Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description focuses on PCR, microarray hybridization, and automated processing. There is no mention of AI or ML in the intended use, device description, or performance studies. The interpretation software appears to be rule-based rather than using AI/ML.

Therapeutic

No
This device is an in vitro diagnostic test designed to detect and identify potentially pathogenic gram-negative bacteria and resistance markers from blood cultures, aiding in the diagnosis of bacterial bloodstream infections. It does not directly treat or prevent a disease, which is characteristic of a therapeutic device.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the iC-GN Assay™ is "a qualitative, multiplexed, in vitro diagnostic test" and is used "to aid in the diagnosis of bacterial bloodstream infections."

SaMD (Software as a Medical Device)

No

The device description clearly outlines hardware components including the iC-Processor™, iC-Reader™, iC-Cassette™, and an iMac® computer, which are integral to the device's function. While software (iC-Report™) is used for interpretation and display, it is part of a larger system that includes physical hardware.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use/Indications for Use: The document explicitly states that the iC-GN Assay™ is a "qualitative, multiplexed, in vitro diagnostic test". It is intended for the detection and identification of potentially pathogenic gram negative bacteria and resistance markers directly from positive blood cultures to aid in the diagnosis of bacterial bloodstream infections. This clearly aligns with the definition of an in vitro diagnostic device, which is used to examine specimens taken from the human body to provide information for diagnosis, treatment, or prevention of disease.
Device Description: The description details a system that processes biological samples (positive blood cultures) using laboratory techniques (PCR, microarray hybridization) to produce results that are interpreted to aid in diagnosis. This is characteristic of an IVD.
Performance Studies: The document describes various performance studies (Method Comparison, Reproducibility, LoD, etc.) which are standard requirements for demonstrating the analytical and clinical performance of an IVD device for regulatory purposes.
Predicate Device: The mention of a predicate device (K132843; Nanosphere, Inc. Verigene® Gram Negative Blood Culture Nucleic Acid Test (GC-GN)) further confirms that this device is being compared to other legally marketed IVD devices.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The iCubate, Inc. iC-GN Assay™ for use on the iC-System™ is a qualitative, multiplexed, in vitro diagnostic test for the detection and identification of potentially pathogenic gram negative bacteria, which may cause bloodstream infection (BSI). The iC-GN Assay™ is performed directly on positive blood cultures, confirmed by Gram stain to contain gram negative bacilli. Cultures demonstrating mixed Gram stain results should not be tested on the assay. The iC-GN Assay™ is validated for use with select BACTEC™, BacT/ALERT® and VersaTREK® blood culture bottles. The iC-GN Assay™ is indicated for use in conjunction with other clinical and laboratory findings, such as culture, to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor bloodstream infections.

The iC-GN Assay™ detects target DNA and identifies the following:

Bacterial Genera and Species
Acinetobacter baumannii complex
Enterobacter cloacae complex
Escherichia coli
Klebsiella oxytoca
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus species
Serratia marcescens

Resistance Markers
KPC (blaKPC)- associated with resistance to carbapenems
NDM (blaNDM)- associated with resistance to carbapenems
CTX-M group 1(blaCTX-M group 1)- associated with resistance to extended spectrum beta-lactams

In mixed growth, the iC-GN Assay™ does not specifically attribute detection of KPC, NDM, or CTX-M group 1 to a specific genera or species.

Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing, identification of organisms not detected by the iC-GN Assay™, differentiation of mixed growth, association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing.

Product codes (comma separated list FDA assigned to the subject device)

PEN

Device Description

The iC-GN Assay™ utilizes polymerase chain reaction (PCR) for the multiplex amplification of specific targets and detects the amplified targets with microarray hybridization. Targets are detected directly from patient positive blood cultures confirmed by Gram stain to contain gram negative bacilli. The iC-GN Assay utilizes proprietary ARM-PCR (Amplicon Rescued Multiplex PCR) technology allowing for multiple targets to be amplified in one reaction. Testing is done in a self-contained, automated, disposable cassette using the iCubate™ processor (iC-Processor™). After the reaction is complete, the cassette is read on the iCubate® reader (iC-Reader™). Results from the iC-Reader™ are interpreted by iC-Report™ software and a final report is displayed on the iMac® computer.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

A method comparison study was performed at five (5) geographically dispersed clinical sites. Sites tested 1002 leftover de-identified specimens from anaerobic blood culture bottles flagged as positive by their respective continuous monitoring blood culture system. Three of the commonly used blood culture systems were included in the study: Thermo Fisher VersaTREK, BD BACTEC and BioMerieux BacT/ALERT.

Patient positive blood cultures confirmed by Gram stain to be positive for gram negative bacilli were enrolled in the study. Any positive blood cultures showing an initial mixed Gram stain were not enrolled or were subsequently withdrawn from the study dataset.

Final performance of the iC-GN Assay organism targets was compared to reference culture followed by MALDI identification per the study protocol. Final performance of the iC-GN Assay resistance marker targets was compared to PCR amplification followed by confirmatory bidirectional sequencing. Phenotypic antimicrobial susceptibility testing (AST) was also performed on all specimens to identify additional samples which required sequencing. Discordant samples were also sequenced.

To supplement performance of observed lower prevalence organisms, 170 contrived samples were prepared using verified strains. Contrived samples were prepared at iCubate using BD BACTEC Plus Aerobic Blood Culture Bottles with 10mL of human blood added (in accordance with BACTEC instructions). Organisms were spiked into bottles at a concentration of 5-30 CFU/bottle and incubated until bottles were flagged as positive. Aliquots of samples were frozen and provided to the sites (frozen) for testing.

Of the 1107 positive blood culture specimens enrolled in the study, a total of 105 specimens were excluded/withdrawn from the study and all subsequent performance analyses. Of the 1002 specimens remaining, 976 were fresh prospective specimens and 26 (2.6%) were frozen prospective specimens.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Reproducibility: Confirmed the site-to-site, operator-to-operator, system-to-system, and lot-to-lot reproducibility of the iC-GN Assay. A representative panel of target organisms and one non-target organism were evaluated at two clinically relevant concentrations: initial bottle positivity and eight hours beyond initial bottle positivity. Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added. Testing was performed by two independent operators at each of three sites, two external and one internal. Each operator tested the eighteen-organism panel in triplicate across five, non-consecutive days. Testing was performed on six iC-GN Cassette lots and multiple iC-Systems. Overall Reproducibility performance was 99.3%.

Limit of Detection (LoD): Determined the lowest concentration (CFU/mL) of analyte that can be detected approximately 95% of the time. For the eleven targets, a panel of twenty-seven representative strains were evaluated, a minimum of three per target. LoD testing was conducted in two phases. In phase II, the approximated 95% performance point determined in phase I was confirmed by testing a minimum of twenty replicates on each of three unique cassette lots. Plating and subsequent colony counts were used to determine organism concentrations.

Bottle Ring: Established the levels of each iC-GN target organism at two clinically relevant concentrations: initial bottle positivity (bottle "ring") and eight hours beyond initial positivity. Twenty-seven representative organisms were evaluated, a minimum of three per iC-GN target. Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System.

Blood Culture Bottle Equivalency: Evaluated commonly used blood culture bottle (BCB) media types to demonstrate that variability in BCB media composition does not interfere with iC-GN Assay performance. Twenty-seven (27) representative iC-GN target organisms plus one non-target organism were tested in thirteen (13) BCB media types. Target organisms were tested near LoD concentrations (2-3×LoD). Each strain was tested in triplicate in each BCB media type. Performance in all bottle types met the acceptance criteria of ≥ 95% performance.

Inclusivity: Demonstrated the inclusivity of the iC-GN Assay. Eighty-two (82) representative strains were evaluated, a minimum of ten strains for each target analyte. Strains were tested at the lowest level of bottle positivity, considered within two hours of bottle "ring." Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System. Each strain was tested in triplicate. Two strains were not detected: Acinetobacter calcoaceticus ATCC 31926 was not detected as A. baumannii complex, and Enterobacter kobei ATCC BAA-260 was not detected as E. cloacae complex. An in silico analysis was also performed for resistance markers.

Exclusivity: Demonstrated the exclusivity of the iC-GN Assay. A comprehensive panel of non-target organisms that may be encountered in positive blood cultures was evaluated. A total of 114 strains were tested. Potential cross-reactivity was evaluated by testing exclusivity panel organisms at the highest possible concentrations, considered eight hours beyond initial bottle positivity or the equivalent. Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System. Each strain was tested in triplicate. Three (3) strains demonstrated reproducible cross-reactivity with iC-GN Assay targets: Acinetobacter haemolyticus cross-reacted with Acinetobacter baumannii complex, Klebsiella variicola cross-reacted with Klebsiella pneumoniae, and Serratia odorifera cross-reacted with Serratia marcescens.

Microbial Interference: Evaluated potential microbial interference by testing high concentrations of gram negative exclusivity organisms in combination with low concentrations of iC-GN target organisms. Sixty (60) gram negative exclusivity strains were tested at the highest possible concentrations. Eight (8) representative iC-GN target organisms were tested at concentrations below the lowest levels of bottle positivity. Each organism combination was tested in triplicate.

Competitive Inhibition: Evaluated iC-GN Assay performance with combinations of target analytes that may be found in mixed positive blood cultures. One target organism was prepared at the lowest level of bottle positivity, while the second target organism was prepared at the highest possible concentration. All organisms were grown in BD BACTEC Plus Aerobic blood cultures bottles with human blood added on the BD BACTEC System. The organisms were combined at a ratio of one part "low" to four parts "high". Each low concentration organism was tested in combination with each high concentration organism in triplicate. All high concentration iC-GN targets were detected. Due to competitive inhibition, low concentration targets were not detected in 1.7% of tests (3/178). When iC-GN target organisms were present at similar concentrations, all targets were detected.

Interfering Substances: Evaluated iC-GN Assay performance in the presence of potentially inhibiting substances that may be encountered in blood culture media. Eight representative target organisms plus one non-target organism were evaluated. Organisms were tested at the lowest levels of bottle positivity. Potential interferents were tested at concentrations exceeding the highest concentrations that may be encountered in blood and blood culture media. Interference testing was performed in BD BACTEC Plus Aerobic blood culture bottle media, which has a sodium polyanetholesulfonate (SPS) concentration of 0.05% w/v. Additional SPS at a concentration greater than 0.05% w/v was found to interfere with the performance of some iC-GN Assay targets, resulting in increased false negative results and positive control check failures.

Method Comparison: A method comparison study was performed at five (5) geographically dispersed clinical sites. Sites tested 1002 leftover de-identified specimens from anaerobic blood culture bottles flagged as positive by their respective continuous monitoring blood culture system.
Key results from method comparison (Overall percent agreement for positive and negative cases):

Acinetobacter baumannii complex: Positive 100% (7/7), Negative 99.9% (994/995)
Enterobacter cloacae complex: Positive 95.0% (57/60), Negative 100% (942/942)
Escherichia coli: Positive 98.4% (486/494), Negative 100% (508/508)
Klebsiella oxytoca: Positive 95.8% (23/24), Negative 99.7% (975/978)
Klebsiella pneumoniae: Positive 96.8% (153/158), Negative 99.3% (838/844)
Proteus mirabilis: Positive 97.9% (46/47), Negative 99.5% (931/936)
Pseudomonas aeruginosa: Positive 95.2% (79/83), Negative 99.8% (917/919)
Serratia marcescens: Positive 100% (29/29), Negative 99.6% (969/973)
CTX-M: Positive 97.0% (65/67), Negative 99.9% (934/935)
KPC: Positive 100% (1/1), Negative 99.9% (1000/1001)
NDM: Positive 0/0, Negative 100% (1002/1002)

Analysis of Mixed Culture Results: In the method comparison study, there were thirty (30) mixed culture specimens that were detected by the iC-GN Assay, culture and MALDI, or both. There were twelve (12) discrepant mixed samples for which iC-GN detected a target that was not detected by the comparator assay. There were four (4) discrepant mixed samples for which the comparator assay detected targets that were not detected by iC-GN.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Not provided as specific metrics but percentage agreement for positive and negative results are shown in Summary of Performance Studies.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K132843

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.3365 Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures.

(a)
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.(b)
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).

