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

The iC-GPC Assay detects organism DNA and identifies the following bacterial species and resistance markers:

Bacterial Species Staphylococcus aureus Staphylococcus epidermidis Streptococcus pneumoniae Enterococcus faecalis Enterococcus faecium

Resistance Markers

mecA- associated with methicillin resistance vanA- associated with vancomycin resistance vanB- associated with vancomycin resistance

The iC-GPC Assay detects the mecA resistance marker, inferring mecA-mediated methicillin resistance, and the vanA and vanB resistance markers, inferring vanAlvanB-mediated vancomycin resistance. In mixed growth, the iC-GPC Assay does not specifically attribute van-mediated vancomycin resistance to either E. faecalis or E. faecium, or mecA-mediated methicillin resistance to either S. aureus or S. epidermidis.

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

Device Description

The iC-GPC Assay utilizes polymerase chain reaction (PCR) for the amplification of specific targets and detects the amplified target DNA with fluorescence-based microarray hybridization. The iC-GPC Assay uses proprietary ARM-PCR (Amplicon Rescued Multiplex PCR) technology allowing for multiple targets to be amplified in one reaction. Targets are detected directly from patient positive blood cultures, confirmed by Gram stain to contain gram positive cocci. Testing is performed within a closed, disposable cassette that contains all the reagents required to complete the iC-GPC Assay, including a universal microarray.

To operate, the user opens the iC-GPC Cassette cap and pipettes an aliquot of the positive blood culture sample into the sample well of the cassette is then inserted into the iC-Processor, which performs the processes of DNA extraction, multiplex amplification, and microarray After processing is complete, the cassette is inserted into the iC-Reader for hybridization. fluorescence-based detection and data analysis. Final results are generated by iC-Report, computer software for data acquisition, analysis, and display.

Extraction, amplification, and hybridization 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-GPC Cassette which communicates with the iC-Processor. To access and pierce the foil-sealed reagent wells located in the bottom well plate of the cassette, the processor manipulates the cassette to move the internal 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 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 4 iC-Cassettes at one time. The results are interpreted via the iC-Report software and displayed for the user on an iMac computer. Raw data and result interpretations are stored within the iMac; raw data is accessible to authorized personnel only and is not available to the end user.

AI/ML Overview

The provided document describes the iC-GPC Assay for use on the iC-System, a qualitative, multiplexed, in vitro diagnostic test for the detection and identification of potentially pathogenic gram-positive bacteria and resistance markers from positive blood cultures. The document details the performance studies conducted to support its substantial equivalence to a legally marketed predicate device (VERIGENE® Gram Positive Blood Culture Nucleic Acid Test (BC-GP)) and its overall analytical and clinical performance.

Here's an breakdown of the acceptance criteria and the studies proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a single, consolidated table with pre-defined thresholds. Instead, it presents performance metrics in various tests (reproducibility, LoD, inclusivity, exclusivity, method comparison). Acceptance for these tests is implied by the successful outcomes. The core performance is summarized in the method comparison study against an FDA-cleared multiplex assay and against traditional culture and susceptibility methods.

Below, I've compiled a table summarizing the key performance metrics from the Method Comparison section, which represents the device's main performance claim for its intended use. The "Acceptance Criteria" here are implicitly High agreement (e.g., >95%) for both positive and negative agreement, as is common for in vitro diagnostics devices aiming for substantial equivalence.

Acceptance Criteria (Implied)	Reported iC-GPC Assay Performance (vs. FDA-Cleared Multiplex Assay)	Reported iC-GPC Assay Performance (vs. Culture & Biochemicals/AST)
Staphylococcus aureus (nuc)
High Positive Agreement	97.0% (258/266) [94.2-98.5%]	95.9% (259/270) [92.9-97.7%]
High Negative Agreement	99.7% (645/647) [98.9-99.9%]	99.8% (610/611) [99.1-100%]
Staphylococcus epidermidis (gseA)
High Positive Agreement	98.3% (231/235) [95.7-99.3%]	96.0% (216/225) [92.6-97.9%]
High Negative Agreement	97.9% (664/678) [96.6-98.8%]	96.5% (633/656) [94.8-97.7%]
Streptococcus pneumoniae (lytA)
High Positive Agreement	85.2% (23/27) [67.5-94.1%] (Fresh: 82.6%)	100% (21/21) [84.5-100%]
High Negative Agreement	99.9% (885/886) [99.4-100%]	99.8% (866/868) [99.2-99.9%]
Enterococcus faecalis (ddl)
High Positive Agreement	96.7% (59/61) [88.8-99.1%]	96.8% (60/62) [89.0-99.1%]
High Negative Agreement	99.9% (851/852) [99.3-100%]	99.9% (828/829) [99.3-100%]
Enterococcus faecium (fcm)
High Positive Agreement	96.6% (28/29) [82.8-99.4%]	96.6% (28/29) [82.8-99.4%]
High Negative Agreement	99.8% (882/884) [99.2-99.9%]	99.7% (859/862) [99.0-99.9%]
mecA (methicillin resistance)
High Positive Agreement	96.1% (274/285) [93.2-97.8%]	90.2% (257/285) [86.2-93.1%]
High Negative Agreement	98.2% (617/628) [96.9-99.0%]	97.0% (551/568) [95.3-98.1%]
vanA (vancomycin resistance)
High Positive Agreement	95.0% (19/20) [76.4-99.1%]	88.0% (22/25) [70.0-95.8%] (vanA/B combined)
High Negative Agreement	99.4% (888/893) [98.7-99.8%]	99.8% (861/863) [99.2-99.9%] (vanA/B combined)
vanB (vancomycin resistance)
High Positive Agreement	100% (2/2) [34.2-100%]	88.0% (22/25) [70.0-95.8%] (vanA/B combined)
High Negative Agreement	99.9% (876/877) [99.4-100%]	99.8% (861/863) [99.2-99.9%] (vanA/B combined)

