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.

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.

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.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied by the "Percent Agreement" values (positive and negative) to reference methods. While specific numeric acceptance thresholds are not explicitly stated as "acceptance criteria," performance metrics of 95% or higher with tight confidence intervals are typically considered strong evidence of meeting performance expectations for such devices. For reproducibility and equivalency, "≥ 95% performance" is explicitly stated as the acceptance criteria.

Metric / Target	Acceptance Criteria (Implied/Explicit)	Reported Device Performance (Positive Percent Agreement)	Reported Device Performance (Negative Percent Agreement)
Reproducibility	≥ 95% performance (explicit)	96.7% - 100% (Overall for individual targets)	96.7% - 100% (Overall for individual targets)
Blood Culture Bottle Equivalency	≥ 95% performance (explicit)	97.9% - 100.0%	98.8% - 100.0%
Method Comparison (Organisms)	High agreement with reference method (implied)
Acinetobacter baumannii complex	High agreement with reference method (implied)	100% (7/7 fresh, 45/45 contrived)	99.9% (968-969/969 fresh, 125/125 contrived)
Enterobacter cloacae complex	High agreement with reference method (implied)	94.5% (52/55 fresh), 100% (5/5 frozen, 17/17 contrived)	100% (921/921 fresh, 153/153 contrived)
Escherichia coli	High agreement with reference method (implied)	98.4% (480/488 fresh), 100% (6/6 frozen, 15/15 contrived)	100% (488/488 fresh, 155/155 contrived)
Klebsiella oxytoca	High agreement with reference method (implied)	95.8% (23/24 fresh), 100% (30/30 contrived)	99.7% (949/952 fresh, 140/140 contrived)
Klebsiella pneumoniae	High agreement with reference method (implied)	96.8% (150/155 fresh), 100% (3/3 frozen, 21/21 contrived)	99.3% (815/821 fresh, 148/149 contrived)
Proteus mirabilis	High agreement with reference method (implied)	97.4% (37/38 fresh), 100% (9/9 frozen, 12/12 contrived)	99.5% (914/919 fresh, 158/158 contrived)
Pseudomonas aeruginosa	High agreement with reference method (implied)	95.1% (78/82 fresh), 100% (1/1 frozen, 10/10 contrived)	99.8% (892/894 fresh, 160/160 contrived)
Serratia marcescens	High agreement with reference method (implied)	100% (29/29 fresh), 100% (20/20 contrived)	99.6% (943/947 fresh, 149/150 contrived)
Method Comparison (Resistance Markers)	High agreement with reference method (implied)
CTX-M	High agreement with reference method (implied)	97.0% (64/66 fresh), 100% (1/1 frozen, 15/15 contrived)	99.9% (909/910 fresh, 155/155 contrived)
KPC	High agreement with reference method (implied)	100% (1/1 fresh), 100% (50/50 contrived)	99.9% (974/975 fresh), 99.2% (119/120 contrived)
NDM	High agreement with reference method (implied)	0/0 (fresh/frozen) - not detected, 100% (50/50 contrived)	100% (976/976 fresh), 100% (120/120 contrived)