Summary

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

June 28, 2019

iCubate, Inc. % Fran White President MDC Associates, LLC 180 Cabot Street Beverly, Massachusetts 01915

Re: K190341

Trade/Device Name: iC-GN iC-Cassette for use on the iC-System Regulation Number: 21 CFR 866.3365 Regulation Name: Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures Regulatory Class: Class II Product Code: PEN Dated: February 11, 2019 Received: February 14, 2019

Dear Fran White:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

1

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

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

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

for

Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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510(k) SUMMARY

Date of Summary: June 22, 2019

Product Name:

iC-GN Assay™ for use on the iC-System™

Sponsor:

iCubate, Inc. 601 Genome Way Huntsville, AL 35806

Correspondent:

MDC Associates, Inc. Fran White, President 180 Cabot Street Beverly, MA 01915 Phone: (978) 705 5011 Fax: (866) 540 3448 Email: regulatory@mdcassoc.com

Common Name:

Gram-Negative Bacteria and Associated Resistance Markers

Regulation Number:

866.3365

Classification:

PEN, Class II

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Substantial Equivalency

| Characteristic | iCubate, Inc.
iC-GN Assay™ for use on the iC-System™
(New Device) | Nanosphere, Inc.
Verigene® Gram Negative Blood
Culture Nucleic Acid Test (GC-GN)
K132843
(Predicate Device) | | | | |
|----------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--|--|--|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Similarities | | | | | | |
| Intended Use | The iCubate, Inc. iC-GN Assay™ for use on
the iC-System™ is a qualitative,
multiplexed, in vitro diagnostic test for the
detection and identification of potentially
pathogenic gram negative bacteria, which
may cause bloodstream infection (BSI). The
iC-GN Assay™ is performed directly on
positive blood cultures, confirmed by Gram
stain to contain gram negative bacilli.
Cultures demonstrating mixed Gram stain
results should not be tested on the assay.
The iC-GN Assay™ is validated for use with
select BACTEC™ , BacT/ALERT® and
VersaTREK® blood culture bottles. The iC-
GN Assay™ is indicated for use in
conjunction with other clinical and
laboratory findings, such as culture, to aid
in the diagnosis of bacterial bloodstream
infections; however, it is not used to
monitor bloodstream infections.

The iC-GN Assay™ detects target DNA and
identifies the following: | The Verigene® Gram Negative
Blood Culture Nucleic Acid Test (BC-
GN), performed using the sample-
to-results Verigene System, is a
qualitative multiplexed in vitro
diagnostic test for the simultaneous
detection and identification of
selected gram-negative bacteria
and resistance markers. BC-GN is
performed directly on blood culture
media using blood culture bottles
identified as positive by a
continuous monitor blood culture
system and which contain gram-
negative bacteria as determined by
Gram stain. BC-GN detects and
identifies the following:
Acinetobacter spp.
Citrobacter spp.
Enterobacter spp.
Proteus spp.
Escherichia coli
Klebsiella pneumoniae
Klebsiella oxytoca
Pseudomonas aeruginosa | | | | |
| | Bacterial Genera
and Species Resistance Markers Acinetobacter
baumannii
complex
Enterobacter
cloacae complex
Escherichia coli
Klebsiella oxytoca
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
Proteus species
Serratia
marcescens KPC (blaKPC)-
associated with
resistance to
carbapenems
NDM (blaNDM)-
associated with
resistance to
carbapenems
CTX-M group
1(blaCTX-M group 1)-
associated with
resistance to
extended spectrum
beta-lactams | | | | | Pseudomonas aeruginosa

BC-GN is indicated for use in
conjunction with other clinical and
laboratory findings to aid in the
diagnosis of bacterial bloodstream
infections; however, is not to be
used to monitor these infections.
Sub-culturing of positive blood
cultures is necessary to recover
organisms for susceptibility testing,
identification of organisms not
detected by BC-GN, differentiation
of mixed growth, association of
antimicrobial resistance marker
genes to a specific organism, or for
epidemiological typing. |
| | In mixed growth, the iC-GN Assay™ does
not specifically attribute detection of KPC,
NDM, or CTX-M group 1 to a specific
genera or species.

Sub-culturing of positive blood cultures is
necessary to recover organisms for | | | | | |

4

| Characteristic | iCubate, Inc.
iC-GN Assay™ for use on the iC-System™
(New Device) | Nanosphere, Inc.
Verigene® Gram Negative Blood
Culture Nucleic Acid Test (GC-GN)
K132843
(Predicate Device) |
|-------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------|
| | susceptibility testing, identification of
organisms not detected by the iC-GN
Assay™, differentiation of mixed growth,
association of antimicrobial resistance
marker genes to a specific organism, or for
epidemiological typing. | |
| Sample Type | Positive Blood Culture | Positive Blood Culture |
| Differences | | |
| INSTRUMENT
REQUIREMENTS | iC-System™ | Verigene System |
| TEST PRINCIPLE | ARM-PCR | Gold nanoparticle probe-based PCR |
| COMPATIBLE BLOOD
CULTURE BOTTLES | BD BACTEC Standard/10 Aerobic/F
BD BACTEC Standard/10 Anaerobic/F
BD BACTEC Plus Aerobic/F
BD BACTEC Plus Anaerobic/F
BD BACTEC Lytic/10 Anaerobic/F
BacT/Alert SA Standard Aerobic
BacT/Alert SN Standard Anaerobic
BacT/Alert FA Aerobic FAN
BacT/Alert FN Anaerobic FAN
BacT/Alert FA Plus Aerobic
BacT/Alert FN Plus Anaerobic
VersaTREK REDOX 1
VersaTREK REDOX 2 | BACTEC™ Plus Aerobic/F
BacT/ALERT FA FAN |
| THROUGHPUT | Four (4) samples/iC-Processor™ | One (1) Sample/Processor |

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Intended Use

The iCubate, Inc. iC-GN Assay™ for use on the iC-System™ is a qualitative, multiplexed, in vitro diagnostic test for the detection and identification of potentially pathogenic gram negative bacteria, which may cause bloodstream infection (BSI). The iC-GN Assay™ is performed directly on positive blood cultures, confirmed by Gram stain gram negative bacilli. Cultures demonstrating mixed Gram stain results should not be tested on the assay. The iC-GN Assay™ is validated for use with select BACTEC™, BacT/ALERT® and VersaTREK® blood culture bottles. The iC-GN Assay™ is indicated for use in conjunction with other clinical and laboratory findings, such as culture, to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor bloodstream infections.

The iC-GN Assay™ detects target DNA and identifies the following:

Bacterial Genera and Species	Resistance Markers
Acinetobacter baumannii complex	KPC (blaKPC)- associated with resistance to carbapenems
Enterobacter cloacae complex	NDM (blaNDM)- associated with resistance to carbapenems
Escherichia coli	CTX-M group 1(blaCTX-M group 1)- associated with resistance to extended spectrum beta-lactams
Klebsiella oxytoca
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus species
Serratia marcescens

In mixed growth, the iC-GN Assay™ does not specifically attribute detection of KPC, NDM, or CTX-M group 1 to a specific genera or species.

Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing, identification of organisms not detected by the iC-GN Assay™, differentiation of mixed growth, association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing.

Limitations

For prescription use only.

Please refer to the iC-GN Assay™ labeling for a more complete list of warnings, precautions and contraindications.

Methodology

The iC-GN Assay™ utilizes polymerase chain reaction (PCR) for the multiplex amplification of specific targets and detects the amplified targets with microarray hybridization. Targets are detected directly from patient positive blood cultures confirmed by Gram stain to contain gram negative bacilli. The iC-GN Assay utilizes proprietary ARM-PCR (Amplicon Rescued Multiplex PCR) technology allowing for multiple targets to be amplified in one reaction. Testing is done in a self-contained, automated, disposable cassette using the iCubate™ processor (iC-Processor™). After the reaction is complete, the cassette is read on the

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iCubate® reader (iC-Reader™). Results from the iC-Reader™ are interpreted by iC-Report™ software and a final report is displayed on the iMac® computer.

To operate, the user opens the iC-Cassette™ cap and pipettes an aliquot of the diluted positive blood culture sample into the sample/PCR well in the bottom well plate of the cassette. Once inoculated, the cassette cap is closed, and all extraction, amplification and detection processes are completed in the cassette, a closed system. Extraction, amplification and detection sequences are defined by an assay script controlled by the iC-Processor™.

The processing script is defined within a barcode label positioned on the top of each iC-Cassette™ which communicates with the iC-Processor™. To access and pierce the foilsealed reagent wells located in the bottom well plate of the cassette, the processor manipulates the cassette to move the cassette pipette horizontally and vertically. The script directs the transfer of reagents between the wells in the bottom well plate and finally to the array within the cassette. The iC-Processor™ is capable of processing four (4) iC-Cassettes™ with random access.

Once processing is complete, the cassette is manually transferred from the iC-Processor™ to the iC-Reader™ where the microarray within the cassette is read. The iC-Reader™ is capable of reading up to four (4) iC-Cassettes™ at one time. The results are interpreted via the iC-Report™ software and displayed for the user on the iMac®. Raw data and result interpretations are stored within the iMac®; raw data is accessible to iCubate® service personnel only and not to the end user.

When finished with a loaded iC-GN Cassette™, it should be disposed as biohazardous waste.

Performance Data

For ease of reference, the following table defines iC-GN target organisms and common acronyms used in the study descriptions.

TABLE 1: iC-GN Assay Targets
Target	Acronym
Acinetobacter baumannii complex	ABX
Enterobacter cloacae complex	ECX
Escherichia coli	EC
Klebsiella oxytoca	KO
Klebsiella pneumoniae	KPN
Proteus mirabilis	PM
Pseudomonas aeruginosa	PA
Serratia marcescens	SM
KPC carbapenemase resistance marker	KPC
NDM carbapenemase resistance marker	NDM
CTX-M group 1 extended spectrum β-lactamase resistance marker	CTXM

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Reproducibility

To confirm the site-to-site, operator-to-operator, system-to-system, and lot-to-lot reproducibility of the iC-GN Assay, a representative panel of target organisms and one nontarget organism were evaluated at two clinically relevant concentrations: initial bottle positivity and eight hours beyond initial bottle positivity. Organisms were grown to the appropriate concentrations in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System. Testing was performed by two independent operators at each of three sites, two external and one internal. Each operator tested the eighteen-organism panel in triplicate across five, non-consecutive days. Testing was performed on six iC-GN Cassette lots and multiple iC-Systems. Performance is based on all expected targets detected and no false positive targets detected. Table 2 below summarizes Reproducibility results stratified by iC-GN target and concentration. Overall Reproducibility performance was 99.3%, confirming that iC-GN Assay performance is reproducible across sites, operators, systems and lots.

