acnes) | Staphylococcus epidermidis |
| Enterococcus | Staphylococcus lugdunensis |
| Enterococcus faecalis | Streptococcus |
| Enterococcus faecium | Streptococcus agalactiae |
| Lactobacillus | Streptococcus anginosus group |
| Listeria | Streptococcus pneumoniae |
| Listeria monocytogenes | Streptococcus pyogenes |
| Antimicrobial Resistance Markers | |
| mecA (methicillin resistance) | vanA (vancomycin resistance) |
| mecC (methicillin resistance) | vanB (vancomycin resistance) |
| Pan Targets | |
| Pan Gram-Negative | Pan Candida |

Materials provided in each kit:

| Product | Item | Components (quantity) | Storage |
| --- | --- | --- | --- |
| ePlex Blood Culture Identification Gram-Positive (BCID-GP) | EA003012 | ePlex BCID-GP Panel Cartridge (12) | 2–8 °C |

# Materials required but not provided:

- GenMark ePlex Instrument and Software
- Pipettes capable of delivering 50μL
- Printer (optional) - See ePlex Operator Manual for compatibility guidelines
- Pipette tips, aerosol resistant, RNase/DNase-free
- Disposable, powder free gloves
- 10% bleach for appropriate surfaces
- 70% ethanol or isopropyl alcohol (or equivalent) for appropriate surfaces
- 1.5mL RNase/DNase-free microcentrifuge tube or equivalent (optional)

# Interpretation of Results

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Results interpretation of the ePlex BCID-GP Panel is performed by the ePlex instrument, and the interpretation of results on the ePlex BCID-GP Panel Detection Report for each targeted analyte is summarized in Table 2 below.

Table 2: Interpretation of Results on the ePlex BCID-GP Panel Detection Report

| Target Result | Explanation | Action |
| --- | --- | --- |
| Detected | The test was completed successfully and the target has generated signal above its defined threshold and the Internal Control was reported as PASS. | All results are displayed on the ePlex BCID-GP Panel Detection Report. Test is valid, report results. |
| Not Detected | The test was completed successfully and the target did not generate signal above its defined threshold and the Internal Control was reported as PASS. | All results are displayed on the ePlex BCID-GP Panel Detection Report. Test is valid, report results. |
| Invalid | The test has not successfully completed and results for this test are not valid. This may be due to an instrument or software error. | No results are displayed on the ePlex BCID-GP Panel Detection Report. Test is not valid, repeat test. |

### Genus and Group Assay Result Interpretation

The ePlex BCID-GP Panel Enterococcus result is based on three assays: the species-specific Enterococcus faecalis and Enterococcus faecium assays and a broad Enterococcus assay. The broad Enterococcus assay will detect Enterococcus faecalis and Enterococcus faecium, however, its primary purpose is to detect non-faecalis/faecium Enterococcus species. If all three assays are negative, the Enterococcus result will be Not Detected. If any of the three assays is positive, the Enterococcus result will be 'Detected'. If only the Enterococcus assay is positive, an unspecified Enterococcus species has been detected. Interpretations of results for Enterococcus are described in Table 3 below.

Table 3: Enterococcus Target Results from ePlex BCID-GP Panel Detection Report

| Enterococcus Result | Enterococcus Assay | Enterococcus faecalis Assay | Enterococcus faecium Assay | Description |
| --- | --- | --- | --- | --- |
| Not Detected | Negative | Negative | Negative | No Enterococcus species Detected |
| Detected | Positive | Positive | Negative | Enterococcus faecalis Detected |
| Detected | Positive | Negative | Positive | Enterococcus faecium Detected |
| Detected | Positive | Positive | Positive | Enterococcus faecalis Detected Enterococcus faecium Detected |
| Detected | Positive | Negative | Negative | Unspecified Enterococcus Detected |

The ePlex BCID-GP Panel Listeria result is based on two assays: the species-specific Listeria monocytogenes assay and a broad Listeria assay. The broad Listeria assay will detect multiple Listeria species including Listeria monocytogenes; however, its primary purpose is

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to detect non-monocytogenes *Listeria* species. If either assay is positive, the *Listeria* result will be 'Detected'. If only the *Listeria* assay is positive, an unspecified *Listeria* species has been detected. Interpretations of *Listeria* results are described in Table 4 below.

Table 4: *Listeria* Target Results from ePlex BCID-GP Panel Detection Report

| *Listeria* Result | *Listeria* Assay | *Listeria monocytogenes* Assay | Description |
| --- | --- | --- | --- |
| Not Detected | Negative | Negative | No *Listeria* species Detected |
| Detected | Positive | Positive | *Listeria monocytogenes* Detected |
| Detected | Positive | Negative | Unspecified *Listeria* Detected |

The ePlex BCID-GP Panel *Staphylococcus* result is based on four assays: the species-specific *Staphylococcus aureus*, *Staphylococcus epidermidis* and *Staphylococcus lugdunensis* assays and a broad *Staphylococcus* assay. The broad *Staphylococcus* assay will detect each of the species targeted by the species-specific assays, but its primary purpose is to detect other *Staphylococcus* species. If all four assays are negative, the *Staphylococcus* result will be 'Not Detected'. If any of the four assays is positive, the *Staphylococcus* result will be 'Detected'. If only the *Staphylococcus* assay is positive, an unspecified *Staphylococcus* has been detected. Interpretations of *Staphylococcus* results are described in Table 5 below.

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Table 5: Staphylococcus Target Results from ePlex BCID-GP Panel Detection Report

| Staphylococcus Result | Staphylococcus Assay | Staphylococcus aureus Assay | Staphylococcus epidermidis Assay | Staphylococcus lugdunensis Assay | Description |
| --- | --- | --- | --- | --- | --- |
| Not Detected | Negative | Negative | Negative | Negative | No Staphylococcus species Detected |
| Detected | Positive | Positive | Negative | Negative | Staphylococcus aureus Detected |
| Detected | Positive | Negative | Positive | Negative | Staphylococcus epidermidis Detected |
| Detected | Positive | Negative | Negative | Positive | Staphylococcus lugdunensis Detected |
| Detected | Positive | Positive | Positive | Negative | Staphylococcus aureus Detected Staphylococcus epidermidis Detected |
| Detected | Positive | Positive | Negative | Positive | Staphylococcus aureus Detected Staphylococcus lugdunensis Detected |
| Detected | Positive | Negative | Positive | Positive | Staphylococcus epidermidis Detected Staphylococcus lugdunensis Detected |
| Detected | Positive | Positive | Positive | Positive | Staphylococcus aureus Detected Staphylococcus epidermidis Detected Staphylococcus lugdunensis Detected |
| Detected | Positive | Negative | Negative | Negative | Unspecified Staphylococcus Detected |

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The ePlex BCID-GP Panel Streptococcus result is based on five assays: the species-specific Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus pneumoniae and Streptococcus pyogenes assays and a broad Streptococcus assay. The broad Streptococcus assay will detect each of the species targeted by the species-specific assays, but its primary purpose is to detect other Streptococcus species. If all five assays are negative, the Streptococcus result will be 'Not Detected'. If any of the five assays is positive, the Streptococcus result will be 'Detected'. If only the Streptococcus assay is positive, an unspecified Streptococcus species has been detected. Interpretations of Streptococcus results are described in Table 6 below.