2. Sample Size and Data Provenance

Test Set Sample Size:
- Clinical Method Comparison Study: 966 positive blood culture specimens initially enrolled, reduced to 913 (879 fresh prospective, 34 frozen prospective) after withdrawals.
- Contrived Samples: An additional 168 contrived samples.
Data Provenance: The general text of the letter indicates "four geographically dispersed clinical sites." Given the context of a US regulatory submission to the FDA, it is highly likely these sites were in the United States. The "Expected Values" table further specifies "U.S. State" data from NY, WI, NM, and FL. The study collected prospective (fresh and frozen) and contrived samples.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the "number of experts" or their "qualifications" for establishing the ground truth for the clinical method comparison test set.

However, for the comparison methods that served as ground truth:

FDA-cleared multiplex assay: This is a pre-existing, validated device, implying its results are considered reliable.
Conventional reference methods: This includes "subculture, identification of isolates using biochemical methods or MALDI-TOF, and phenotypic antimicrobial susceptibility testing (AST)." These methods are standard clinical laboratory practices performed by trained microbiologists and clinical laboratory scientists. The qualification of these individuals would adhere to standard laboratory accreditation and personnel requirements.

4. Adjudication Method for the Test Set

The document details discordant analysis for the method comparison studies:

Against FDA-Cleared Multiplex Assay: PCR and bidirectional sequencing were performed for all specimens with positive results for resistance markers by either the iC-GPC Assay or the FDA-cleared multiplex assay. These results were used to analyze and investigate discordant results. This implies a form of 2+1 adjudication or investigation where if the iC-GPC and comparator disagreed, an independent gold standard (PCR/sequencing) was used to resolve.
Against Conventional Reference Methods: PCR and bi-directional sequencing were not used for discordant analysis between iC-GPC and conventional reference method results. This suggests that the conventional methods served as the direct reference, without further independent adjudication for discrepancies with the iC-GPC assay.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

This is not a multi-reader multi-case (MRMC) study. The iC-GPC Assay is an in vitro diagnostic device for laboratory use that provides automated results, not a diagnostic imaging AI algorithm that assists human readers. Therefore, there is no human-in-the-loop performance measurement or effect size reported here regarding human reader improvement with AI assistance.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, this entire submission revolves around the standalone performance of the iC-GPC Assay, which is an automated system (iC-System) that performs DNA extraction, multiplex amplification, hybridization, and detection to generate results. The reported performance metrics (positive/negative agreement, LoD, inclusivity, exclusivity) are a direct measure of the algorithm's (device's) performance.

7. Type of Ground Truth Used

For Analytical Studies (LoD, Inclusivity, Exclusivity, Reproducibility, etc.): Ground truth was established by known concentrations/strains of organisms, obtained from characterized sources (e.g., ATCC strains, well-characterized clinical isolates) and confirmed by methods such as colony counts.
For Clinical Method Comparison Study:
- Primary Ground Truth 1: An FDA-cleared multiplex assay.
- Secondary Ground Truth 2: Conventional laboratory reference methods (subculture, biochemical identification, MALDI-TOF, and phenotypic antimicrobial susceptibility testing (AST)).
- Discordant Resolution (for resistance markers in comparison to FDA-Cleared Multiplex Assay): PCR and bi-directional sequencing as an independent, highly accurate molecular method.

8. Sample Size for the Training Set

The document does not provide information on the training set size for the iC-GPC Assay's development. This FDA 510(k) submission focuses on the validation of the device through performance studies (test sets) rather than detailing the specifics of its development or "training" phase. For in vitro diagnostics that use molecular techniques, the "training" analogous to machine learning models usually involves extensive research and development to optimize primer and probe design, reaction conditions, and analytical algorithms based on large collections of known positive and negative samples, but these are typically internal R&D activities not explicitly reported as "training set size" in the final validation summary.