TABLE 2: iC-GN Assay Reproducibility Performance by Target
Target/Concentration	Overall
Performance	Overall
Performance %
[95% CI]	False
Negatives	False
Positives	PC Check
Failures	System
Failures
A. baumannii complex
Bottle Ring	90/90	100.0
[95.91-100.0]	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)
A. baumannii complex
Bottle Ring + 8 hours	87/90	96.7
[90.65-98.86]	0/90
(0.00%)	3/90
(3.33%)	0/90
(0.00%)	0/90
(0.00%)
E. cloacae complex
Bottle Ring	86/88	97.7
[92.09-99.37]	1/88
(1.14%)	1/88
(1.14%)	2/90
(2.22%)	0/90
(0.00%)
E. cloacae complex
Bottle Ring + 8 hours	90/90	100.0
[95.91-100.0]	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)
E. coli
Bottle Ring	90/90	100.0
[95.91-100.0]	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)
E. coli
Bottle Ring + 8 hours	89/89	100.0
[95.86-100.0]	0/89
(0.00%)	0/89
(0.00%)	0/90
(0.00%)	1/90
(1.11%)
K. oxytoca
Bottle Ring	89/90	98.9
[93.97-99.80]	0/90
(0.00%)	1/90
(1.11%)	0/90
(0.00%)	0/90
(0.00%)
K. oxytoca
Bottle Ring + 8 hours	89/89	100.0
[95.86-100.0]	0/89
(0.00%)	0/89
(0.00%)	1/90
(1.11%)	0/90
(0.00%)
K. pneumoniae
Bottle Ring	90/90	100.0
[95.91-100.0]	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)	0/90
(0.00%)
K. pneumoniae
Bottle Ring + 8 hours	89/89	100.0
[95.86-100.0]	0/89
(0.00%)	0/89
(0.00%)	1/90
(1.11%)	0/90
(0.00%)
Proteus species
Bottle Ring	89/89	100.0
[95.86-100.0]	0/89
(0.00%)	0/89
(0.00%)	1/90
(1.11%)	0/90
(0.00%)
Proteus species
Bottle Ring + 8 hours	88/88	100.0
[95.92-100.0]	0/88
(0.00%)	0/88
(0.00%)	0/90
(0.00%)	2/90
(2.22%)

8

TABLE 2: iC-GN Assay Reproducibility Performance by Target
Target/Concentration	Overall
Performance	Overall
Performance %
[95% CI]	False
Negatives	False
Positives	PC Check
Failures	System
Failures
P. aeruginosa	88/89	98.9	1/89	0/89	1/90	0/90
Bottle Ring		[93.91-99.80]	(1.12%)	(0.00%)	(1.11%)	(0.00%)
P. aeruginosa	89/90	98.9	1/90	0/90	0/90	0/90
Bottle Ring + 8 hours		[93.97-99.80]	(1.11%)	(0.00%)	(0.00%)	(0.00%)
S. marcescens	87/89	97.8	0/89	2/89	1/90	0/90
Bottle Ring		[92.17-99.38]	(0.00%)	(2.25%)	(1.11%)	(0.00%)
S. marcescens	87/89	97.8	0/89	2/89	1/90	0/90
Bottle Ring + 8 hours		[92.17-99.38]	(0.00%)	(2.25%)	(1.11%)	(0.00%)
CTX-M group 1	90/90	100.0	0/90	0/90	0/90	0/90
Bottle Ring		[95.91-100.0]	(0.00%)	(0.00%)	(0.00%)	(0.00%)
CTX-M group 1	89/89	100.0	0/89	0/89	0/90	1/90
Bottle Ring + 8 hours		[95.86-100.0]	(0.00%)	(0.00%)	(0.00%)	(1.11%)
KPC	90/90	100.0	0/90	0/90	0/90	0/90
Bottle Ring		[95.91-100.0]	(0.00%)	(0.00%)	(0.00%)	(0.00%)
KPC	89/89	100.0	0/89	0/89	1/90	0/90
Bottle Ring + 8 hours		[95.86-100.0]	(0.00%)	(0.00%)	(1.11%)	(0.00%)
NDM	89/89	100.0	0/89	0/89	1/90	0/90
Bottle Ring		[95.86-100.0]	(0.00%)	(0.00%)	(1.11%)	(0.00%)
NDM	89/90	98.9	1/90	0/90	0/90	0/90
Bottle Ring + 8 hours		[93.97-99.80]	(1.11%)	(0.00%)	(0.00%)	(0.00%)

Limit of Detection (LoD)

A study was performed to determine | A. baumannii complex | CDC-83 | A. baumannii complex | ATCC 23055 | TABLE 3: iC-GN Assay LoD Results | | Target | Strain | E. cloacae complex | Z101 | | CDC-164 | | ATCC 700323 | E. coli | ATCC 43895 | | ATCC BAA-2326 | | CDC-55 | K. oxytoca | Z115 | | ATCC 13182 | | CDC-147 | K. pneumoniae | ATCC 35657 | | CDC-40 | | CDC-42 | | KPC-2 | Proteus species | LACNY 11 | | Z050 | | CDC-59 | | Z028 | P. aeruginosa | Z129 | | Z139 | | CDC-231 | | CDC-250 | S. marcescens | ATCC 43297 | | ATCC 21212 | | CDC-91 | CTX-M group 1 | | CDC-40 (CTX-M-15) | | CDC-42 (CTX-M-15) | KPC | CDC-147 (KPC-3) | | KPC-2 | | CDC-231 (KPC-5) | NDM | CDC-83 (NDM-1) | | CDC-55 (NDM-1) | | CDC-250 (NDM-1) the limit of detection for each iC-GN Assay target, defined as the lowest concentration (CFU/mL) of analyte that can be detected approximately 95% of the time. For the eleven targets detected by the iC-GN Assay, a panel of twenty-seven representative strains were evaluated, a minimum of three per target. For complex and genus level targets, at least two representative species were evaluated. LoD testing was conducted in two phases, the first to narrow the range for LoD analysis. In phase II, the approximated 95% performance point determined in phase I was confirmed by testing a minimum of twenty replicates on each of three unique cassette lots. Plating and subsequent colony counts were used to determine organism concentrations. The final limit of detection for each target, provided in Table 3 below, was defined as the concentration that produced a positive result ≥ 95% but A. baumannii complex | 307-0294 | $5.3 \times 10^5$ | |
| $5.2 \times 10^6$ | $5.3 \times 10^5 - 5.2 \times 10^6$ |
| $9.0 \times 10^5$ | |
| | |
| Concentration (CFU/mL) | Defined Target LoD (CFU/mL) |
| $5.0 × 10^6$ | $4.9 × 10^5-5.5 × 10^6$ |
| $5.5 × 10^6$ | |
| $4.9 × 10^5$ | |
| $7.7 × 10^5$ | |
| $7.9 × 10^5$ | $7.7 × 10^5-8.4 × 10^5$ |
| $8.4 × 10^5$ | |
| $6.2 × 10^5$ | |
| $5.4 × 10^5$ | $5.4 × 10^5-1.1 × 10^6$ |
| $1.1 × 10^6$ | |
| $1.9 × 10^6$ | |
| $3.6 × 10^6$ | |
| $1.9 × 10^6$ | $6.0 × 10^5-4.2 × 10^6$ |
| $4.2 × 10^6$ | |
| $6.0 × 10^5$ | |
| $1.1 × 10^6$ | |
| $9.9 × 10^5$ | $6.9 × 10^5-1.1 × 10^6$ |
| $7.6 × 10^5$ | |
| $6.9 × 10^5$ | |
| $1.2 × 10^6$ | $5.0 × 10^5-1.2 × 10^6$ |
| $5.0 × 10^5$ | |
| $6.9 × 10^5$ | |
| $7.2 × 10^5$ | |
| $8.1 × 10^5$ | $6.4 × 10^5-8.1 × 10^5$ |
| $6.4 × 10^5$ | |
| ATCC BAA-2326 (CTX-M-15) | $7.9 × 10^5$ | |
| $2.3 × 10^6$ | $7.9 × 10^5-2.3 × 10^6$ |
| $1.9 × 10^6$ | |
| $2.3 × 10^6$ | |
| $4.2 × 10^6$ | $1.5 × 10^5-4.2 × 10^6$ |
| $1.5 × 10^5$ | |
| $5.2 × 10^6$ | |
| $4.0 × 10^6$ | $3.3 × 10^5-5.2 × 10^6$ |
| $3.3 × 10^5$ | |

9

Bottle Ring

A study was performed to establish the levels of each iC-GN target organism at two clinically relevant concentrations: initial bottle positivity (bottle "ring") and eight hours beyond initial positivity. Twenty-seven representative organisms were evaluated, a minimum of three per iC-GN target. Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System. Within two hours of initial bottle positivity, the bottles were removed for plating and subsequent colony counts to determine organism concentrations. The bottles were then returned to the incubator and approximately eight hours after initial bottle positivity, the bottles were again removed for plating and subsequent colony counts to determine organism concentrations. Three bottles were grown for each

10

strain, and the average concentrations at initial bottle positivity and eight hours beyond initial bottle positivity are provided in Table 4 below. The concentrations at initial bottle positivity, representative of the lowest levels that may be observed in a clinical setting, are above the limits of detection determined for each strain.

TABLE 4: iC-GN Target Organism Concentrations at Bottle “Ring”
Organism	Strain ID	Initial Bottle Positivity
Average Concentration
(CFU/mL)	Bottle Positivity + 8
Average Concentration
(CFU/mL)
Acinetobacter baumannii	307-0294	4.24 × 108	8.27 × 108
Acinetobacter baumannii	CDC-83	3.39 × 108	7.23 × 108
Acinetobacter
calcoaceticus	ATCC 23055	6.78 × 107	2.93 × 108
Enterobacter cloacae	Z101	2.17 × 108	1.97 × 109
Enterobacter cloacae	CDC-164	5.62 × 108	2.31 × 109
Enterobacter hormaechei	ATCC 700323	4.36 × 108	2.75 × 109
Escherichia coli	ATCC 43895	1.50 × 108	9.48 × 108
Escherichia coli	ATCC BAA-2326	6.23 × 108	1.52 × 109
Escherichia coli	CDC-55	4.93 × 108	1.51 × 109
Klebsiella oxytoca	Z115	5.32 × 108	2.07 × 109
Klebsiella oxytoca	ATCC 13182	4.16 × 108	4.52 × 109
Klebsiella oxytoca	CDC-147	9.67 × 108	1.31 × 109
Klebsiella pneumoniae	ATCC 35657	9.78 × 108	1.08 × 109
Klebsiella pneumoniae	CDC-40	2.16 × 108	1.36 × 109
Klebsiella pneumoniae	CDC-42	2.55 × 108	1.10 × 109
Klebsiella pneumoniae	KPC-2	7.70 × 108	1.66 × 109
Klebsiella pneumoniae	LACNY 11	5.43 × 107	1.67 × 109
Proteus mirabilis	Z050	1.71 × 108	7.40 × 108
Proteus mirabilis	CDC-59	7.37 × 107	8.10 × 108
Proteus penneri	Z028	8.88 × 107	4.33 × 108
Proteus vulgaris	Z129	4.37 × 107	5.00 × 108
Pseudomonas aeruginosa	Z139	9.18 × 107	1.37 × 1010
Pseudomonas aeruginosa	CDC-231	3.26 × 108	7.98 × 108
Pseudomonas aeruginosa	CDC-250	1.64 × 108	8.97 × 108
Serratia marcescens	ATCC 43297	8.55 × 108	2.03 × 109
Serratia marcescens	ATCC 21212	1.07 × 108	8.83 × 108
Serratia marcescens	CDC-91	7.28 × 108	1.67 × 109

Blood Culture Bottle Equivalency

Commonly used blood culture bottle (BCB) media types were evaluated to demonstrate that variability in BCB media composition does not interfere with iC-GN Assay performance. Twenty-seven (27) representative iC-GN target organisms plus one non-target organism were tested in thirteen (13) BCB media types. Target organisms were tested near LoD

11

concentrations (2-3×LoD). Each strain was tested in triplicate in each BCB media type. Target performance is based on all expected targets detected and no false positive targets detected. Non-target performance is based on all expected negative results. In the event of a false negative result, the strain was retested in replicates of ten. In the event of a false positive result or other failure, the strain was retested in triplicate. The results of iC-GN BCB equivalency testing are summarized in Table 5 below. Performance in all bottle types met the acceptance criteria of ≥ 95% performance; all bottle types are validated for use with the iC-GN Assay.