Table 6: Streptococcus Target Results from ePlex BCID-GP Panel Detection Report

| Streptococcus Result | Streptococcus Assay | Streptococcus agalactiae Assay | Streptococcus anginosus group Assay | Streptococcus pneumoniae Assay | Streptococcus pyogenes Assay | Description |
| --- | --- | --- | --- | --- | --- | --- |
| Negative | Negative | Negative | Negative | Negative | Negative | No Streptococcus species Detected |
| Detected | Detected | Detected | Negative | Negative | Negative | Streptococcus agalactiae Detected |
| Detected | Detected | Negative | Detected | Negative | Negative | Streptococcus anginosus group Detected |
| Detected | Detected | Negative | Negative | Detected | Negative | Streptococcus pneumonia Detected |
| Detected | Detected | Negative | Negative | Negative | Detected | Streptococcus pyogenes Detected |
| Detected | Detected | Detected | Detected | Negative | Negative | Streptococcus agalactiae Detected Streptococcus anginosus group Detected |
| Detected | Detected | Detected | Negative | Positive | Negative | Streptococcus agalactiae Detected Streptococcus pneumoniae Detected |
| Detected | Detected | Detected | Negative | Negative | Positive | Streptococcus agalactiae |

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| Streptococcus Result | Streptococcus Assay | Streptococcus agalactiae Assay | Streptococcus anginosus group Assay | Streptococcus pneumoniae Assay | Streptococcus pyogenes Assay | Description |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | | | Detected Streptococcus pyogenes Detected |
| Detected | Detected | Negative | Detected | Detected | Negative | Streptococcus anginosus group Detected Streptococcus pneumoniae Detected |
| Detected | Detected | Negative | Detected | Negative | Detected | Streptococcus anginosus group Detected Streptococcus pyogenes Detected |
| Detected | Detected | Negative | Negative | Detected | Detected | Streptococcus pneumoniae Detected Streptococcus pyogenes Detected |
| Detected | Detected | Detected | Detected | Detected | Negative | Streptococcus agalactiae Detected Streptococcus anginosus group Detected Streptococcus pneumoniae Detected |

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| Streptococcus Result | Streptococcus Assay | Streptococcus agalactiae Assay | Streptococcus anginosus group Assay | Streptococcus pneumoniae Assay | Streptococcus pyogenes Assay | Description |
| --- | --- | --- | --- | --- | --- | --- |
| Detected | Detected | Detected | Detected | Negative | Detected | Streptococcus agalactiae Detected Streptococcus anginosus group Detected Streptococcus pyogenes Detected |
| Detected | Detected | Detected | Negative | Detected | Detected | Streptococcus agalactiae Detected Streptococcus pneumoniae Detected Streptococcus pyogenes Detected |
| Detected | Detected | Negative | Detected | Detected | Detected | Streptococcus anginosus group Detected Streptococcus pneumoniae Detected Streptococcus pyogenes Detected |
| Detected | Detected | Detected | Detected | Detected | Detected | Streptococcus agalactiae Detected Streptococcus anginosus Detected Streptococcus pneumoniae Detected Streptococcus pyogenes Detected |

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| Streptococcus Result | Streptococcus Assay | Streptococcus agalactiae Assay | Streptococcus anginosus group Assay | Streptococcus pneumoniae Assay | Streptococcus pyogenes Assay | Description |
| --- | --- | --- | --- | --- | --- | --- |
| Detected | Detected | Negative | Negative | Negative | Negative | Unspeciated Streptococcus Detected |

Test results for resistance genes are only reported when an associated organism assay is positive in the same sample. See Table 7 for organisms specifically associated with the four resistance markers on the ePlex BCID-GP Panel.

Table 7: Resistance Marker Organism Associations

| Resistance Gene Result | Associated Targets |
| --- | --- |
| mecA and/or mecC | Any Staphylococcus assay (Staphylococcus, S. aureus, S. epidermidis, S. lugdunensis) |
| vanA and/or vanB | Any Enterococcus assay (Enterococcus, E. faecalis, E. faecium) |

The ePlex BCID-GP Panel Pan Gram-Negative result is based on a broad assay that covers most gram-negative organisms which include but are not limited to Acinetobacter, Bacteroides, Enterobacteriaceae, Neisseria, Pseudomonas, Serratia and Stenotrophomonas maltophilia, as shown in Table 8.

Table 8: Pan Gram-Negative Target Results from ePlex BCID-GP Panel Detection Report

| Pan Gram-Negative Result | Description |
| --- | --- |
| Not Detected | No gram-negative organism detected. |
| Detected | One or more gram-negative organisms detected: genera include but are not limited to Acinetobacter, Bacteroides, Enterobacteriaceae, Neisseria, Pseudomonas, Serratia, Stenotrophomonas maltophilia. Additional testing for identification is recommended. |

The ePlex BCID-GP Panel Pan Candida result indicates the presence of one or more of the following Candida species targets: Candida albicans, Candida glabrata, Candida krusei, or Candida parapsilosis as shown in Table 9.

Table 9: Pan Candida Target Results from ePlex BCID-GP Panel Detection Report

| Pan Candida Result | Description |
| --- | --- |
| Not Detected | No specified Candida species detected. |
| Detected | One or more of the following Candida organisms has been detected: Candida albicans, Candida glabrata, Candida krusei and/or Candida parapsilosis. Additional testing for identification is recommended. |

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# ePlex BCID-GP Panel Test Reports

Several different reports are available on the ePlex System. Results are provided in a printable format and may be viewed electronically or exported for additional analysis. Reports can be customized with account specific information such as the address, logo and institutional specific footers on each report.

# Detection Report

The ePlex BCID-GP Panel Detection Report includes the results for each individual sample run on the ePlex System. The Summary section indicates the overall test result and lists all detected targets in that sample. The Results section includes a list of all targets on the panel with an individual result for each target. Results are reported as Detected, Not Detected, or Invalid (displayed as a red x); results for the Internal Control are reported as PASS, FAIL, INVALID, or N/A.

# External Control Report

The ePlex BCID-GP Panel External Control Report is generated for an external control that has been pre-defined in the ePlex BCID-GP Panel Software. For more information on defining external controls on the ePlex System, refer to the ePlex Operator Manual.

The Summary section indicates the overall result (PASS or FAIL status) and lists all detected targets for that external control. The Results section includes a list of all panel targets with the result, expected result and PASS/FAIL status for each. Results are reported as Detected, Not Detected, or Invalid (displayed as a red x). A target is reported as PASS if the actual result matches the expected result (as defined for that control); a target is reported as FAIL if the actual result does not match the expected result. If the actual result for each target matches the expected result (all targets reported as PASS), the overall result for the external control is reported as PASS in the Summary section. If the actual result for any target does not match the expected result, the overall result for the external control is reported as FAIL in the Summary section.

# Summary Report

The Summary Report allows the operator to use searchable criteria to create customized reports, using specified targets, dates, range of dates, sample, external control, test bay, or operator.