2. Sample size used for the test set and the data provenance

Method Comparison Study (Clinical Study):
- Total specimens enrolled: 1107
- Specimens included in performance analysis: 1002
  - Fresh prospective specimens: 976
  - Frozen prospective specimens (retrospectively tested): 26 (2.6%)
- Contrived samples: 170
- Data Provenance: Five geographically dispersed clinical sites in the U.S. (NY, WI, NM, FL, IN). The specimens were "leftover de-identified specimens from anaerobic blood culture bottles flagged as positive." This indicates a retrospective collection of clinical samples taken from patients, which were then de-identified and used for the study.
Reproducibility Study:
- Eighteen-organism panel tested in triplicate across five, non-consecutive days by two independent operators at each of three sites. This implies approximately 18 organisms * 3 replicates * 5 days * 2 operators * 3 sites = 1620 tests, but the table breaks down performance by target with denominators of ~90, suggesting a more specific number per target/concentration.
Limit of Detection (LoD) Study:
- Twenty-seven representative strains, a minimum of three per target. Tested in a minimum of twenty replicates (for confirmation phase) on each of three unique cassette lots.
Inclusivity Study:
- Eighty-two (82) representative strains, a minimum of ten strains for each target analyte. Each strain tested in triplicate.
Exclusivity Study:
- A total of 114 strains. Each strain tested in triplicate (retested in replicates of 3 or 10 for discordant results).
Microbial Interference Study:
- Sixty (60) gram negative exclusivity strains in combination with eight (8) iC-GN target organisms. Each combination tested in triplicate (retested in replicates of 3 or 10 for discordant results).
Competitive Inhibition Study:
- One target organism (low concentration) combined with a second target organism (high concentration). Each combination tested in triplicate (retested in replicates of 10 for false negatives).
Interfering Substances Study:
- Eight representative target organisms plus one non-target organism. Organism/interferent combination tested in triplicate (retested in replicates of 10 or 3 for discordant results).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document does not specify the number or qualifications of experts used to establish the ground truth. It states that the ground truth for the clinical method comparison was based on:

"reference culture followed by MALDI identification" for organism targets.
"PCR amplification followed by confirmatory bidirectional sequencing" for resistance markers.
"Phenotypic antimicrobial susceptibility testing (AST)" was also performed to identify samples requiring sequencing.

While these are standard laboratory methods, the involvement of specific "experts" (e.g., microbiologists, infectious disease specialists) beyond performing these standard laboratory procedures is not detailed.

4. Adjudication method for the test set

The document does not describe a formal adjudication method (like 2+1 or 3+1). For the method comparison study, discordant samples were "sequenced" (for resistance markers) or further analyzed by PCR/bi-directional sequencing for organism targets. For other performance studies (inclusivity, exclusivity, microbial interference), discordant results led to retesting in replicates (3 or 10). This indicates a re-testing approach for discrepancies rather than a multi-expert consensus.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. The iC-GN Assay is an in vitro diagnostic (IVD) device, specifically a molecular diagnostic test for identifying microorganisms and resistance markers from positive blood cultures. It does not involve "human readers" interpreting images or data in a way that would typically be evaluated in an MRMC study for AI assistance. Its performance is compared against laboratory reference methods.

6. If a standalone (i.e. algorithm only without human-in-the loop performance) was done

Yes, the studies presented evaluate the standalone performance of the iC-GN Assay. The assay is an automated system (iC-System) that detects and identifies targets directly from positive blood cultures using PCR and microarray hybridization. The results are interpreted by software (iC-Report™) and displayed for the user. The performance data (reproducibility, LoD, inclusivity, exclusivity, microbial interference, competitive inhibition, and method comparison) all reflect the device's inherent analytical and clinical performance without a human interpreting the primary result, but rather the system's output. The human role is operational (loading samples, reading the final report), not interpretive of the raw data.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The ground truth used for the clinical method comparison study was established using:

Reference culture followed by MALDI identification: This is a combination of microbiological culture methods and advanced biochemical analysis to identify bacterial species.
PCR amplification followed by confirmatory bidirectional sequencing: This is a molecular method used to confirm the presence and identity of specific genes (e.g., resistance markers) or bacterial species through DNA sequencing.
Phenotypic antimicrobial susceptibility testing (AST): Used to guide which resistance markers might need further sequencing for confirmation.

For other analytical studies (Inclusivity, Exclusivity, LoD, etc.), the ground truth was based on the known identity and concentration of the characterized bacterial strains used.

8. The sample size for the training set

The document does not provide details about a "training set" in the context of an algorithm or AI development. This device is a molecular diagnostic test, not an AI/ML-based diagnostic that would typically involve a separate training, validation, and test set for algorithm development. The studies described are performance validation studies for the finished medical device.