TABLE 5: iC-GN Assay BCB Equivalency Results
BCB Media Type	Overall
Performance
(%)	False
Negatives
(%)	False
Positives
(%)	PC
Check
Failures
(%)	System
Failures
(%)
BACTEC Standard Aerobic	93/94
(98.9%)	1/94
(1.1%)	0/94
(0.0%)	3/97
(3.1%)	0/97
(0.0%)
BACTEC Standard Anaerobic	85/86
(98.8%)	0/86
(0.0%)	1/86
(1.2%)	0/87
(0.0%)	1/87
(1.1%)
BACTEC Plus Aerobic	93/94
(98.9%)	1/94
(1.1%)	0/94
(0.0%)	2/97
(2.1%)	1/97
(1.1%)
BACTEC Plus Anaerobic	95/96
(98.6%)	1/96
(1.0%)	0/96
(0.0%)	2/100
(2.0%)	2/100
(2.0%)
BACTEC Lytic/10 Anaerobic	81/81
(100.0%)	0/81
(0.0%)	0/81
(0.0%)	0/81
(0.0%)	0/81
(0.0%)
BACT/ALERT SA Standard Aerobic	97/99
(98.0%)	1/99
(1.0%)	1/99
(1.0%)	4/103
(3.9%)	0/103
(0.0%)
BACT/ALERT SN Standard
Anaerobic	87/88
(98.9%)	0/88
(0.0%)	1/88
(1.1%)	2/90
(2.2%)	0/90
(0.0%)
BACT/ALERT FA Aerobic FAN	94/96
(97.9%)	0/96
(0.0%)	2/96
(2.1%)	1/97
(1.0%)	0/97
(0.0%)
BACT/ALERT FN Anaerobic FAN	92/94
(97.9%)	0/94
(0.0%)	2/94
(2.1%)	2/97
(2.1%)	1/97
(1.0%)
BACT/ALERT FA Plus Aerobic	94/95
(98.9%)	1/95
(1.1%)	0/95
(0.0%)	1/97
(1.1%)	1/97
(1.1%)
BACT/ALERT FN Plus Anaerobic	87/87
(100.0%)	0/87
(0.0%)	0/87
(0.0%)	2/90
(2.2%)	1/90
(1.1%)
VersaTREK REDOX 1	81/81
(100.0%)	0/81
(0.0%)	0/81
(0.0%)	0/81
(0.0%)	0/81
(0.0%)
VersaTREK REDOX 1	92/93
(98.9%)	1/93
(1.1%)	0/93
(0.0%)	1/94
(1.1%)	0/94
(0.0%)

An increased rate of false positive Proteus results was observed in some lots of BD BACTEC blood culture bottles. The high rate of false positive results observed prompted an

12

investigation by the manufacturer, BD Life Sciences. The false positives are due to the presence of nucleic acids or non-viable organisms present in the culture media at concentrations near or above the target's limit of detection. While the observed contamination was resolved at the time of publication, positive Proteus results observed in BD BACTEC media types should be confirmed using alternative methods.

Inclusivity

To demonstrate the inclusivity of the iC-GN Assay, eighty-two (82) representative strains were evaluated, a minimum of ten strains for each target analyte. Strains were tested at the lowest level of bottle positivity, considered within two hours of bottle "ring." Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System. Each strain was tested in triplicate. Performance is based on all expected targets detected and no false positive targets detected. In the event of a false negative result, the strain was retested in replicates of ten. In the event of a false positive result or other failure, the strain was retested in triplicate. The results of iC-GN Inclusivity testing are summarized in Table 6 below. Two strains were not detected by the iC-GN Assay: Acinetobacter calcoaceticus ATCC 31926 was not detected as A. baumannii complex and Enterobacter kobei ATCC BAA-260 was not detected as E. cloacae complex. An in silico analysis was also performed, and the predicted reactivity of each resistance marker detected by the iC-GN Assay is summarized in Tables 7-9 below.

TABLE 6: iC-GN Assay Inclusivity Results
Organism	Strain	Targets	Performance
Acinetobacter baumannii	ATCC 19606	ABX	5/5
Acinetobacter baumannii	NCIMB 12457	ABX	3/3
Acinetobacter baumannii	CDC-36	ABX	3/3
Acinetobacter baumannii	CDC-37	ABX, NDM-1	3/3
Acinetobacter baumannii	CDC-45	ABX	3/3
Acinetobacter baumannii	CDC-52	ABX	3/3
Acinetobacter baumannii	CDC-56	ABX	3/3
Acinetobacter baumannii	CDC-88	ABX, NDM-1	3/3
Acinetobacter baumannii	CDC-101	ABX	3/3
Acinetobacter calcoaceticus	ATCC 14987	ABX	3/3
Acinetobacter calcoaceticus	ATCC 31926	ABX	2/111
Enterobacter cloacae	ATCC BAA-1143	ECX	3/3
Enterobacter cloacae	ATCC BAA-2341	ECX, KPC	3/3
Enterobacter cloacae	NCTC 10005	ECX	14/162
Enterobacter cloacae	NCTC 13464	ECX	3/3
Enterobacter cloacae	CDC-32	ECX, KPC-3	3/3
Enterobacter cloacae	CDC-38	ECX, CTX-M-15, NDM-1	3/3
Enterobacter cloacae	CDC-65	ECX	3/3
Enterobacter cloacae	CDC-163	ECX, CTX-M-15, KPC-2	3/3
Enterobacter asburiae	ATCC 35923	ECX	3/3
Enterobacter hormaechei	ATCC 49162	ECX	3/3
TABLE 6: iC-GN Assay Inclusivity Results
Organism	Strain	Targets	Performance
Enterobacter kobei	ATCC BAA-260	ECX	0/133
Escherichia coli	ATCC 10536	EC	3/3
Escherichia coli	ATCC BAA-2469	EC, NDM-1	3/3
Escherichia coli	NCTC 9001	EC	3/3
Escherichia coli	NCTC 10538	EC	5/5
Escherichia coli	NCTC 13476	EC	3/3
Escherichia coli	CDC-48	EC, CTX-M-15, NDM-1	3/3
Escherichia coli	CDC-61	EC, KPC-3	3/3
Escherichia coli	CDC-104	EC, KPC-4	7/84
Escherichia coli	CDC-119	EC, CTX-M-15, NDM-1	3/3
Escherichia coli	CDC-162	EC, CTX-M-15, NDM-7	3/3
Klebsiella oxytoca	ATCC 8724	KO	3/3
Klebsiella oxytoca	ATCC 43086	KO	3/3
Klebsiella oxytoca	ATCC 43165	KO	3/3
Klebsiella oxytoca	ATCC 43863	KO	3/3
Klebsiella oxytoca	ATCC 49134	KO	3/3
Klebsiella oxytoca	ATCC 49334	KO	3/3
Klebsiella oxytoca	ATCC 51817	KO	3/3
Klebsiella oxytoca	ATCC 700324	KO	3/3
Klebsiella oxytoca	NCTC 11686	KO	3/3
Klebsiella oxytoca	CDC-71	KO	3/3
Klebsiella pneumoniae	ATCC-13882	KPN	3/3
Klebsiella pneumoniae	ATCC BAA-1705	KPN, KPC-2	3/3
Klebsiella pneumoniae	NCTC 9633	KPN	3/3
Klebsiella pneumoniae	NCTC 13438	KPN, KPC-3	3/3
Klebsiella pneumoniae	NCTC 13443	KPN, CTX-M-15, NDM-1	3/3
Klebsiella pneumoniae	CDC-44	KPN, CTX-M-15	3/3
Klebsiella pneumoniae	CDC-46	KPN, CTX-M-15	5/5
Klebsiella pneumoniae	CDC-49	KPN, CTX-M-15, NDM-1	3/3
Klebsiella pneumoniae	CDC-66	KPN, CTX-M-15	3/3
Klebsiella pneumoniae subsp.
ozaenae	ATCC 11296	KPN	3/3
Proteus mirabilis	ATCC 7002	Proteus	3/3
Proteus mirabilis	ATCC 21100	Proteus	3/3
Proteus mirabilis	ATCC 43071	Proteus	3/3
Proteus mirabilis	NCIMB 13283	Proteus	3/3
Proteus mirabilis	CDC-155	Proteus, KPC-6	3/3
Proteus mirabilis	CDC-156	Proteus, KPC-2	3/3
Proteus mirabilis	CDC-159	Proteus, NDM-1	3/3
Proteus penneri	ATCC 33519	Proteus	3/3
Proteus vulgaris	ATCC 9484	Proteus	3/3
Proteus vulgaris	ATCC 29905	Proteus	3/3
Pseudomonas aeruginosa	ATCC 10145	PA	3/3
Pseudomonas aeruginosa	ATCC 19429	PA	3/3
TABLE 6: iC-GN Assay Inclusivity Results
Organism	Strain	Targets	Performance
Pseudomonas aeruginosa	ATCC BAA-1744	PA	3/3
Pseudomonas aeruginosa	CDC-54	PA	3/3
Pseudomonas aeruginosa	CDC-64	PA	3/3
Pseudomonas aeruginosa	CDC-90	PA, KPC-5	3/3
Pseudomonas aeruginosa	CDC-94	PA	3/3
Pseudomonas aeruginosa	CDC-105	PA	3/3
Pseudomonas aeruginosa	CDC-108	PA	3/3
Pseudomonas aeruginosa	CDC-246	PA, NDM-1	5/5
Serratia marcescens	ATCC 8100	SM	3/3
Serratia marcescens	ATCC 13880	SM	3/3
Serratia marcescens	ATCC 14041	SM	3/3
Serratia marcescens	ATCC 14756	SM	3/3
Serratia marcescens	ATCC 29634	SM	3/3
Serratia marcescens	ATCC 29635	SM	3/3
Serratia marcescens	ATCC 43861	SM	3/3
Serratia marcescens	ATCC 43862	SM	3/3
Serratia marcescens	NCTC 9743	SM	3/3
Serratia marcescens	CDC-99	SM	3/3

13

14

1. 2/2 false negative ABX in initial testing. 7/9 false negative ABX in repeat testing. See limitation.
1. 1/3 false positive ABX in initial testing. 1/3 false positive ABX in repeated in replicates of 10, 10/10 repeats passed.
1. 3/3 false negative ECX in initial testing. 10/10 false negative ECX in repeat testing. See limitation.
1/3 processor error in initial testing. 1/3 false positive KPN in repeat testing. Strain repeated in 4) triplicate, 3/3 repeats passed.

TABLE 7: Predicted (in silico) Reactivity for CTX-M group 1
Associated Target Organism	Variant Detected	Associated Target Organism	Variant Detected
Acinetobacter baumannii complex	CTX-M-3	Klebsiella oxytoca	CTX-M-3
	CTX-M-15		CTX-M-15
	CTX-M-1		CTX-M-35
	CTX-M-3		CTX-M-36
	CTX-M-15		CTX-M-162
Enterobacter cloacae complex	CTX-M-22	Klebsiella pneumoniae	CTX-M-1
	CTX-M-37		CTX-M-3
	CTX-M-55		CTX-M-15
	CTX-M-167		CTX-M-22
	CTX-M-177		CTX-M-28
	CTX-M-187		CTX-M-32
	CTX-M-224		CTX-M-54
	Escherichia coli		CTX-M-1
		CTX-M-2
		CTX-M-3
		CTX-M-4
		CTX-M-5
		CTX-M-6
		CTX-M-7
TABLE 7: Predicted (in silico) Reactivity for CTX-M group 1
Associated Target Organism	Variant Detected	Associated Target Organism	Variant Detected
	CTX-M-8		CTX-M-133
	CTX-M-9		CTX-M-135
	CTX-M-10		CTX-M-138
	CTX-M-11		CTX-M-139
	CTX-M-12		CTX-M-173
	CTX-M-15		CTX-M-176
	CTX-M-28		CTX-M-183
	CTX-M-29		CTX-M-188
	CTX-M-32		CTX-M-197
	CTX-M-33		CTX-M-204
	CTX-M-36		CTX-M-208
	CTX-M-42		CTX-M-210
	CTX-M-55		CTX-M-220
	CTX-M-58		CTX-M-15
	CTX-M-69		CTX-M-66
	CTX-M-71		CTX-M-116
	CTX-M-79	Proteus species	CTX-M-136
	CTX-M-82		CTX-M-164
	CTX-M-90		CTX-M-167
	CTX-M-101		CTX-M-212
	CTX-M-102		CTX-M-1
	CTX-M-103	Pseudomonas aeruginosa	CTX-M-15
	CTX-M-109		CTX-M-32
	CTX-M-117		CTX-M-3
	CTX-M-120		CTX-M-15
	CTX-M-125	Serratia marcescens	CTX-M-22
	CTX-M-127		CTX-M-55
	CTX-M-128		CTX-M-221
	CTX-M-131
	CTX-M-132
	CTX-M-134
	CTX-M-137
	CTX-M-138
	CTX-M-139
	CTX-M-140
	CTX-M-141
	CTX-M-142
	CTX-M-143
	CTX-M-146
	CTX-M-158
	CTX-M-163
	CTX-M-166
	CTX-M-167
	CTX-M-170
	CTX-M-172
	CTX-M-175
	CTX-M-178
	CTX-M-179
	CTX-M-180
	CTX-M-181
	CTX-M-182
TABLE 7: Predicted (in silico) Reactivity for CTX-M group 1
Associated Target Organism	Variant Detected	Associated Target Organism	Variant Detected
	CTX-M-186
	CTX-M-188
	CTX-M-189
	CTX-M-193
	CTX-M-194
	CTX-M-202
	CTX-M-203
	CTX-M-207
	CTX-M-211
	CTX-M-216
	CTX-M-218
	CTX-M-222
	CTX-M-226