# J. Substantial Equivalence Information:

1. Predicate device name(s):

BioFire Diagnostics; FilmArray Blood Culture Identification (BCID) Panel

2. Predicate 510(k) number(s):

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k130914

# 3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device: ePlex BCID-GP Panel (k181663) | Predicate: FilmArray BCID Panel (k130914) |
| Indication for Use | The GenMark ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-positive bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GP Panel is capable of detecting a wide variety of gram-negative bacteria (Pan Gram-Negative assay) and several Candida species (Pan Candida assay). The ePlex BCID-GP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-positive organism. The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GP Panel: Bacillus cereus group, Bacillus subtilis group, Corynebacterium, Cutibacterium acnes (Propionibacterium acnes), Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus, Streptococcus agalactiae (GBS), Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes (GAS), mecA, mecC, vanA and vanB. The ePlex BCID-GP Panel contains assays | The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with the FilmArray Instrument. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrates the presence of organisms as determined by Gram stain. The following gram-positive bacteria, gram-negative bacteria, and yeast are identified using the FilmArray BCID Panel: Enterococci, Listeria monocytogenes, commonly encountered Staphylococci (including specific differentiation of Staphylococcus aureus), commonly encountered Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, commonly encountered Enterobacteriaceae (including specific differentiation of the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. The FilmArray BCID Panel also contains assays for the detection of genetic determinants of resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (blaKPC) to aid in the identification of potentially antimicrobial resistant organisms in positive |

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| Similarities | | |
| --- | --- | --- |
| Item | Device: ePlex BCID-GP Panel (k181663) | Predicate: FilmArray BCID Panel (k130914) |
| | for the detection of genetic determinants associated with resistance to methicillin (mecA and mecC) and vancomycin (vanA and vanB) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. The ePlex BCID-GP Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include a broad Pan Gram-Negative assay as well as a Pan Candida assay, which is designed to detect four of the most prevalent Candida species: Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GP Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not | blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenems exist. FilmArray BCID is indicated as an aid in the diagnosis of specific agents of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID is not intended to monitor treatment for bacteremia or fungemia. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Enterococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays. |

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| Similarities | | |
| --- | --- | --- |
| Item | Device: ePlex BCID-GP Panel (k181663) | Predicate: FilmArray BCID Panel (k130914) |
| | detected by ePlex BCID-GP Panel and for susceptibility testing, differentiation of mixed growth and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of blood stream infection. | |
| Analyte | DNA | DNA |
| Sample Processing | Automated by instrument | Automated by instrument |
| Controls | Each cartridge includes internal controls that monitor performance of each step of the testing process, including extraction, amplification and detection of targets. | Two controls are included in each reagent pouch to control for sample processing and both stages of PCR and melt analysis. |

| Differences | | |
| --- | --- | --- |
| Item | Device: ePlex BCID-GP Panel (k181663) | Predicate: FilmArray BCID Panel (k130914) |
| Specimen Type | Gram-Positive Blood Culture | Gram-Positive & Gram-Negative Blood Culture |
| Test Principles | Reagents on cartridge include: sample lysis and nucleic acid extraction, PCR amplification and hybridization-based electrochemical detection reagents. | The FilmArray BCID pouch contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex PCR with DNA melt analysis. |
| Instrumentation and Hardware | GenMark ePlex Instrument & Single Use Cartridge | FilmArray Instrument and assay pouch |
| Software Interface Result Reporting | • GenMark ePlex System Software • GenMark ePlex BCID-GP Panel Software | The FilmArray Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism and antimicrobial resistance gene on the panel. |

### K. Standard/Guidance Document Referenced

- Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures (May 2015)
- CLSI MM17-A, Vol. 28, No. 9, Verification and Validation of Multiplex Nucleic Acid Assays
- CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition (June 2012)

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- CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline – Second Edition (November 2005)
- CLSP EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline (May 2013)

# L. Test Principle:

The ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is based on the principle of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization.

Nucleic acid extraction from biological samples occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to specific target sequence/region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets.

# M. Performance Characteristics:

# 1. Analytical performance:

# a. Analytical Sensitivity:

The limit of detection (LoD) of the ePlex Blood Culture Identification Gram-Positive Panel was assessed with by testing pooled analytes at a range of concentrations (CFU/mL). Each pool was comprised of an organism mix combined together in negative blood matrix. LoD was determined using qualified stocks of organisms with documented CFU/mL that were pooled together and serially diluted to create organism mixes at the test concentrations. Each assay's LoD was determined by testing at least two strains or species. The Pan Gram-Negative assay was evaluated with 6 species due to its broad specificity. Testing was initially done using 3 test concentrations per multi-organism mix. Testing was performed with a minimum of two ePlex consumable lots using multiple instruments and

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covered a minimum of two testing days. A minimum of 20 replicates per organism mix were tested at each concentration.

A summary of the determined LoD with the overall detection rate is shown in Table 10 below. The target LoD was conservatively established based on the observed testing outcomes. The species tested for each genus level call or antibiotic resistance marker is noted in parentheses, where appropriate.

Table 10: BCID-GP Panel LoD Results Summary

| BCID-GP Panel | Organism Strain ID | Strain | LoD Concentration (CFU/mL) |
| --- | --- | --- | --- |
| B. cereus group | Bacillus cereus | ATCC 21769 | \( 1 \times 10^{5} \) |
| | Bacillus thuringiensis | ATCC 35646 | \( 1 \times 10^{5} \) |
| B. subtilis group | Bacillus subtilis | ATCC 55614 | \( 1 \times 10^{6} \) |
| | Bacillus atrophaeus | ATCC 51189 | \( 1 \times 10^{6} \) |
| Corynebacterium | Corynebacterium striatum | ATCC 43735 | \( 1 \times 10^{6} \) |
| | Corynebacterium jeikeium | ATCC 43217 | \( 1 \times 10^{7} \) |
| C. acnes (P. acnes) | Cutibacterium acnes (P. acnes) | ATCC 33179 | \( 1 \times 10^{7} \) |
| | Cutibacterium acnes (P. acnes) | ATCC 6919 | \( 1 \times 10^{8} \) |
| Enterococcus | Enterococcus faecium | ATCC BAA-2316 | \( 1 \times 10^{5} \) |
| | Enterococcus faecium | ATCC BAA-2317 | \( 1 \times 10^{6} \) |
| | Enterococcus raffinosus | ATCC 49464 | \( 1 \times 10^{6} \) |
| E. faecium | Enterococcus faecium | ATCC BAA-2316 | \( 1 \times 10^{5} \) |
| | Enterococcus faecium | ATCC BAA-2317 | \( 1 \times 10^{6} \) |
| E. faecalis | Enterococcus faecalis | ATCC 51575 | \( 1 \times 10^{6} \) |
| | Enterococcus faecalis | ATCC 700802 | \( 1 \times 10^{6} \) |
| Lactobacillus | Lactobacillus paracasei | ATCC 25598 | \( 1 \times 10^{5} \) |
| | Lactobacillus casei | ATCC 334 | \( 1 \times 10^{5} \) |
| Listeria | Listeria seeligeri | ATCC 35967 | \( 1 \times 10^{5} \) |
| | Listeria monocytogenes | ATCC 10890 | \( 1 \times 10^{5} \) |
| | Listeria monocytogenes | ATCC 19111 | \( 1 \times 10^{6} \) |
| L. monocytogenes | Listeria monocytogenes | ATCC 10890 | \( 1 \times 10^{5} \) |
| | Listeria monocytogenes | ATCC 19111 | \( 1 \times 10^{5} \) |
| Micrococcus | Micrococcus luteus | ATCC 19212 | \( 1 \times 10^{6} \) |
| | Micrococcus luteus | ATCC 10240 | \( 1 \times 10^{7} \) |
| Staphylococcus | Staphylococcus aureus | ATCC BAA-2313 | \( 1 \times 10^{4} \) |
| | Staphylococcus aureus | ATCC BAA-2312 | \( 1 \times 10^{5} \) |
| | Staphylococcus epidermidis | ATCC 35983 | \( 1 \times 10^{5} \) |
| | Staphylococcus epidermidis | ATCC 35984 | \( 1 \times 10^{5} \) |
| | Staphylococcus lugdunensis | NRS 879 | \( 1 \times 10^{5} \) |
| | Staphylococcus lugdunensis | ATCC 49576 | \( 1 \times 10^{6} \) |
| | Staphylococcus haemolyticus | NRS 62 | \( 1 \times 10^{7} \) |