9. How the ground truth for the training set was established

As there's no explicit mention of a "training set" for an algorithm, the method for establishing its ground truth is not described. The ground truth for the verification and validation studies (as detailed in point 7) was established through standard microbiological and molecular laboratory techniques, which served as the comparator for assessing device performance.

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

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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 BloodCulture Nucleic Acid Test (GC-GN)K132843(Predicate Device)
Similarities
Intended Use	The iCubate, Inc. iC-GN Assay™ for use onthe iC-System™ is a qualitative,multiplexed, in vitro diagnostic test for thedetection and identification of potentiallypathogenic gram negative bacteria, whichmay cause bloodstream infection (BSI). TheiC-GN Assay™ is performed directly onpositive blood cultures, confirmed by Gramstain to contain gram negative bacilli.Cultures demonstrating mixed Gram stainresults should not be tested on the assay.The iC-GN Assay™ is validated for use withselect BACTEC™ , BacT/ALERT® andVersaTREK® blood culture bottles. The iC-GN Assay™ is indicated for use inconjunction with other clinical andlaboratory findings, such as culture, to aidin the diagnosis of bacterial bloodstreaminfections; however, it is not used tomonitor bloodstream infections.The iC-GN Assay™ detects target DNA andidentifies the following:	The Verigene® Gram NegativeBlood Culture Nucleic Acid Test (BC-GN), performed using the sample-to-results Verigene System, is aqualitative multiplexed in vitrodiagnostic test for the simultaneousdetection and identification ofselected gram-negative bacteriaand resistance markers. BC-GN isperformed directly on blood culturemedia using blood culture bottlesidentified as positive by acontinuous monitor blood culturesystem and which contain gram-negative bacteria as determined byGram stain. BC-GN detects andidentifies the following:Acinetobacter spp.Citrobacter spp.Enterobacter spp.Proteus spp.Escherichia coliKlebsiella pneumoniaeKlebsiella oxytocaPseudomonas aeruginosa
	Bacterial Generaand Species Resistance Markers AcinetobacterbaumanniicomplexEnterobactercloacae complexEscherichia coliKlebsiella oxytocaKlebsiellapneumoniaePseudomonasaeruginosaProteus speciesSerratiamarcescens KPC (blaKPC)-associated withresistance tocarbapenemsNDM (blaNDM)-associated withresistance tocarbapenemsCTX-M group1(blaCTX-M group 1)-associated withresistance toextended spectrumbeta-lactams		Pseudomonas aeruginosaBC-GN is indicated for use inconjunction with other clinical andlaboratory findings to aid in thediagnosis of bacterial bloodstreaminfections; however, is not to beused to monitor these infections.Sub-culturing of positive bloodcultures is necessary to recoverorganisms for susceptibility testing,identification of organisms notdetected by BC-GN, differentiationof mixed growth, association ofantimicrobial resistance markergenes to a specific organism, or forepidemiological typing.
	In mixed growth, the iC-GN Assay™ doesnot specifically attribute detection of KPC,NDM, or CTX-M group 1 to a specificgenera or species.Sub-culturing of positive blood cultures isnecessary to recover organisms for

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Characteristic	iCubate, Inc.iC-GN Assay™ for use on the iC-System™(New Device)	Nanosphere, Inc.Verigene® Gram Negative BloodCulture Nucleic Acid Test (GC-GN)K132843(Predicate Device)
	susceptibility testing, identification oforganisms not detected by the iC-GNAssay™, differentiation of mixed growth,association of antimicrobial resistancemarker genes to a specific organism, or forepidemiological typing.
Sample Type	Positive Blood Culture	Positive Blood Culture
Differences
INSTRUMENTREQUIREMENTS	iC-System™	Verigene System
TEST PRINCIPLE	ARM-PCR	Gold nanoparticle probe-based PCR
COMPATIBLE BLOODCULTURE BOTTLES	BD BACTEC Standard/10 Aerobic/FBD BACTEC Standard/10 Anaerobic/FBD BACTEC Plus Aerobic/FBD BACTEC Plus Anaerobic/FBD BACTEC Lytic/10 Anaerobic/FBacT/Alert SA Standard AerobicBacT/Alert SN Standard AnaerobicBacT/Alert FA Aerobic FANBacT/Alert FN Anaerobic FANBacT/Alert FA Plus AerobicBacT/Alert FN Plus AnaerobicVersaTREK REDOX 1VersaTREK REDOX 2	BACTEC™ Plus Aerobic/FBacT/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.