15

16

TABLE 8: Predicted (in silico) Reactivity for KPC
Associated Target Organism	Variant Detected	Associated Target Organism	Variant Detected
Acinetobacter baumannii
complex	KPC-2		KPC-1
	KPC-3		KPC-2
	KPC-10		KPC-3
Enterobacter cloacae
complex	KPC-1	Klebsiella pneumoniae	KPC-4
	KPC-2		KPC-5
	KPC-3		KPC-6
	KPC-4		KPC-7
	KPC-13		KPC-8
	KPC-18
	KPC-47
Escherichia coli	KPC-2
	KPC-3
	KPC-12
	KPC-18
	KPC-20
	KPC-21
	KPC-28
Klebsiella oxytoca	KPC-2
	KPC-3
Proteus species	KPC-1
	KPC-2
Pseudomonas aeruginosa	KPC-2
	KPC-5
Serratia marcescens	KPC-2
			KPC-34
			KPC-35
			KPC-36
			KPC-37
			KPC-38
			KPC-39
			KPC-42
			KPC-43
			KPC-59

17

TABLE 9: Predicted (in silico) Reactivity for NDM
Associated Target Organism	Variant Detected	Associated Target Organism	Variant Detected
Acinetobacter baumannii
complex	NDM-1	Klebsiella oxytoca	NDM-1
	NDM-2		NDM-3
	NDM-3		NDM-4
	NDM-4
	NDM-5	Klebsiella pneumoniae	NDM-1
	NDM-7		NDM-3
	NDM-14		NDM-4
Enterobacter cloacae
complex	NDM-1		NDM-5
	NDM-4		NDM-6
	NDM-5		NDM-7
	NDM-7		NDM-9
	NDM-22	NDM-10
		NDM-16
Escherichia coli	NDM-1	Proteus species	NDM-23
	NDM-2		NDM-28
	NDM-3	NDM-1	Pseudomonas aeruginosa
	NDM-4
	NDM-5	NDM-1
	NDM-6	NDM-5	Serratia marcescens
	NDM-7
	NDM-9	NDM-1
	NDM-11	NDM-4
	NDM-12	NDM-12
	NDM-13
	NDM-15
	NDM-16
	NDM-17
	NDM-18
NDM-19
NDM-20
NDM-21
NDM-27

Exclusivity

To demonstrate the exclusivity of the iC-GN Assay, a comprehensive panel of non-target organisms that may be encountered in positive blood cultures was evaluated. A total of 114 strains were tested including organisms phylogenetically related to iC-GN target organisms as well as common blood culture contaminants. Potential cross-reactivity was evaluated by testing exclusivity panel organisms at the highest possible concentrations, considered eight hours beyond initial bottle positivity or the equivalent. Organisms were grown in BD BACTEC Plus Aerobic blood culture bottles with human blood added on the BD BACTEC System. Each strain was tested in triplicate. Performance is based on the observation of all expected negative results. In the event of a false positive result or other failure, the organism was retested in replicates of three (3) or ten (10). Exclusivity results are presented in Table 10 below. Three (3) strains demonstrated reproducible cross-reactivity with iC-GN Assay targets: Acinetobacter haemolyticus cross-reacted with Acinetobacter baumannii complex, Klebsiella

18

variicola cross-reacted with Klebsiella pneumoniae, and Serratia odorifera cross-reacted with Serratia marcescens.

TABLE 10: iC-GN Assay Exclusivity Results
Organism	Strain	Concentration
(CFU/mL)	Performance
Acinetobacter haemolyticus	ATCC 19002	$7.20 \times 10^8$	0/31
Acinetobacter Iwoffi	Z141	$2.45 \times 10^8$	3/3
Acinetobacter radioresistens	ATCC 43998	$5.20 \times 10^7$	3/3
Acinetobacter schindleri	ATCC BAA618	$3.50 \times 10^8$	3/3
Acinetobacter ursingii	ATCC BAA617	$3.80 \times 10^8$	3/3
Aerococcus viridans	Z219	$2.24 \times 10^7$	3/3
Aeromonas hydrophila	Z161	$8.10 \times 10^8$	3/3
Alcaligenes faecalis	Z218	$9.70 \times 10^8$	3/3
Aspergillus niger	Z105	$1.62 \times 10^8$	3/3
Bacillus cereus	Z091	ND	3/3
Bacteroides fragilis	Z029	$8.40 \times 10^9$	3/3
Brevundimonas vesicularis	ATCC 11426	$3.80 \times 10^8$	5/5
Burkholderia cepacia	ATCC 25416	$5.40 \times 10^8$	3/3
Campylobacter coli	Z293	$3.90 \times 10^8$	3/3
Campylobacter jejuni	Z086	$4.60 \times 10^8$	11/132
Candida albicans	Z006	ND	3/3
Candida glabrata	Z007	$3.20 \times 10^7$	3/3
Candida krusei	Z009	$1.90 \times 10^7$	3/3
Candida parapsilosis	Z011	$9.00 \times 10^6$	3/3
Candida tropicalis	Z012	$3.50 \times 10^7$	4/4
Cedecea davisae	ATCC 33431	$6.20 \times 10^8$	3/3
Citrobacter amalonaticus	Z051	$8.4 \times 10^8$	3/3
Citrobacter braakii	ATCC 51113	$4.90 \times 10^8$	3/3
Citrobacter freundii	Z064	$2.25 \times 10^8$	3/3
Citrobacter koseri	Z039	$1.14 \times 10^9$	3/3
Citrobacter sedlakii	ATCC 51115	$9.80 \times 10^8$	2/2
Clostridium difficile (NAP-1 toxigenic)	NAP1	$4.87 \times 10^7$	4/4
Clostridium difficile (non-toxigenic)	Z228	$5.93 \times 10^7$	3/3
Clostridium novyi*	Z179	$1.14 \times 10^7$	5/5
Corynebacterium amycolatum	Z284	$9.26 \times 10^8$	3/3
Corynebacterium genitalium	Z328	$1.35 \times 10^8$	3/3
Corynebacterium jeikeium	Z232	$8.50 \times 10^8$	4/4
Corynebacterium striatum	MCW000	$2.07 \times 10^9$	5/63
Cronobacter muytjensii	ATCC 51329	$2.79 \times 10^8$	3/3
Cronobacter sakazakii	ATCC 29544	$6.90 \times 10^8$	3/3
Cryptococcus neoformans	Serotype A	$2.15 \times 10^8$	3/3
Edwardsiella tarda	Z183	$8.70 \times 10^7$	4/54
Enterobacter aerogenes	Z052	$1.77 \times 10^9$	5/5
Enterobacter amnigenus	ATCC 51816	$7.50 \times 10^8$	3/3
Enterococcus avium	Z171	$2.58 \times 10^8$	5/65
TABLE 10: iC-GN Assay Exclusivity Results
Organism	Strain	Concentration
(CFU/mL)	Performance
Enterococcus casseliflavus	Z002	$2.44 \times 10^9$	4/4
Enterococcus cecorum	Z208	$1.03 \times 10^9$	5/6
Enterococcus faecalis	ATCC 51299	$2.13 \times 10^9$	3/3
Enterococcus faecium	ATCC 700221	$7.20 \times 10^8$	3/3
Enterococcus gallinarum	Z209	$1.35 \times 10^9$	3/3
Enterococcus hirae	Z193	$2.37 \times 10^8$	3/3
Enterococcus raffinosus	ATCC 49427	$5.40 \times 10^8$	3/3
Escherichia fergusonii	ATCC 35469	$8.70 \times 10^8$	3/3
Escherichia hermannii	Z184	$1.01 \times 10^9$	5/5
Escherichia vulneris	ATCC 33821	$7.50 \times 10^8$	3/3
Fusobacterium varium	Z361	$2.49 \times 10^9$	3/3
Hafnia alvei	ATCC 51815	$1.37 \times 10^9$	3/3
Haemophilus influenzae	ATCC 10211	$3.09 \times 10^9$	3/3
Haemophilus parainfluenzae	ATCC 9796	$1.33 \times 10^8$	3/3
Klebsiella variicola	ATCC 31488	$4.40 \times 10^8$	0/3 7
Kluyvera ascorbata (KPC+)	CDC-0144	$1.40 \times 10^9$	3/3
Kocuria kristinae	Z250	$7.20 \times 10^7$	3/3
Kytococcus schroeteri	ATCC
BAA2410	$1.50 \times 10^7$	3/3
Lactobacillus acidophilus	Z048	$6.00 \times 10^8$	3/3
Lactobacillus plantarum	17-5	$5.30 \times 10^8$	3/3
Lactobacillus reuteri	Z333	$5.80 \times 10^7$	5/5
Lactococcus lactis	Z169	$9.30 \times 10^7$	3/3
Leclercia adecarboxylata	ATCC 23216	$1.01 \times 10^9$	3/3
Leminorella grimontii	Z364	$4.00 \times 10^9$	3/3
Leuconostoc mesenteroides	Z197	$4.00 \times 10^7$	5/5
Listeria monocytogenes	ATCC 19115	$2.03 \times 10^9$	3/3
Micrococcus luteus	Z100	$1.80 \times 10^8$	3/3
Moraxella catarrhalis	ATCC 25238	$1.27 \times 10^9$	3/3
Morganella morganii	ATCC 25830	$1.23 \times 10^9$	3/3
Neisseria gonorrhoeae	ATCC 19424	ND	3/3
Neisseria lactamica	ATCC 23970	$2.90 \times 10^8$	3/3
Neisseria meningitidis	Serotype A	$2.55 \times 10^8$	5/5
Neisseria mucosa	ATCC 49233	$5.80 \times 10^8$	4/4
Neisseria sicca	ATCC 9913	$1.43 \times 10^8$	3/3
Pantaea agglomerans	ATCC 27155	$2.00 \times 10^6$	3/3
Pasturella multocida	ATCC 12945	$2.84 \times 10^9$	2/2
Pediococcus pentosaceus	Z226	$1.91 \times 10^8$	3/3
Planococcus citreus	ATCC 14404	$1.95 \times 10^8$	3/3
Pluralibacter gergoviae	ATCC 33028	$1.27 \times 10^9$	3/3
Propionibacterium acnes	Z144	$7.90 \times 10^8$	5/5
Providencia alcalifaciens	Z292	$3.10 \times 10^9$	3/3
Providencia rettgeri	Z370	$2.20 \times 10^9$	3/3
Providencia stuartii	Z212	$1.70 \times 10^9$	2/2
TABLE 10: iC-GN Assay Exclusivity Results
Organism	Strain	Concentration
(CFU/mL)	Performance
Pseudomonas fluorescens	ATCC 13525	2.43 x 108	3/3
Pseudomonas luteola	ATCC 43273	1.09 × 108	3/3
Pseudomonas mendocina	ATCC 25411	1.23 x 109	3/3
Pseudomonas nitroreducens	ATCC 33634	5.30 × 108	3/3
Pseudomonas oryzihabitans	ATCC 43272	1.70 x 107	4/58
Pseudomonas putida	Z030	3.30 x 108	3/3
Pseudomonas stutzeri	ATCC 17588	6.20 x 108	3/3
Raoultella planitcola	ATCC 33558	1.25 x 109	3/3
Rothia mucilaginosus	Z033	5.50 × 107	3/3
Salmonella enterica	ATCC
BAA1715	2.23 x 109	3/3
Serratia fonticola	ATCC 29844	1.18 x 109	3/3
Serratia liquefaciens	ATCC 27592	1.24 x 109	11/129
Serratia odorifera	ATCC 33077	2.19 × 109	11/1310
Serratia rubidaea	ATCC 19278	1.54 x 108	5/5
Staphylococcus aureus	ATCC 700699	4.30 x 107	3/3
Staphylococcus capitis	Z192	2.13 x 108	3/3
Staphylococcus epidermidis	ATCC 700566	5.90 x 107	3/3
Staphylococcus haemolyticus	Z067	2.70 x 107	5/5
Staphylococcus hominis	Z031	9.90 × 107	3/3
Staphylococcus intermedius	Z112	3.30 x 107	3/3
Staphylococcus lugdunensis	Z097	2.43 x 108	3/3
Staphylococcus schleiferi	Z294	2.52 x 109	3/3
Stenotrophomonas maltophilia	ATCC BAA84	1.74 x 109	5/5
Streptococcus agalactiae	Z019	5.80 × 108	3/3
Streptococcus anginosus	Z179	9.50 × 108	3/3
Streptococcus bovis	Z167	8.00 × 108	3/3
Streptococcus dysgalactiae	Z068	2.65 x 108	3/3
Streptococcus intermedius	Z126	1.40 x 107	5/611
Streptococcus pneumoniae	ATCC 6301	3.80 × 108	5/612
Streptococcus pyogenes	Z018	4.80 x 107	3/3
Veillonella parvula	Z379	6.70 x 109	5/613

19

20

3/3 false positive A. baumannii complex in initial testing. See limitation.