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| BCID-GP Panel | Organism Strain ID | Strain | LoD Concentration (CFU/mL) |
| --- | --- | --- | --- |
| S. aureus | Staphylococcus aureus | ATCC BAA-2313 | \( 1 \times 10^{5} \) |
| | Staphylococcus aureus | ATCC BAA-2312 | \( 1 \times 10^{5} \) |
| S. epidermidis | Staphylococcus epidermidis | ATCC 35983 | \( 1 \times 10^{5} \) |
| | Staphylococcus epidermidis | ATCC 35984 | \( 1 \times 10^{5} \) |
| S. lugdunensis | Staphylococcus lugdunensis | NRS 879 | \( 1 \times 10^{5} \) |
| | Staphylococcus lugdunensis | ATCC 49576 | \( 1 \times 10^{5} \) |
| Streptococcus | Streptococcus pneumoniae | ATCC BAA-475 | \( 1 \times 10^{5} \) |
| | Streptococcus pneumoniae | ATCC 10357 | \( 1 \times 10^{5} \) |
| | Streptococcus gordonii | ATCC 10558 | \( 1 \times 10^{6} \) |
| | Streptococcus agalactiae | ATCC 12401 | \( 1 \times 10^{6} \) |
| | Streptococcus agalactiae | ATCC 13813 | \( 1 \times 10^{7} \) |
| S. agalactiae | Streptococcus agalactiae | ATCC 12401 | \( 1 \times 10^{5} \) |
| | Streptococcus agalactiae | ATCC 13813 | \( 1 \times 10^{6} \) |
| S. anginosus | Streptococcus intermedius | ATCC 27335 | \( 1 \times 10^{4} \) |
| | Streptococcus anginosus | ATCC 9895 | \( 1 \times 10^{6} \) |
| S. pneumoniae | Streptococcus pneumoniae | ATCC BAA-475 | \( 1 \times 10^{5} \) |
| | Streptococcus pneumoniae | ATCC 10357 | \( 1 \times 10^{5} \) |
| S. pyogenes | Streptococcus pyogenes | ATCC 12384 | \( 1 \times 10^{5} \) |
| | Streptococcus pyogenes | ATCC 49399 | \( 1 \times 10^{5} \) |
| Pan Gram-Negative | Stenotrophomonas maltophilia | ATCC 13636 | \( 1 \times 10^{6} \) |
| | Enterobacter cloacae | ATCC 13047 | \( 1 \times 10^{6} \) |
| | Escherichia coli | ATCC 4157 | \( 1 \times 10^{6} \) |
| | Klebsiella pneumoniae | ATCC BAA-1706 | \( 1 \times 10^{6} \) |
| | Serratia marcescens | ATCC 8100 | \( 1 \times 10^{6} \) |
| | Proteus mirabilis | ATCC 43071 | \( 1 \times 10^{6} \) |
| | Acinetobacter baumannii | NCTC13302 | \( 1 \times 10^{7} \) |
| | Neisseria meningitidis | ATCC 13113 | \( 1 \times 10^{7} \) |
| | Pseudomonas aeruginosa | ATCC 15442 | \( 1 \times 10^{7} \) |
| Pan Candida | Candida albicans | ATCC 24433 | \( 1 \times 10^{6} \) |
| | Candida glabrata | ATCC 66032 | \( 1 \times 10^{6} \) |
| mecA | Staphylococcus epidermidis | ATCC 35983 | \( 1 \times 10^{5} \) |
| | Staphylococcus epidermidis | ATCC 35984 | \( 1 \times 10^{5} \) |
| mecC | Staphylococcus aureus | ATCC BAA-2313 | \( 1 \times 10^{4} \) |
| | Staphylococcus aureus | ATCC BAA-2312 | \( 1 \times 10^{4} \) |
| vanA | Enterococcus faecium | ATCC BAA-2316 | \( 1 \times 10^{4} \) |
| | Enterococcus faecium | ATCC BAA-2317 | \( 1 \times 10^{5} \) |
| vanB | Enterococcus faecalis | ATCC 51575 | \( 1 \times 10^{5} \) |
| | Enterococcus faecalis | ATCC 700802 | \( 1 \times 10^{5} \) |

b. Reproducibility:

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A multisite reproducibility study of the ePlex BCID-GP Panel was conducted at three testing sites (two external sites and one internal site) across major potential sources of variability, such as site-to-site, lot-to-lot, day-to-day, and operator-to-operator. One ePlex instrument per study site with four towers was employed in this reproducibility study. Two operators performed testing at each site on six days (five nonconsecutive days) with three unique lots of ePlex BCID-GP Panel cartridges. A reproducibility study panel consisting of four panel members; including 9 on-panel organisms and three antibiotic resistance genes representing 15 targets at two concentrations and one negative mix including an off-panel organism were tested in triplicate. Concentrations in the positive mixes reflected those observed at time of bottle positivity plus 8 hours (BP+8) and time of bottle positivity (BP) and one mix containing an off-panel organism grown to bottle positivity, which is expected to yield a negative result.

Summary results for the ePlex BCID-GP Panel reproducibility study are provided in Table 11 below.