Limitations

For prescription use only.

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

Methodology

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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.

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	OverallPerformance	OverallPerformance %[95% CI]	FalseNegatives	FalsePositives	PC CheckFailures	SystemFailures
A. baumannii complexBottle 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 complexBottle 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 complexBottle 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 complexBottle 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. coliBottle 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. coliBottle 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. oxytocaBottle 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. oxytocaBottle 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. pneumoniaeBottle 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. pneumoniaeBottle 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 speciesBottle 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 speciesBottle 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%)

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TABLE 2: iC-GN Assay Reproducibility Performance by Target
Target/Concentration	OverallPerformance	OverallPerformance %[95% CI]	FalseNegatives	FalsePositives	PC CheckFailures	SystemFailures
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 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 < 100% of the time.

TABLE 3: iC-GN Assay LoD Results
Target	Strain	Concentration(CFU/mL)	Defined Target LoD(CFU/mL)
A. baumannii complex	307-0294	$5.3 \times 10^5$
A. baumannii complex	CDC-83	$5.2 \times 10^6$	$5.3 \times 10^5 - 5.2 \times 10^6$
A. baumannii complex	ATCC 23055	$9.0 \times 10^5$
TABLE 3: iC-GN Assay LoD Results
Target	Strain	Concentration (CFU/mL)	Defined Target LoD (CFU/mL)
E. cloacae complex	Z101	$5.0 × 10^6$	$4.9 × 10^5-5.5 × 10^6$
	CDC-164	$5.5 × 10^6$
	ATCC 700323	$4.9 × 10^5$
E. coli	ATCC 43895	$7.7 × 10^5$
	ATCC BAA-2326	$7.9 × 10^5$	$7.7 × 10^5-8.4 × 10^5$
	CDC-55	$8.4 × 10^5$
K. oxytoca	Z115	$6.2 × 10^5$
	ATCC 13182	$5.4 × 10^5$	$5.4 × 10^5-1.1 × 10^6$
	CDC-147	$1.1 × 10^6$
K. pneumoniae	ATCC 35657	$1.9 × 10^6$
	CDC-40	$3.6 × 10^6$
	CDC-42	$1.9 × 10^6$	$6.0 × 10^5-4.2 × 10^6$
	KPC-2	$4.2 × 10^6$
Proteus species	LACNY 11	$6.0 × 10^5$
	Z050	$1.1 × 10^6$
	CDC-59	$9.9 × 10^5$	$6.9 × 10^5-1.1 × 10^6$
	Z028	$7.6 × 10^5$
P. aeruginosa	Z129	$6.9 × 10^5$
	Z139	$1.2 × 10^6$	$5.0 × 10^5-1.2 × 10^6$
	CDC-231	$5.0 × 10^5$
	CDC-250	$6.9 × 10^5$
S. marcescens	ATCC 43297	$7.2 × 10^5$
	ATCC 21212	$8.1 × 10^5$	$6.4 × 10^5-8.1 × 10^5$
	CDC-91	$6.4 × 10^5$
CTX-M group 1	ATCC BAA-2326 (CTX-M-15)	$7.9 × 10^5$
	CDC-40 (CTX-M-15)	$2.3 × 10^6$	$7.9 × 10^5-2.3 × 10^6$
	CDC-42 (CTX-M-15)	$1.9 × 10^6$
KPC	CDC-147 (KPC-3)	$2.3 × 10^6$
	KPC-2	$4.2 × 10^6$	$1.5 × 10^5-4.2 × 10^6$
	CDC-231 (KPC-5)	$1.5 × 10^5$
NDM	CDC-83 (NDM-1)	$5.2 × 10^6$
	CDC-55 (NDM-1)	$4.0 × 10^6$	$3.3 × 10^5-5.2 × 10^6$
	CDC-250 (NDM-1)	$3.3 × 10^5$