2/3 false positive E. coli in initial testing. 10/10 repeats negative. 2)
1/3 false positive S. marcescens in initial testing. 3/3 repeats negative. 3)
1. 1/3 positive control check failure in initial testing. 1/3 false positive S. marcescens in repeat testing.
1. 1/3 false positive S. marcescens in initial testing. 3/3 repeats negative.
1. 1/3 false positive E. coli in initial testing. 3/3 repeats negative.
1. 3/3 false positive K. pneumoniae in initial testing. See limitation.
1/3 false positive S. marcescens in initial testing. 2/2 repeats negative. 8)
1/3 false positive S. marcescens in initial testing. 9/9 repeats negative. ತಿ)
1. 1/3 false positive S. marcescens in initial testing. 1/10 false positive S. marcescens in repeat testing. See limitation.
1. 1/3 false positive S. marcescens in initial testing. 3/3 repeats negative.

21

1. 1/3 false positive S. marcescens in initial testing. 3/3 repeats negative.
1. 1/3 false positive E. coli in initial testing. 3/3 repeats negative.

Microbial Interference

Potential microbial interference was evaluated by testing high concentrations of gram negative exclusivity organisms in combination with low concentrations of iC-GN target organisms. A total of sixty (60) gram negative exclusivity strains were tested at the highest possible concentrations, considered eight hours beyond initial bottle positivity or the equivalent. Eight (8) representative iC-GN target organisms were tested at concentrations below the lowest levels of bottle positivity. Each organism combination was tested in triplicate. Performance was based on all expected targets detected and no false positive targets detected. In the event of a false negative result, the combination was retested in replicates of ten (10). In the event of a false positive result or other failure, the combination was retested in replicates of three (3) or ten (10). Microbial interference results are presented in Table 11 below.

Organism	ABX	ECX	EC	KO	KPN	PM	PA	SM	CTX-M-15	KPC-2	NDM-1
A. Iwoffi	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
A. radioresistens	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
A. schindleri	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
A. ursingii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
A. hydrophila	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
A. faecalis	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
B. fragilis	3/3	3/3	3/3	3/3	3/3	4/51	3/3	3/3	3/3	3/3	3/3
B. vesicularis	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
B. cepacia	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. coli	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. jejuni	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. davisae	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. amalonaticus	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. braakii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. freundii	3/3	3/3	3/3	3/3	3/3	2/2	3/3	3/3	3/3	3/3	3/3
C. koseri	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. sedlakii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. muytjensii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
C. sakazakii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
E. tarda	3/3	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	5/5
E. aerogenes	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
E. amnigenus	3/3	3/3	5/5	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3
E. fergusonii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
F. hermannii	3/3	3/3	5/5	3/3	13/13	3/3	3/3	3/3	5/5	12/132	3/3
TABLE 11: iC-GN Assay Microbial Interference Results
Organism	ABX	ECX	EC	KO	KPN	PM	PA	SM	CTX-M-15	KPC-2	NDM-1
E. vulneris	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
F. varium	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
H. alvei	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
H. influenzae	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
H.
parainfluenzae	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
K. ascorbata	10/133	3/3	11/124	3/3	3/3	3/3	3/3	3/3	10/124	3/3	3/3
L. adecarboxylata	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
L. grimontii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
M. catarrhalis	12/135	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
M. morganii	3/3	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	5/5
N. gonorrhoeae	5/5	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
N. lactamica	3/3	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	5/5
N. meningitidis	3/3	3/3	5/5	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3
N. mucosa	5/5	3/3	5/5	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3
N. sicca	5/66	3/3	3/3	5/5	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. agglomerans	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. multocida	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. gergoviae	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. alcalifaciens	5/5	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. rettgeri	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. stuartii	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. fluorescens	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. luteola	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. mendocina	3/3	3/3	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3
P. nitroreducens	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
P. oryzihabitans	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	5/5	3/3
P. putida	3/3	3/3	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	5/5
P. stutzeri	3/3	5/5	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	5/5
R. planitcola	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
S. enterica	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
S. fonticola	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
S. liquefaciens	3/3	5/5	3/3	3/3	3/3	3/3	3/3	3/3	3/3	13/13	3/3
S. odorifera	3/3	3/3	3/3	12/138	3/3	3/3	3/3	3/3	3/3	3/3	3/3
S. rubidaea	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
S. maltophilia	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
V. parvula	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3

22

1/3 array registration error in initial testing. 1/3 false positive E. cloacae complex in repeat testing.
1/3 false negative KPC in initial testing. 10/10 repeats passed.
3/3 false negative A. baumannii complex in initial testing; concentration was determined to be below the target limit of detection. 10/10 repeats passed.

23

1. 1/2 false negative CTX-M in initial testing. 1/10 false positive K. pneumoniae in repeat testing.
1. 1/3 false negative A. baumannii complex in initial testing. 10/10 repeats passed.
ର) 1/3 false positive E. coli in initial testing. 3/3 repeats passed.
1. 1/3 false negative K. pneumoniae in initial testing. 10/10 repeats passed.
1. 1/3 false positive S. marcescens in initial testing. 10/10 repeats passed.

Competitive Inhibition

iC-GN Assay performance was evaluated with combinations of target analytes that may be found in mixed positive blood cultures. One target organism was prepared at the lowest level of bottle positivity, considered within two hours of bottle "ring", while the second target organism was prepared at the highest possible concentration, considered eight hours after initial bottle positivity. All organisms were grown in BD BACTEC Plus Aerobic blood cultures bottles with human blood added on the BD BACTEC System. The organisms were combined at a ratio of one part "low" to four parts "high". Each low concentration organism was tested in combination with each high concentration organism in triplicate. Performance was based on all expected targets detected. In the event of a false negative result, the organism combination was retested in replicates of ten (10) at the same "low" and "high" organism ratio. In the event of a reproducible false negative result, the organism combination was retested in replicates of ten (10) at a ratio of one part "low" to one part "high." Competitive inhibition results are presented in Table 12 below. All high concentration iC-GN targets were detected. Due to competitive inhibition, low concentration targets were not detected in 1.7% of tests (3/178). When iC-GN target organisms were present at similar concentrations, all targets were detected.

TABLE 12: iC-GN Assay Competitive Inhibition Performance
		Target Performance
Low	High Organism	Low	Low	High	High
Organism		Organism	Marker	Organism	Marker
	ECX	3/3	NA	3/3	NA
	EC (CTX-M-15+)	3/3	NA	3/3	3/3
	KO	3/3	NA	3/3	NA
A. baumannii	KPN (KPC-2+)	3/3	NA	3/3	3/3
	PM	3/3	NA	3/3	NA
	PA (NDM-1+)	3/3	NA	3/3	3/3
	SM	3/3	NA	3/3	NA
E. cloacae	ABX	3/3	NA	3/3	1 FP KPC
	EC (CTX-M-15+)	3/3	NA	3/3	3/3
	KO	3/3	NA	3/3	NA
	KPN (KPC-2+)	3/3	NA	3/3	3/3
	PM	3/3	NA	3/3	NA
	PA (NDM-1+)	3/3	NA	3/3	3/3
	SM	3/3	NA	3/3	NA
E. coli	ABX	3/3	3/3	3/3	NA
TABLE 12: iC-GN Assay Competitive Inhibition Performance		Target Performance
Low
Organism	High Organism	Low
Organism	Low
Marker	High
Organism	High
Marker
(CTX-M-15+)	ECX	3/3	3/3	3/3	NA
(CTX-M-15+)	KO	3/3	3/3	3/3	NA
(CTX-M-15+)	KPN (KPC-2+)	3/3	3/3	3/3	3/3
(CTX-M-15+)	PM	3/3	3/3	3/3	NA
(CTX-M-15+)	PA (NDM-1+)	3/3	3/3	3/3	3/3
(CTX-M-15+)	SM	3/3	3/3	3/3	NA
(CTX-M-15+)	ABX	3/3	NA	3/3	NA
K. oxytoca	ECX	3/3	NA	3/3	NA
	EC (CTX-M-15+)	3/3	NA	3/3	3/3
	KPN (KPC-2+)	3/3	NA	3/3	3/3
	PM	3/3	NA	3/3	NA
	PA (NDM-1+)	3/3	NA	3/3	3/3
	SM	3/3	3/3	3/3	NA
	ABX	3/3	3/3	3/3	NA
K.
pneumoniae
(KPC-2+)	ECX	3/3	3/3	3/3	NA
	EC (CTX-M-15+)	3/3	3/3	3/3	3/3
	KO	3/3	3/3	3/3	NA
	PM	3/3	3/3	3/3	NA
	PA (NDM-1+)	3/3	3/3	3/3	3/3
	SM	3/3	3/3	3/3	NA
	ABX	3/3	NA	3/3	NA
P. mirabilis	ECX	3/3	NA	3/3	NA
	EC (CTX-M-15+)	3/3	NA	3/3	3/3
	KO	5/5	NA	5/5	NA
	KPN (KPC-2+)	3/3	NA	3/3	3/3
	PA (NDM-1+)	3/3	NA	3/3	3/3
	SM	3/3	NA	3/3	NA
	ABX	3/3	3/3	3/3	NA
P. aeruginosa
(NDM-1+)	ECX	3/3	3/3	3/3	NA
	EC (CTX-M-15+)	12/13	11/13	13/13	13/13
	EC (CTX-M-15+) 1:1	10/10	10/10	10/10	10/10
	KO	3/3	3/3	3/3	NA
	KPN (KPC-2+)	3/3	3/3	3/3	3/3
	PM	3/3	3/3	3/3	NA
	SM	3/3	3/3	3/3	NA
S. marcescens	ABX	3/3	NA	3/3	NA
	ECX	3/3	NA	3/3	NA
	EC (CTX-M-15+)	3/3	NA	3/3	3/3
S. marcescens	KO	3/3	NA	3/3	NA
	TABLE 12: iC-GN Assay Competitive Inhibition Performance
	Target Performance
Low
Organism	High Organism	Low
Organism	Low
Marker	High
Organism	High
Marker
	KPN (KPC-2+)	3/3	NA	3/3	3/3
	PM	3/3	NA	3/3	NA
	PA (NDM-1+)	3/3	NA	3/3	3/3

24

25

Interfering Substances

iC-GN Assay performance was evaluated in the presence of potentially inhibiting substances that may be encountered in blood culture media. Eight representative target organisms plus one non-target organism were evaluated. Organisms were tested at the lowest levels of bottle positivity, considered within two hours of bottle "ring." Potential interferents were tested at concentrations exceeding the highest concentrations that may be encountered in blood and blood culture media (Table 12). Target performance is based on all expected targets detected and no false positive targets detected. Non-target performance is based on all negative results. In the event of a false negative result, the organism/interferent combination was retested in replicates of ten (10). In the event of a false positive result or other failure, the organism/interferent combination was retested in triplicate. If the discordant result was observed in repeat testing, the combination was retested at a decreased inhibitor concentration. Interference results are presented in Table 13 below. Interference testing was performed in BD BACTEC Plus Aerobic blood culture bottle media, which has a sodium polyanetholesulfonate (SPS) concentration of 0.05% w/v. Additional SPS at a concentration greater than 0.05% w/v was found to interfere with the performance of some iC-GN Assay targets, resulting in increased false negative results and positive control check failures.