Table 11: Summary of Reproducibility Results

| BCID-GP Test Result | Organism Test Concentration | Site | Agreement with Expected Results | | |
| --- | --- | --- | --- | --- | --- |
| | | | Agreed/N | % | 95% CI |
| *Corynebacterium striatum* | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Bottle Positive | 1 | 35/36 | 97.2 | (85.8-99.5) |
| | | 2 | 35/36 | 97.2 | (85.8-99.5) |
| | | 3 | 33/36 | 91.7 | (78.2-97.1) |
| | | **All** | **103/108** | **95.4** | **(89.6-98.0)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 107/108 | 99.1 | (94.9-99.8) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **322/323** | **99.7** | **(98.3-99.9)** |
| *Cutibacterium acnes* *(Propionibacterium acnes)* | Bottle Positive + 8 Hours | 1 | 32/36 | 88.9 | (74.7-95.6) |
| | | 2 | 33/36 | 91.7 | (78.2-97.1) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **101/108** | **93.5** | **(87.2-96.8)** |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 33/36 | 91.7 | (78.2-97.1) |
| | | 3 | 35/36 | 97.2 | (85.8-99.5) |
| | | **All** | **104/108** | **96.3** | **(90.9-98.6)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **323/323** | **100** | **(98.8-100)** |

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| Enterococcus | Bottle Positive + 8 Hours | 1 | 72/72 | 100 | (94.9-100) |
| --- | --- | --- | --- | --- | --- |
| | | 2 | 72/72 | 100 | (94.9-100) |
| | | 3 | 72/72 | 100 | (94.9-100) |
| | | **All** | **216/216** | **100** | **(98.3-100)** |
| | Bottle Positive | 1 | 72/72 | 100 | (94.9-100) |
| | | 2 | 72/72 | 100 | (94.9-100) |
| | | 3 | 72/72 | 100 | (94.9-100) |
| | | **All** | **216/216** | **100** | **(98.3-100)** |
| | Negative | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 35/35 | 100 | (90.1-100) |
| | | **All** | **107/107** | **100** | **(96.5-100)** |
| Enterococcus faecalis | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **323/323** | **100** | **(98.8-100)** |
| Enterococcus faecium | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 107-107 | 100 | (96.5-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **323/323** | **100** | **(98.8-100)** |
| Lactobacillus | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.1-100) |
| | | 2 | 36/36 | 100 | (89.8-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.5-100)** |
| | Bottle Positive | 1 | 36/36 | 100 | (89.8-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.1-100) |
| | | **All** | **108/108** | **100** | **(96.5-100)** |
| | Negative | 1 | 108/108 | 100 | (90.4-100) |

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| | | 2 | 108/108 | 100 | (89.8-100) |
| --- | --- | --- | --- | --- | --- |
| | | 3 | 107/107 | 100 | (90.1-100) |
| | | All | 323/323 | 100 | (96.5-100) |
| Staphylococcus | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 35/36 | 97.2 | (85.8-99.5) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 107/108 | 99.1 | (94.9-99.8) |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 107/108 | 99.1 | (94.9-99.8) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | All | 322/323 | 99.7 | (98.3-99.9) |
| Staphylococcus aureus | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | All | 323/323 | 100 | (98.8-100) |
| Streptococcus | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 35/36 | 97.2 | (85.8-99.5) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 107/108 | 99.1 | (94.9-99.8) |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 107/108 | 99.1 | (94.9-99.8) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | All | 322/323 | 99.7 | (98.3-99.9) |
| Streptococcus anginosus group | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 35/36 | 97.2 | (85.8-99.5) |

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| | Negative | 3 | 36/36 | 100 | (90.4-100) |
| --- | --- | --- | --- | --- | --- |
| | | **All** | **107/108** | **99.1** | **(94.9-99.8)** |
| | | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **323/323** | **100** | **(98.8-100)** |
| **Pan Candida** | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **323/323** | **100** | **(98.8-100)** |
| **Pan Gram-Negative** | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 107/108 | 99.1 | (94.9-99.8) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **322/323** | **99.7** | **(98.3-99.9)** |
| **mecA** | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | **All** | **108/108** | **100** | **(96.6-100)** |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | **All** | **323/323** | **100** | **(98.8-100)** |
| **vanA** | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |

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| | | All | 108/108 | 100 | (96.6-100) |
| --- | --- | --- | --- | --- | --- |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | All | 323/323 | 100 | (98.8-100) |
| vanB | Bottle Positive + 8 Hours | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Bottle Positive | 1 | 36/36 | 100 | (90.4-100) |
| | | 2 | 36/36 | 100 | (90.4-100) |
| | | 3 | 36/36 | 100 | (90.4-100) |
| | | All | 108/108 | 100 | (96.6-100) |
| | Negative | 1 | 108/108 | 100 | (96.6-100) |
| | | 2 | 108/108 | 100 | (96.6-100) |
| | | 3 | 107/107 | 100 | (96.5-100) |
| | | All | 323/323 | 100 | (98.8-100) |

c. Linearity/assay reportable range:

Not applicable

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

### Assay Controls

Internal Controls:

Each ePlex BCID- GP Panel cartridge includes internal controls that monitor performance of each step of the testing process, including extraction, amplification and detection of targets.

Each amplification reaction on the cartridge has an internal control and in each reaction either the internal control or a target must generate signal above the defined threshold for a valid test result. Internal control results are interpreted by the ePlex Software and displayed on the ePlex BCID-GP Panel Reports as Internal Control with a result of PASS, FAIL, N/A or INVALID. Table 12 includes details on the interpretation of Internal Control results.

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Table 12: Internal Control Results

| Internal Control Result | Explanation | Action |
| --- | --- | --- |
| PASS | Signal above threshold has been detected from each amplification reaction. The test was completed and internal controls were successful, indicating valid results were generated. | All results are displayed on the ePlex BCID-GP Panel Detection Report. Test is valid, report results. |
| FAIL | Signal above threshold has not been detected from at least one amplification reaction. The test was completed but internal controls were not detected, indicating that results may not be valid. | No results are displayed on the ePlex BCID-GP Panel Detection Report. Test is not valid, repeat the test using a new cartridge. |
| N/A | The internal control in every amplification reaction does not generate signal above the threshold, but a target in every amplification reaction does generate signal above the threshold. The test was completed and internal controls were not successful, however detection of signal above the threshold for a target in every amplification reaction indicates valid results were generated. | All results are displayed on the ePlex BCID-GP Panel Detection Report. Test is valid, report results. |
| INVALID | An error has occurred during processing that prevents analysis of signal data. The test has not successfully completed and results for this test are not valid. This may be due to an instrument or software error. | No results are displayed on the ePlex BCID-GP Panel Detection Report. Test is not valid, repeat the test using a new cartridge. |

Recommended External Controls: External controls are not provided with the ePlex BCID Gram-Positive Panel, but are recommended in the package insert. Positive and negative external controls should be tested with each new lot of reagents or monthly, whichever occurs first. Blood culture medium can be used as the negative control. Previously characterized positive samples or blood culture medium spiked with well characterized organisms can be used as the external positive control. External controls should be run in accordance with laboratory protocols and accrediting organizations, as applicable.

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## Specimen Stability

A specimen stability study was performed to confirm the stability of specimens stored under four different temperature conditions over multiple time points which vary based on storage temperature. Organism mixes whose members were at a concentration approximating bottle positivity were stored at ≤-70°C, ≤-20°C, 2° - 8°C, and ambient temperature over various time points described in Table 13 below.