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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 PositivityAverage Concentration(CFU/mL)	Bottle Positivity + 8Average Concentration(CFU/mL)
Acinetobacter baumannii	307-0294	4.24 × 108	8.27 × 108
Acinetobacter baumannii	CDC-83	3.39 × 108	7.23 × 108
Acinetobactercalcoaceticus	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

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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	OverallPerformance(%)	FalseNegatives(%)	FalsePositives(%)	PCCheckFailures(%)	SystemFailures(%)
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 StandardAnaerobic	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

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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

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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 baumanniicomplex	KPC-2		KPC-1
	KPC-3		KPC-2
	KPC-10		KPC-3
Enterobacter cloacaecomplex	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 baumanniicomplex	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 cloacaecomplex	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	ATCCBAA2410	$1.50 \times 10^7$	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	ATCCBAA1715	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
LowOrganism	High Organism	LowOrganism	LowMarker	HighOrganism	HighMarker
(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
LowOrganism	High Organism	LowOrganism	LowMarker	HighOrganism	HighMarker
	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 RelevantConcentration	TestConcentration
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

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TABLE 13: Interfering Substances Test Panel
Interference Compound	Clinically RelevantConcentration	TestConcentration
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
InterferenceCompound	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
ConjugatedBilirubin	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
UnconjugatedBilirubin	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 GenomicDNA	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

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TABLE 14: iC-GN Assay Interfering Substances Performance
	Target Performance
InterferenceCompound	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.

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Site Code	UnresolvediC-GN Error	IncompleteReferenceMethod	OutsideFresh StabilityWindow	Didn't MeetInclusionCriteria	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

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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: Acinetobacterbaumannii complex (ppa)
SpecimenType	N=	Percent Agreement	ComparatorMethod
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)
SpecimenType	N=	Percent Agreement	ComparatorMethod
		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)

SpecimenType	N=	Percent Agreement		ComparatorMethod
		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

	SpecimenType	N=	Percent Agreement		ComparatorMethod
			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

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Table 22: iC-GN Assay Performance: Klebsiella pneumoniae(parC)
Specimen Type	N=	Percent AgreementPositive(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: Pseudomonasaeruginosa (algD)
	SpecimenType	N=	Percent Agreement		ComparatorMethod
			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: Proteusmirabilis (rpoB)
	SpecimenType	N=	Percent Agreement		ComparatorMethod
			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)
	SpecimenType	N=	Percent Agreement		ComparatorMethod
			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)

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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
SpecimenType	N=	Percent Agreement	ComparatorMethod
		Positive(95% CI)	Negative(95% CI)
Prospective	Fresh	976	100%1/1(20.7-100)	99.9%974/975(99.4-100)	PCR/Bi-directionalsequencing
	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

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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					TotalTargetsDetectedby iC-GN	No ofDiscrepantTargets	DiscrepantResults (TargetsNot Detected byculture/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. cloacaecomplex	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. cloacaecomplex	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. cloacaecomplex	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. cloacaecomplex	P. aeruginosa		2	1	P. aeruginosa
TGH	4132	E. cloacaecomplex	K. pneumoniae		2	0
IU	5025	A. baumanniicomplex	K. pneumoniae		2	1	A. baumanniicomplex
IU	5031	E. coli	K. pneumoniae		2	0
IU	5042	E. cloacaecomplex	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. StateTOTAL n	NY355	WI188	NM156	FL209	IN94	TOTAL1002
Acinetobacterbaumannii complex	POSITIVE n	1	1	0	3	3	8
Acinetobacterbaumannii complex	% Positivity	0.3%	0.5%	0.0%	1.4%	3.2%	0.8%
Acinetobacterbaumannii 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
Enterobactercloacae 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%
Klebsiellapneumoniae	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%
Pseudomonasaeruginosa	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.

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).