TABLE 13: Interfering Substances Test Panel
Interference Compound	Clinically Relevant
Concentration	Test
Concentration
Hemoglobin	1-2 g/L	10 g/L
Conjugated Bilirubin	0.1-0.4 mg/dL	10 mg/dL
Unconjugated Bilirubin	0.1-0.8 mg/dL	10 mg/dL
Protein (γ-globulin + albumin)	0.7-1.7 g/dL	4 g/dL
Triglyceride	300-500 mg/dL	1500 mg/dL
Human Genomic DNA	NA	1 × 106 cells/mL
Sodium Polyanetholesulfonate (SPS)	0.02-0.05% w/v	0.1% w/v
Cefepime	16 µg/mL	80 µg/mL

26

TABLE 13: Interfering Substances Test Panel
Interference Compound	Clinically Relevant
Concentration	Test
Concentration
Ceftriaxone	16 µg/mL	80 µg/mL
Fluconazole	25 µg/mL	100 µg/mL
Gentamicin	20 µg/mL	80 µg/mL
Meropenem	16 µg/mL	80 µg/mL
Piperacillin	32 µg/mL	160 µg/mL
Vancomycin	20 µg/mL	100 µg/mL

TABLE 14: iC-GN Assay Interfering Substances Performance
	Target Performance
Interference
Compound	ABX	ECX	EC	KO	KPN	PM	PA	SM	SE	KPC
-2	NDM-
1	CTX-M
-15
Hemoglobin	3/3	3/3	3/3	3/3	3/3	3/3	14/14	3/3	5/5	3/3	14/14	3/3
Conjugated
Bilirubin	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Unconjugated
Bilirubin	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Protein (γ-
globulin +
albumin)	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Triglyceride	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Human Genomic
DNA	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
SPS (0.1%)	3/121	3/3	3/3	3/3	3/3	3/3	2/122	3/3	4/4	3/3	8/123	3/3
SPS (0.5%)	3/3	--	--	--	--	--	3/3	--	--	--	3/3	--
Cefepime	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Ceftriaxone	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Fluconazole	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Gentamicin	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
Meropenem	3/3	3/3	5/5	3/3	3/3	5/64	3/3	3/3	3/3	3/3	3/3	5/5
Piperacillin	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3

27

TABLE 14: iC-GN Assay Interfering Substances Performance
	Target Performance
Interference
Compound	ABX	ECX	EC	KO	KPN	PM	PA	SM	SE	KPC
-2	NDM-
1	CTX-M
-15
Vancomycin	3/3	3/3	3/3	3/3	5/5	3/3	3/3	3/3	3/3	5/5	3/3	3/3

1. 2/3 false negative A. baumannii complex in initial testing. 7/9 false negative A. baumannii complex in repeat testing.
1. 3/3 false negative P. aeruginosa in initial testing. 7/9 false negative P. aeruginosa in repeat testing.
1. 4/9 false negative NDM in repeat testing.
1. 1/3 false positive K. pneumoniae in initial testing. 3/3 repeats passed.

Method Comparison

A method comparison study was performed at five (5) geographically dispersed clinical sites. Sites tested 1002 leftover de-identified specimens from anaerobic blood culture bottles flagged as positive by their respective continuous monitoring blood culture system. Three of the commonly used blood culture systems were included in the study: Thermo Fisher VersaTREK, BD BACTEC and BioMerieux BacT/ALERT.

Patient positive blood cultures confirmed by Gram stain to be positive for gram negative bacilli were enrolled in the study. Any positive blood cultures showing an initial mixed Gram stain were not enrolled or were subsequently withdrawn from the study dataset.

Final performance of the iC-GN Assay organism targets was compared to reference culture followed by MALDI identification per the study protocol. Final performance of the iC-GN Assay resistance marker targets was compared to PCR amplification followed by confirmatory bidirectional sequencing. Phenotypic antimicrobial susceptibility testing (AST) was also performed on all specimens to identify additional samples which required sequencing. Discordant samples were also sequenced.

To supplement performance of observed lower prevalence organisms, 170 contrived samples were prepared using verified strains. Contrived samples were prepared at iCubate using BD BACTEC Plus Aerobic Blood Culture Bottles with 10mL of human blood added (in accordance with BACTEC instructions). Organisms were spiked into bottles at a concentration of 5-30 CFU/bottle and incubated until bottles were flagged as positive. Aliquots of samples were frozen and provided to the sites (frozen) for testing.

Of the 1107 positive blood culture specimens enrolled in the study, a total of 105 specimens were excluded/withdrawn from the study and all subsequent performance analyses. Of the 1002 specimens remaining, 976 were fresh prospective specimens and 26 (2.6%) were frozen prospective specimens.

The total specimens excluded from the iC-GN Assay Method Comparison Study (n=105) are listed by site and reason for exclusion in the table below. The most common reasons for exclusion included incomplete reference testing and repeat iC-GN errors.

28

| Site Code | Unresolved
iC-GN Error | Incomplete
Reference
Method | Outside
Fresh Stability
Window | Didn't Meet
Inclusion
Criteria | Total Withdrawn |
|-----------|---------------------------|-----------------------------------|--------------------------------------|--------------------------------------|-----------------|
| LAC | 2 | 26 | 0 | 0 | 28 |
| IU | 0 | 10 | 0 | 0 | 10 |
| MCW | 2 | 23 | 0 | 2 | 27 |
| TC | 4 | 25 | 5 | 0 | 34 |
| TGH | 1 | 3 | 1 | 1 | 6 |
| Total | 9* | 87 | 6 | 3 | 105 |

Table 15: Withdrawn Summary

*Please Note: the nine (9) samples that were excluded from performance analysis due to Unresolved iC-GN are included in the calculation of instrument errors; please refer to Table 17: No-Calls

Gender Demographics

Gender was reported when available for all clinical samples collected for the study. of the 1002 clinical samples included in performance analysis 47.3% were males and 52.6% were female; gender was not provided for 1/1002. The table below summarizes this data.

Site	MALES		FEMALES		Not Provided		Clinical Samples
	#	%	#	%	#	%
LAC	156	43.9%	199	56.1%	0	-	355
IU	41	43.6%	53	56.4%	0	-	94
MCW	109	58.0%	78	41.5%	1	0.5%	188
TC	71	45.5%	85	54.5%	0	-	156
TGH	97	46.4%	112	53.6%	0	-	209
Total	474	47.3%	527	52.6%	1	0.1%	1002

Table 16: Gender Stratification

Error Rate

Throughout the course of the study, an initial error rate of 2.9 % (34/1181) was observed. Reasons for error included the following: Positive controls check failure (27), Array registration error (6), and Processor/System error (1). When an error was observed, repeat testing was performed with the iC-GN Assay per the protocol. Upon repeat testing, the error rate was reduced to 0.8% (9/1181).

Table 17: NO-Calls
Internal Positive Control Failure		Instrument Errors		Total Non-Reportable Rate
Initial	Final	Initial	Final	Initial	Final
#fail/#total	#fail/#total	#fail/#total	#fail/#total	#fail/#total	#fail/#total
(95% CI)	(95% CI)	(95% CI)	(95% CI)	(95% CI)	(95% CI)
2.3%	0.8%	0.6%	-	2.9%	0.8%
27/1181	9/1181	7/1181	0/1181	34/1181	9/1181
[1.6-3.3%]	[0.4-1.4%]	[0.3-1.2%]	[0.0-0.3%]	[2.1-4.0%]	[0.4-1.4%]

Table 17: No-Calls

29

When performance of the iC-GN Assay was compared to reference culture followed by MALDI identification or PCR/bi-directional sequencing, there was no apparent difference in performance noted between the five study sites or between the three blood culture systems. Performance for all positive bottle types/systems combined is presented in the tables below for detection of the iC-GN Assay targets as compared to culture and MALDI or PCR/bidirectional sequencing. Results are stratified by prospectively tested fresh specimens, prospectively collected/retrospectively tested frozen specimens and contrived specimens.

| Table 18: iC-GN Assay Performance: Acinetobacter

baumannii complex (ppa)
Specimen
Type	N=	Percent Agreement	Comparator
Method
Prospective		Positive
(95% CI)	Negative
(95% CI)
	Fresh	976	100%
7/7
(64.6-100)	99.9%
968-969**
(99.4-100)	Culture &
MALDI
	Frozen	26	-
0/0

```
          | 100%
```

26/26
(87.1-100) | |
| | TOTAL | 1002 | 100%
7/7
(64.6-100) | 99.9%
994/995
(99.4-100) | |
| Contrived | | 170 | 100%
45/45
(92.1-100) | 100%
125/125
(97.0-100) | |

**1/1 false positive observed was negative for A. baumannii complex by PCR/bi-directional sequencing

Table 20: iC-GN Assay Performance: Escherichia coli (uidA)
Specimen
Type	N=	Percent Agreement	Comparator
Method
		Positive
(95% CI)	Negative
(95% CI)
Prospective	Fresh	976	98.4%
480/488*
(96.8-99.2)	100%
488/488
(99.2-100)	Culture &
MALDI
	Frozen	26	100%
6/6
(61.0-100)	100%
20/20
(83.9-100)
	TOTAL	1002	98.4%
486/494
(96.8-99.2)	100%
508/508
(99.2-100)
Contrived	170	100%
15/15
(79.6-100)	100%
155/155
(97.6-100)

*4/8 false negatives observed were negative for E. coli by PCR/bidirectional sequencing; 3/8 were positive for E. coli by PCR/bidirectional sequencing; 1/8 was not available for sequencing

Table 19: iC-GN Assay Performance: Enterobacter cloacae complex (ramA)

| Specimen
Type | N= | Percent Agreement | | Comparator
Method | |
|------------------|--------|-----------------------------|--------------------------------|-------------------------------|--------------------|
| | | Positive
(95% CI) | Negative
(95% CI) | | |
| Prospective | Fresh | 976 | 94.5%
52/55*
(85.1-98.1) | 100%
921/921
(99.6-100) | Culture &
MALDI |
| | Frozen | 26 | 100%
5/5
(56.6-100) | 100%
21/21
(84.5-100) | |
| | TOTAL | 1002 | 95.0%
57/60
(86.3-98.3) | 100%
942/942
(99.6-100) | |
| Contrived | 170 | 100%
17/17
(81.6-100) | 100%
153/153
(97.6-100) | | |

*1/3 false negatives observed was negative for E. cloacae complex by PCR/bi-directional sequencing; 2/3 were positive for E. cloacae complex by PCR/bi-directional sequencing

Table 21: iC-GN Assay Performance: Klebsiella oxytoca

| | Specimen
Type | N= | Percent Agreement | | Comparator
Method |
|-------------|------------------|------|--------------------------------|-----------------------------------|----------------------|
| | | | Positive
(95% CI) | Negative
(95% CI) | |
| Prospective | Fresh | 976 | 95.8%
23/24*
(79.8-99.3) | 99.7%
949/952**
(99.1-99.9) | Culture &
MALDI |
| | Frozen | 26 | -
0/0

```
             | 100%
```

26/26
(87.1-100) | |
| | TOTAL | 1002 | 95.8%
23/24
(79.8-99.3) | 99.7%
975/978
(99.1-99.9) | |
| | Contrived | 170 | 100%
30/30
(88.6-100) | 100%
140/140
(97.3-100) | |

*1/1 false negative observed was negative for K. oxytoca by PCR/bi-directional sequencing

**3/3 false positives observed were negative for K. oxytoca by PCR/bi-directional sequencinq

30

| Table 22: iC-GN Assay Performance: Klebsiella pneumoniae

(parC)
Specimen Type	N=	Percent Agreement
Positive
(95% CI)	Negative
(95% CI)	Comparator Method
Prospective	Fresh	976	96.8%
150/155*
(92.7-98.6)	99.3%
815/821**
(98.4-99.7)
	Frozen	26	100%
3/3
(43.9-100)	100%
23/23
(85.7-100)	Culture &
MALDI
	TOTAL	1002	96.8%
153/158
(92.8-98.6)	99.3%
838/844
(98.4-99.7)
Contrived		170	100%
21/21
(84.5-100)	99.3%
148/149
(96.3-99.9)

*3/5 false neqatives observed were negative for K. pneumoniae by PCR/bi-directional sequencing; 2/3 were positive for K. pneumoniae by PCR/bi-directional sequencing