Table 13: Storage Conditions and Testing Time Points

| Storage Condition | Time Points | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Ambient | T0 | 1 day | 3 days | 5 days | 7 days | 14 days | 30 days | -- |
| Refrigerated (2-8°C) | -- | 1 day | 3 days | 5 days | 7 days | 14 days | 30 days | -- |
| Frozen (≤-20°C) | 1 week | 2 weeks | 1 mo. | 3 mo.* | 6 mo. | 12 mo. | 18 mo. | 24 mo. |
| Frozen (≤-70°C) | 1 week | 2 weeks | 1 mo. | 3 mo.* | 6 mo. | 12 mo. | 18 mo. | 24 mo. |
| Bottle Incubator | Bottle positivity | 12 hours post positivity | -- | -- | -- | -- | -- | -- |

*Mix 1 was tested up to 3 months; Mix 2 was tested up to 1 month.

These organism mixes represent each of the eight multiplex amplification pools on the BCID-GP Panel and include common gram-positive bacteria, drug resistance genes, bottle contaminants (Lactobacillus, Corynebacterium, Cutibacterium), gram-negative (Klebsiella pneumoniae) and fungal (Candida albicans) targets. Twenty replicates were tested at the beginning of the study when organism mixes were freshly made (time point T0). Ten replicates were tested for each additional temperature and time point.

## Ambient Storage Condition

All Mix 1 and Mix 2 analytes showed positivity rates of >95% when stored under ambient temperature for up to 1 month. The positivity rates and mean signals (nA) are summarized for Mix 1 and Mix 2 in Table 14 and Table 15, respectively.

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**Table 14: Mix 1 Ambient Storage Sample Stability**

| Time Point | n | Metric | Mix 1 Analytes Ambient Storage | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | *C. acnes* | *E. faecalis** | *L. casei* | *Staphylococcus* | *S. epidermidis* | *vanB* |
| Day 0 | 20 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 276.0 | 897.2 | 423.8 | 484.0 | 496.6 | 486.7 |
| Day 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 309.3 | 794.3 | 396.6 | 492.4 | 361.4 | 447.7 |
| Day 3 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 448.4 | 915.0 | 515.9 | 635.7 | 447.0 | 507.8 |
| Day 5 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 438.1 | 901.5 | 489.7 | 673.0 | 491.7 | 526.0 |
| Week 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 449.8 | 979.8 | 533.1 | 657.4 | 540.6 | 526.5 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 295.2 | 996.1 | 479.4 | 624.2 | 520.0 | 452.3 |
| Month 1 | 20 | % Detected | 95 | 95 | 95 | 95 | 95 | 95 |
| | | Mean Signal (nA) | 374.1 | 980.7 | 492.1 | 642.2 | 582.8 | 486.2 |

\* The *Enterococcus* genus call is not sensitive to *Enterococcus faecalis* and is therefore forced by the software logic in the presence of *Enterococcus faecalis* signal as detected. Due to this, signal and detection rate for *Enterococcus* are not analyzed in this table.

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Table 15: Mix 2 Ambient Storage Sample Stability

| Time point | n | Metric | Mix 2 Analytes Ambient Storage | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Enterococcus | E. faecium | Staph | S. aureus | Streptococcus | S. pneumoniae | C albicans | K. pneumo | vanA |
| Day 0 | 20 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 230.1 | 548.0 | 511.3 | 458.9 | 766.0 | 669.0 | 281.1 | 135.8 | 741.8 |
| Day 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 294.1 | 580.8 | 606.5 | 641.1 | 829.5 | 705.4 | 308.1 | 143.2 | 900.0 |
| Day 3 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 292.9 | 592.0 | 654.8 | 751.2 | 876.3 | 756.4 | 335.4 | 141.2 | 854.5 |
| Day 5 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 303.3 | 586.0 | 603.3 | 716.2 | 865.2 | 753.4 | 334.1 | 170.9 | 931.7 |
| Week 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 322.9 | 608.5 | 722.3 | 537.1 | 813.2 | 739.5 | 358.7 | 177.5 | 950.1 |
| Week 2 | 20 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 95 | 100 |
| | | Mean Signal (nA) | 323.7 | 632.0 | 733.0 | 465.2 | 800.5 | 714.4 | 338.5 | 214.7 | 971.5 |
| Month 1 | 20 | % Detected | 95 | 95 | 100 | 100 | 95 | 100 | 95 | 100 | 95 |
| | | Mean Signal (nA) | 262.4 | 478.0 | 672.9 | 284.0 | 720.3 | 593.0 | 278.2 | 198.8 | 865.7 |

All Mix 1 and Mix 2 analytes showed positivity rates of ≥95% when stored under refrigerated conditions (2°C-8°C) for up to 1 month. The positivity rates and mean signals (nA) are summarized for Mix 1 and Mix 2 in Table 16 and Table 17, respectively.

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Table 16: Mix 1, 2-8°C Storage Sample Stability

| Time Point | n | Metric | Mix 1 Analytes 2°C-8°C Storage | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | C. acnes | E. faecalis* | L. casei | Staphylococcus | S. epidermidis | vanB |
| Day 1 | 20 | % Detected | 95 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 377.9 | 909.5 | 489.8 | 578.7 | 515.3 | 473.4 |
| Day 3 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 341.1 | 830.7 | 446.7 | 496.2 | 451.5 | 466.7 |
| Day 5 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 427.8 | 900.4 | 500.2 | 566.2 | 461.2 | 493.6 |
| Week 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 298.5 | 778.6 | 418.9 | 488.7 | 414.7 | 503.3 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 316.8 | 1015.2 | 522.5 | 586.0 | 443.4 | 475.0 |
| Month 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 414.8 | 1134.2 | 563.7 | 685.1 | 721.7 | 514.7 |

* The Enterococcus genus call is not sensitive to Enterococcus faecalis and is therefore forced by the software logic in the presence of Enterococcus faecalis signal as detected. Due to this, signal and detection rate for Enterococcus are not analyzed in this table.

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Table 17: Mix 2, 2°C-8°C Storage Sample Stability

| Time Point | n | Metric | Mix 2 Analytes 2°C-8°C Storage | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Enterococcus | E. faecium | Staphylococcus | S. aureus | Streptococcus | S. pneumoniae | C. albicans | K. pneumoniae | vanA |
| Day 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 310.7 | 553.0 | 582.6 | 620.7 | 809.1 | 710.4 | 316.9 | 138.2 | 890.5 |
| Day 3 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 321.1 | 582.7 | 574.7 | 621.7 | 876.7 | 732.9 | 329.5 | 180.9 | 870.2 |
| Day 5 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 302.9 | 589.1 | 597.5 | 623.6 | 869.2 | 753.2 | 334.3 | 119.0 | 863.6 |
| Week 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 270.8 | 564.9 | 582.7 | 334.0 | 706.2 | 655.5 | 307.4 | 176.5 | 889.6 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 282.7 | 593.2 | 611.2 | 400.0 | 698.3 | 596.1 | 301.5 | 153.5 | 951.0 |
| Month 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 270.6 | 526.0 | 604.2 | 312.8 | 681.3 | 596.5 | 311.1 | 164.2 | 891.3 |

### Frozen (≤-20°C) Storage Condition

All Mix 1 and Mix 2 analytes showed positivity rates of 100% when stored under frozen conditions ( \( \leq-20^{\circ}C \) ) for up to 1 month. The positivity rates and mean signals (nA) are summarized in Table 18 and Table 19, respectively. Mix 1 was also tested at the subsequent 3 month time point and continued to show 100% positivity for all analytes.