**6/6 false positives observed were neqative for K. pneumoniae by PCR/bi-directional sequencing

| Table 24: iC-GN Assay Performance: Pseudomonas

aeruginosa (algD)
	Specimen
Type	N=	Percent Agreement		Comparat
or
Method
			Positive
(95% CI)	Negative
(95% CI)
Prospective	Fresh	976	95.1%
78/82*
(88.1-98.1)	99.8%
892/894**
(99.2-99.9)	Culture &
MALDI
	Frozen	26	100%
1/1
(20.7-100)	100%
25/25
(86.7-100)
	TOTAL	1002	95.2%
79/83
(88.3-98.1)	99.8%
917/919
(99.2-99.9)
Contrived		170	100%
10/10
(72.2-100)	100%
160/160
(97.7-100)

*4/4 false negatives observed were positive for P. aeruginosa by PCR/bi-directional sequencing

**2/2 false positives observed were negative for P. aeruginosa by PCR/bi-directional sequencing

Nineteen (19) samples were excluded from Proteus mirabilis performance analysis due to confirmed Proteus contamination within the BD BACTEC Bottles leaving a total of 983 evaluable specimens.

| Table 23: iC-GN Assay Performance: Proteus

mirabilis (rpoB)
	Specimen
Type	N=	Percent Agreement		Comparator
Method
			Positive
(95% CI)	Negative
(95% CI)
	Fresh	957	97.4%
37/38*
(86.5-99.5)	99.5%
914/919**
(98.7-99.8)
Prospective	Frozen	26	100%
9/9
(70.1-100)	100%
17/17
(81.6-100)	Culture &
MALDI
	TOTAL	983	97.9%
46/47
(88.9-99.6)	99.5%
931/936
(98.8-99.8)
Contrived		170	100%
12/12
(75.8-100)	100%
158/158
(97.6-100)

*1/1 false neqative observed was positive for P. mirabilis by PCR/bi-directional sequencinq

**3/5 false positives observed were negative for P. mirabilis by PCR/bi-directional sequencing; 2/5 were not available for sequencing

marcescens (gyrB)
	Specimen
Type	N=	Percent Agreement		Comparator
Method
			Positive
(95% CI)	Negative
(95% CI)
Prospective	Fresh	976	100%
29/29
(88.3-100)	99.6%
943/947**
(98.9-99.8)	Culture &
MALDI
	Frozen	26	-
0/0

```
          | 100%
```

26/26
(87.1-100) | |
| | TOTAL | 1002 | 100%
29/29
(88.3-100) | 99.6%
969/973
(98.9-99.8) | |
| | Contrived | 170 | 100%
20/20
(83.9-100) | 99.3%
149/150
(96.3-99.9) | |

**1/4 false positives observed was positive for S. marcescens by PCR/bi-directional sequencing; 3/4 were negative for S. marcescens by PCR/bi-directional sequencing

Table 25: iC-GN Assay Performance: Serratia cescens (avrB)

31

Table 26: iC-GN Assay Performance: CTX-M
Specimen Type	N=	Percent Agreement		Comparator Method
		Positive (95% CI)	Negative (95% CI)
Prospective	Fresh	976	97.0%
64/66
(89.6-99.2)	99.9%
909/910
(99.4-100)	PCR/Bi-directional sequencing
	Frozen	26	100%
1/1
(20.7-100)	100%
25/25
(86.7-100)
	TOTAL	1002	97.0%
65/67
(89.8-99.2)	99.9%
934/935
(99.4-100)
Contrived	170	100%
15/15
(79.6-100)	100%
155/155
(97.6-100)

Table 27: iC-GN Assay Performance: KPC
Specimen
Type	N=	Percent Agreement	Comparator
Method
		Positive
(95% CI)	Negative
(95% CI)
Prospective	Fresh	976	100%
1/1
(20.7-100)	99.9%
974/975
(99.4-100)	PCR/Bi-
directional
sequencing
	Frozen	26	-
0/0

```
          | 100%
```

26/26
(87.1-100) | |
| | TOTAL | 1002 | 100%
1/1
(20.7-100) | 99.9%
1000/1001
(99.4-100) | |
| Contrived | | 170 | 100%
50/50
(92.9-100) | 99.2%
119/120
(95.4-99.9) | |

Table 28: iC-GN Assay Performance: NDM
Specimen Type	N=	Percent Agreement		Comparator Method
		Positive (95% CI)	Negative (95% CI)
Prospective	Fresh	976	0/0	100%
976/976
(99.6-100)	PCR/Bi-directional sequencing
	Frozen	26	0/0	100%
26/26
(87.1-100)
	TOTAL	1002	0/0	100%
1002/1002
(99.6-100)
Contrived	170	170	100%
50/50
(92.9-100)	100%
120/120
(96.9-100)

Analysis of Mixed Culture Results:

In the method comparison study, there were thirty (30) mixed culture specimens that were detected by the iC-GN Assay, culture and MALDI, or both. The tables below list the mixed target combinations detected by iC-GN and the comparator method in the clinical study. There were twelve (12) discrepant mixed samples for which iC-GN detected a target that was not detected by the comparator assay. There were four (4) discrepant mixed samples for which the comparator assay detected targets that were not detected by iC-GN. Due to competitive

32

inhibition, target organisms present at low concentrations may not be detected by the iC-GN Assay when a second target organism is present at higher concentrations.

TABLE 29: Multiple Organism Detections by iC-GN as Compared to Culture/MALDI
Multiple Detections by iC-GN					Total
Targets
Detected
by iC-GN	No of
Discrepant
Targets	Discrepant
Results (Targets
Not Detected by
culture/MALDI)
Site	ID	Target 1	Target 2	Target 3
LAC	1102	E. coli	K. pneumoniae		2	0
LAC	1118	E. coli	K. pneumoniae		2	0
LAC	1141	E. coli	K. pneumoniae		2	0
LAC	1220	E. cloacae
complex	E. coli		2	0
LAC	1236	E. coli	K. oxytoca	K. pneumoniae	3	1	K. oxytoca
LAC	1285	K. pneumoniae	S. marcescens		2	1	S. marcescens
LAC	1307	E. coli	K. oxytoca		2	0
LAC	1378	E. coli	K. pneumoniae		2	1	K. pneumoniae
LAC	1382	K. oxytoca	K. pneumoniae		2	1	K. oxytoca
MCW	2023	E. coli	K. pneumoniae		2	0
MCW	2032	E. cloacae
complex	K. oxytoca		2	0
MCW	2038	K. oxytoca	K. pneumoniae		2	0
MCW	2041	E. coli	P. mirabilis		2	0
MCW	2104	E. coli	S. marcescens		2	1	S. marcescens
MCW	2193	K. pneumoniae	S. marcescens		2	2	K. pneumoniae, S.
marcescens
TC	3015	K. oxytoca	P. aeruginosa		2	2	K. oxytoca, P.
aeruginosa
TC	3096	E. coli	K. pneumoniae		2	1	K. pneumoniae
TC	3131	E. cloacae
complex	K. pneumoniae		2	0
TC	3183	K. pneumoniae	S. marcescens		2	1	S. marcescens
TGH	4031	E. coli	P. mirabilis		2	1	P. mirabilis
TGH	4037	E. coli	P. aeruginosa		2	0
TGH	4124	E. cloacae
complex	P. aeruginosa		2	1	P. aeruginosa
TGH	4132	E. cloacae
complex	K. pneumoniae		2	0
IU	5025	A. baumannii
complex	K. pneumoniae		2	1	A. baumannii
complex
IU	5031	E. coli	K. pneumoniae		2	0
IU	5042	E. cloacae
complex	K. pneumoniae		2	0

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TABLE 29: Multiple Organism Detections by Culture/MALDI as Compared to iC-GN
Multiple Detections by culture/MALDI				Total Targets Detected by Culture	Discrepant Targets	Discrepant Targets (Targets Not Detected by iC-GN)
Site	ID	Target 1	Target 2
LAC	1102	K. pneumoniae	E. coli	2	0
LAC	1118	K. pneumoniae	E. coli	2	0
LAC	1141	E. coli	K. pneumoniae	2	0
LAC	1220	E. cloacae complex	E. coli	2	0
LAC	1236	K. pneumoniae	E. coli	2	0
LAC	1268	P. aeruginosa	P. mirabilis	2	1	P. aeruginosa
LAC	1307	E. coli	K. oxytoca	2	0
LAC	1338	E. coli	K. pneumoniae	2	1	K. pneumoniae
MCW	2023	K. pneumoniae	E. coli	2	0
MCW	2032	K. oxytoca	E. cloacae complex	2	0
MCW	2038	K. pneumoniae	K. oxytoca	2	0
MCW	2041	P. mirabilis	E. coli	2	0
TC	3006	E. coli	K. pneumoniae	2	1	K. pneumoniae
TC	3131	E. cloacae complex	K. pneumoniae	2	0
TGH	4007	E. coli	P. aeruginosa	2	1	P. aeruginosa
TGH	4037	E. coli	P. aeruginosa	2	0
TGH	4132	K. pneumoniae	E. cloacae complex	2	0
IU	5031	E. coli	K. pneumoniae	2	0
IU	5042	K. pneumoniae	E. cloacae complex	2	0

Expected Values:

A total of 1002 prospectively collected fresh and frozen blood culture specimens were obtained from five geographically dispersed clinical sites. The number and percentage of positive cases (positivity rate) determined by the iC-GN Assay stratified by U.S. state for each of the organisms and resistance markers detected by the assay are presented below. Overall, the iC-GN Assay detected at least one organism in 89% (901/1002) prospectively collected specimens and at least one resistance marker in 6.8% (68/1002) prospectively collected specimens. Expected values are presented in the table below.

TABLE 30: Positivity by the iC-GN Assay as Observed in the Clinical Study
Organism	U.S. State
TOTAL n	NY
355	WI
188	NM
156	FL
209	IN
94	TOTAL
1002
Acinetobacter
baumannii complex	POSITIVE n	1	1	0	3	3	8
Acinetobacter
baumannii complex	% Positivity	0.3%	0.5%	0.0%	1.4%	3.2%	0.8%
Acinetobacter
baumannii complex	POSITIVE n	27	11	5	13	1	57

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TABLE 30: Positivity by the iC-GN Assay as Observed in the Clinical Study
	U.S. State	NY	WI	NM	FL	IN	TOTAL
Organism	TOTAL n	355	188	156	209	94	1002
Enterobacter
cloacae complex	% Positivity	7.6%	5.9%	3.2%	6.2%	1.1%	5.7%
	POSITIVE n	182	74	94	88	48	486
Escherichia coli	% Positivity	51.3%	39.4%	60.3%	42.1%	51.1%	48.5%
	POSITIVE n	9	7	2	4	4	26
Klebsiella oxytoca	% Positivity	2.5%	3.7%	1.3%	1.9%	4.3%	2.6%
Klebsiella
pneumoniae	POSITIVE n	51	32	28	33	15	159
	% Positivity	14.4%	15.4%	17.3%	15.8%	16.0%	15.9%
Proteus mirabilis	POSITIVE n	21	9	1	13	7	51
	% Positivity	5.9%	4.8%	0.6%	6.2%	7.4%	5.1%
Pseudomonas
aeruginosa	POSITIVE n	24	19	5	25	8	81
	% Positivity	6.8%	10.1%	3.2%	12.0%	8.5%	8.1%
Serratia marcescens	POSITIVE n	9	8	2	8	6	33
	% Positivity	2.5%	4.3%	1.3%	3.8%	6.4%	3.3%
Resistance Marker	TOTAL n	355	188	156	209	94	1002
KPC	POSITIVE n	0	2	0	0	0	2
	% Positivity	0.0%	1.1%	0.0%	0.0%	0.0%	0.2%
NDM	POSITIVE n	0	0	0	0	0	0
	% Positivity	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
CTX-M	POSITIVE n	12	22	7	15	10	66
	% Positivity	3.4%	11.7%	4.5%	7.2%	10.6%	6.6%

Statement of Safety and Effectiveness

The data presented clearly demonstrates the safety and efficacy of the iC-GN Assay™ for use on the iC-System as compared to the reference method when the product Instructions for Use are followed.