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Table 18: Mix 1, ≤-20°C Storage Sample Stability

| Time Point | n | Metric | Mix 1 Analytes ≤-20°C Storage | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | C. acnes | E. faecalis* | L. casei | Staphylococcus | S. epidermidis | vanB |
| Week 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 447.0 | 986.2 | 537.1 | 651.5 | 504.6 | 545.1 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 299.7 | 943.0 | 458.3 | 553.9 | 445.6 | 467.9 |
| Month 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 373.2 | 934.2 | 490.5 | 581.8 | 661.4 | 474.3 |
| Month 3 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 214.7 | 853.6 | 440.2 | 637.3 | 584.0 | 505.4 |

* The Enterococcus genus call is not sensitive to Enterococcus faecalis and is therefore forced by the software logic in the presence of Enterococcus faecalis signal as detected. Due to this, signal and detection rate for Enterococcus are not analyzed in this table.

Table 19: Mix 2, ≤-20°C Storage Sample Stability

| Time Point | n | Metric | Mix 2 Analytes ≤-20°C Storage | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Enterococcus | E. faecium | Staphylococcus | S. aureus | Streptococcus | S. pneumoniae | C. albicans | K. pneumoniae | vanA |
| Week 1 | 20 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 95 | 100 |
| | | Mean Signal (nA) | 282.8 | 559.4 | 608.2 | 504.0 | 763.9 | 663.5 | 316.8 | 174.9 | 873.6 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 282.7 | 589.6 | 558.8 | 445.4 | 807.2 | 671.3 | 305.0 | 197.8 | 951.3 |
| Month 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 281.0 | 482.2 | 614.4 | 317.8 | 813.4 | 630.4 | 261.3 | 155.3 | 847.4 |

# Frozen (≤-70°C) Storage Condition

Mix 1 analytes showed positivity rates of ≥95% when stored under frozen conditions (≤-70°C) for up to 3 months with the exception of C. acnes and L. casei, which observed a positivity rate of 90% on the first week of storage at ≤-70°C. Per the study acceptance criteria, subsequent time points were evaluated. At all other tested time points up to 3 months, C. acnes and L. casei showed ≥95% positive detection indicating that the lower detection rates at the week 1

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time point were not due to specimen stability issues. The observed positivity rates and mean signals (nA) for Mix 1 analytes are summarized in Table 20 below.

Table 20: Mix 1, ≤-70°C Storage Sample Stability

| Time Point | n | Metric | Mix 1 Analytes ≤-70°C Storage | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | C. acnes | E. faecalis* | L. casei | Staphylococcus | S. epidermidis | vanB |
| Week 1 | 20 | % Detected | 90 | 100 | 90 | 100 | 100 | 95 |
| | | Mean Signal (nA) | 328.8 | 796.8 | 448.6 | 465.6 | 367.4 | 473.9 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 275.3 | 795.4 | 385.8 | 505.3 | 420.9 | 414.6 |
| Month 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 308.3 | 872.4 | 428.5 | 490.3 | 590.9 | 469.8 |
| Month 3 | 20 | % Detected | 95 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 206.3 | 795.0 | 405.4 | 514.7 | 481.8 | 500.2 |

* The Enterococcus genus call is not sensitive to Enterococcus faecalis and is therefore forced by the software logic in the presence of Enterococcus faecalis signal as detected. Due to this, signal and detection rate for Enterococcus are not analyzed in this table.

All Mix 2 analytes showed positivity rates of 100% when stored under frozen conditions ( \( \leq-70^{\circ}C \) ) for up to 1 month. The positivity rates and mean signals (nA) for Mix 2 analytes are summarized in Table 21.

Table 21: Mix 2, ≤-70°C Storage Sample Stability

| Time Point | n | Metric | Mix 2 Analytes ≤-70°C Storage | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Enterococcus | E. faecium | Staphylococcus | S. aureus | Streococcus | S. pneumoniae | C albicans | K. pneumoniae | vanA |
| Week 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 295.6 | 587.2 | 591.5 | 466.9 | 733.6 | 677.2 | 318.6 | 143.0 | 893.8 |
| Week 2 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 278.8 | 507.3 | 555.2 | 431.2 | 793.7 | 643.4 | 297.5 | 167.0 | 885.2 |
| Month 1 | 10 | % Detected | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| | | Mean Signal (nA) | 267.2 | 498.5 | 609.1 | 349.2 | 775.5 | 617.6 | 306.6 | 182.3 | 862.9 |

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## Bottle Ring and 12 Hours Post Bottle Ring

The positivity rates and mean signals were calculated for the two organisms (Staphylococcus lugdunensis and Streptococcus pyogenes) grown to bottle positivity and incubated 12 hours post positivity. The positivity rates for Staphylococcus lugdunensis and Streptococcus pyogenes at bottle ring and 12 hours post bottle ring was 100% for both organisms. Results are summarized in Table 22.

Table 22: Bottle Ring and 12 hours Post Bottle Ring

| Blood culture | Metric | Bottle ring | | Bottle ring + 12 hrs | |
| --- | --- | --- | --- | --- | --- |
| | | Staphylococcus | S. lugdunensis | Staphylococcus | S. lugdunensis |
| Staphylococcus lugdunensis | % Detected | 100% | 100% | 100% | 100% |
| | Mean Signal (nA) | 617.7 | 833.8 | 537.8 | 800.1 |
| Blood culture | Metric | Streptococcus | S. pyogenes | Streptococcus | S. pyogenes |
| Streptococcus pyogenes | % Detected | 100% | 100% | 100% | 100% |
| | Mean Signal (nA) | 591.8 | 936.9 | 666.1 | 1045.9 |

Results demonstrated that specimens can be stored up to 1 month under the following temperature conditions without adversely impacting the performance of the BCID-GP Panel:

- Ambient temperature
- Refrigerated (2°C-8°C)
- Frozen (≤-20°C and ≤-70°C)

Specimens can also be tested when incubated up to 12 hours after bottle ring in a continuously monitoring blood culture device.

The specimen stability claims are summarized in the table below.

| Storage Condition | Specimen Stability |
| --- | --- |
| Ambient | 1 month |
| Refrigerated (2°C-8°C) | 1 month |
| Frozen (≤-20°C) | 1 month |
| Frozen (≤-70°C) | 1 month |
| Post Bottle Ring | 12 hours |

## Freeze-Thaw Study

A study was performed to assess the tolerance of the ePlex BCID-GP to correctly identify specimens containing bacterial and fungal organisms that have gone through one or two freeze/thaw cycles prior to testing. One hundred and ten positive clinical samples that were tested fresh during the BCID-GP Panel Clinical Performance Study were selected to best represent a variety of organisms in clinical samples. The samples were frozen at ≤-

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70°C and then thawed once or twice prior to re-testing with the BCID-GP Panel.

The positive agreement with the fresh condition at each freeze-thaw cycle was calculated and was above 95% for each condition. Table 23 below summarizes the concordance at each freeze-thaw cycle with a 95% confidence level.

Table 23: Concordance of Results with Fresh Condition

| | 1x Freeze Thaw | 2x Freeze Thaw |
| --- | --- | --- |
| Concordant Samples | 98 | 101 |
| Discordant Samples | 4 | 1 |
| Total Samples Tested | 102 | 102 |
| Concordance | 96.08% | 99.02% |
| CI Low | 90.35% | 94.65% |
| CI High | 98.46% | 99.83% |

Results demonstrate that the BCID-GP Panel can detect the same organisms in samples that were tested fresh and in samples that have been frozen and thawed up to 2 times.

### In-Cartridge Sample Stability Study

An analytical study was carried out to demonstrate performance under the following conditions:

1) Condition 1 (Open Pouch): the cartridge is stable for 2 hours after the pouch has been opened and the unloaded cartridge is exposed to the environment.
2) Condition 2 (In-Consumable Sample Stability): the cartridge can be stored at room temperature for at least 2 hours after the sample has been loaded prior to running the cartridge in an ePlex bay.

A test mix of five organisms was used in the study. The selected organisms represent each of the eight multiplex primer pools on the BCID-GP Panel and produce nine target results. Each organism was tested at a concentration approximating bottle positivity. A description of the test mix used for this study is summarized in the Table below.

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Table 24: Representative Organisms Contained in Test Mix

| Organism in Test Mix | Concentration in Mix (CFU/mL) | Primer Pool | ePlex BCID-GP Panel Expected Result |
| --- | --- | --- | --- |
| *Streptococcus pneumoniae* | 4 x 10^{7} | 5 | *Streptococcus pneumoniae* |
| | | | *Streptococcus** |
| *Enterococcus faecium/vanA* | 4 x 10^{7} | 1, 3, 4 | *Enterococcus faecium* |
| | | | *Enterococcus** |
| | | | *vanA* |
| *Candida albicans* | 1 x 10^{6} | 2 | Pan *Candida* |
| *Klebsiella pneumoniae* | 5 x 10^{8} | 7 | Pan Gram-Negative |
| *Staphylococcus aureus* | 2 x 10^{7} | 6, 8 | *Staphylococcus aureus* |
| | | | *Staphylococcus** |

* Software logic defines that when a Staphylococcus, Streptococcus or Enterococcus species is detected, the corresponding genus call is detected.

The open pouch stability study compared 20 replicates of cartridges that were kept in closed pouches until the cartridges were loaded with sample vs. 20 replicates of cartridges where the pouches were opened and the cartridges were exposed to ambient room temperature conditions for up to 2 hours before being loaded with sample.

To demonstrate that the consumable can be held at room temperature for at least 2 hours after the sample has been loaded (prior to running the consumable on an ePlex bay), 20 consumables were loaded with the organism mix, within 20 minutes of opening the pouch. The consumables were kept for 2 hours at room temperature and then run in the ePlex instrument.

A control condition was run wherein 20 replicates of the organism mix were loaded within 20 minutes of pouch opening and the consumables were run on the ePlex instrument within 20 minutes of sample loading. The data from the test conditions were compared to the control condition and all cartridges were run on the ePlex instrument following packing insert instructions.

The results of the study which assessed the interim storage conditions are summarized in Table 25. All representative targets were detected in all replicates (20/20) in the control and the two test conditions (Open Pouch Stability Condition and In-Consumable Sample Stability Condition).

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**Table 25: Detection Rates for All Conditions Tested**

| Detection Rate | | | |
| --- | --- | --- | --- |
| Target | Control Condition | Open Pouch Stability Condition | In-Consumable Sample Stability Condition |
| *Enterococcus* | 100% | 100% | 100% |
| *Enterococcus faecium* | 100% | 100% | 100% |
| *Staphylococcus* | 100% | 100% | 100% |
| *Staphylococcus aureus* | 100% | 100% | 100% |
| *Streptococcus* | 100% | 100% | 100% |
| *Streptococcus pneumoniae* | 100% | 100% | 100% |
| Pan *Candida* | 100% | 100% | 100% |
| Pan Gram-Negative | 100% | 100% | 100% |
| vanA | 100% | 100% | 100% |

The mean signal, standard deviation (SD), and mean minus 2SD were calculated for each target and test condition (Table 26). Mean minus 2SD values were all well above the assigned cutoff for each target/condition.

**Table 26: Signals Remain Above Assay Cut-Offs in All Test Conditions**

| Target | Test Condition | Mean Signal (nA) | Std Dev (nA) | Mean minus 2SD (nA) | Target Cutoff (nA) |
| --- | --- | --- | --- | --- | --- |
| *Enterococcus* | Control | 313.80 | 70.76 | 172.29 | 20 |
| | In-Consumable Sample Stability Test | 297.93 | 85.45 | 127.02 | |
| | Open Pouch Stability Test | 329.48 | 43.90 | 241.68 | |
| *Enterococcus faecium* | Control | 561.66 | 89.84 | 381.98 | 20* |
| | In-Consumable Sample Stability Test | 539.41 | 113.24 | 312.92 | |
| | Open Pouch Stability Test | 574.44 | 74.49 | 425.46 | |
| *Staphylococcus* | Control | 704.29 | 109.10 | 486.09 | 20 |
| | In-Consumable Sample Stability Test | 694.93 | 119.17 | 456.59 | |
| | Open Pouch Stability Test | 679.21 | 129.69 | 419.83 | |
| *Staphylococcus aureus* | Control | 673.25 | 225.07 | 223.10 | 15 |
| | In-Consumable Sample Stability Test | 681.33 | 201.87 | 277.59 | |
| | Open Pouch Stability Test | 739.48 | 269.42 | 200.63 | |
| *Streptococcus* | Control | 682.97 | 187.04 | 308.88 | 30 |
| | In-Consumable Sample Stability Test | 652.05 | 202.44 | 247.17 | |

36

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| Target | Test Condition | Mean Signal (nA) | Std Dev (nA) | Mean minus 2SD (nA) | Target Cutoff (nA) |
| --- | --- | --- | --- | --- | --- |
| *Streptococcus pneumoniae* | Open Pouch Stability Test | 785.78 | 132.81 | 520.15 | 20 |
| | Control | 715.97 | 118.33 | 479.30 | |
| | In-Consumable Sample Stability Test | 682.68 | 132.63 | 417.41 | |
| | Open Pouch Stability Test | 757.41 | 90.83 | 575.76 | |
| *Pan Candida* | Control | 350.90 | 68.35 | 214.20 | 40 |
| | In-Consumable Sample Stability Test | 338.80 | 76.92 | 184.96 | |
| | Open Pouch Stability Test | 347.09 | 68.26 | 210.56 | |
| *Pan Gram-Negative* | Control | 261.41 | 88.85 | 83.70 | 40 |
| | In-Consumable Sample Stability Test | 248.32 | 50.92 | 146.49 | |
| | Open Pouch Stability Test | 296.43 | 66.03 | 164.37 | |
| *VanA* | Control | 935.81 | 102.70…