9. How the Ground Truth for the Training Set Was Established

As the document does not detail a "training set," it also does not describe how its ground truth was established. However, for a molecular diagnostic assay like this, the underlying "ground truth" for developing and optimizing the assay's targets and reaction conditions would typically be established through:

Known, characterized bacterial strains and clinical isolates: Identified and confirmed using a combination of standard microbiological methods (culture, Gram stain, biochemical tests, phenotypic susceptibility testing) and advanced molecular methods (16S rRNA gene sequencing, whole-genome sequencing) to determine precise species and resistance marker presence.
Synthetic DNA/RNA targets: Used for initial assay design and analytical performance testing.
Bioinformatics analysis: To select highly specific gene targets (like gseA, nuc, lytA, ddl, fcm, mecA, vanA, vanB) that are unique to the target organisms and resistance mechanisms.

This developmental phase would involve rigorous, iterative testing and refinement against a comprehensive panel of both target and non-target organisms to ensure high sensitivity and specificity before formal validation studies like those presented in the 510(k) summary are conducted.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

August 8, 2017

ICUBATE, INC. C/O FRAN WHITE MDC ASSOCIATES, LLC 180 CABOT STREET BEVERLY, MA 01915

Re: K163390

Trade/Device Name: iC-GPC Assay, 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: PAM, NSU Dated: July 14, 2017 Received: July 17, 2017

Dear Ms. 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. The general controls provisions of the Act include requirements for annual registration. Iisting 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 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 Parts 801 and 809); medical device reporting (reporting of

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medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely,

Steven R. Gitterman -S for

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

Enclosure

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Indications for Use

510(k) Number (if known) K163390

Device Name iC-GPC Assay for use on the iC-System

Indications for Use (Describe)

The iC-GPC Assay detects organism DNA and identifies the following bacterial species and resistance markers:

Bacterial Species Staphylococcus aureus Staphylococcus epidermidis Streptococcus pneumoniae Enterococcus faecalis Enterococcus faecium

Resistance Markers

mecA- associated with methicillin resistance vanA- associated with vancomycin resistance vanB- associated with vancomycin resistance

Type of Use (Select one or both, as applicable)
× Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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510(k) SUMMARY

Date of Summary:	August 2, 2017
Product Name	iC-GPC Assay™ for use on the iC-System™
Sponsor	iCubate®601 Genome WayHuntsville, AL 35806

Correspondent MDC Associates, LLC Fran White, President 180 Cabot Street Beverly, MA 01915 Phone: (978) 705-5011

Device Trade or Proprietary Name

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

Common Name

Gram positive bacteria and their resistance markers

Regulation

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

Fax: (800) 498-9121

Product Codes

PAM, NSU

Classification

Class II

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Substantial Equivalency

Characteristic	iC-GPC Assay™ for use on the iC-System™(New Device)	VERIGENE® Gram Positive Blood Culture NucleicAcid Test (BC-GP) (K122514)(Primary Predicate Device)
Similarities
Intended Use	The iCubate iC-GPC Assay™ for use on the iC-System™ is a qualitative, multiplexed, in vitrodiagnostic test for the detection andidentification of potentially pathogenic grampositive bacteria, which may cause bloodstreaminfection (BSI). The iC-GPC Assay™ is performeddirectly on positive blood cultures, confirmed byGram Stain to contain gram positive cocci.Cultures demonstrating mixed Gram stain resultsshould not be tested on the assay. The iC-GPCAssay™ is validated for use with select BACTEC™,BacT/ALERT® and VersaTREK® blood culturebottles. The iC-GPC Assay™ is indicated for use inconjunction with other clinical and laboratoryfindings, such as blood culture isolateidentification and antimicrobial susceptibilitytesting, to aid in the diagnosis of bacterialbloodstream infections; however, it is not used tomonitor bloodstream infections.The iC-GPC Assay™ detects organism DNA andidentifies the following bacterial species andresistance markers:	The Verigene® Gram Positive Blood Culture NucleicAcid Test (BC-GP) performed using the sample-to-result Verigene System is a qualitative, multiplexed in vitro diagnostic test for the simultaneous detectionand identification of potentially pathogenic gram-positive bacteria which may cause bloodstreaminfection (BSI). BC-GP is performed directly onpositive blood culture using BACTEC™ Plus Aerobic/Fand BacT/ALERT FA FAN® Aerobic blood culturebottles, which contain gram positive bacteria. BC-GPis indicated for use in conjunction with other clinicaland laboratory findings, such as culture, to aid in thediagnosis of bacterial bloodstream infections;however, it is not used to monitor bloodstreaminfections.BC-GP detects and identifies the following bacterialgenera and species:Staphylococcus spp.Staphylococcus aureusStaphylococcus epidermidisStaphylococcus lugdunensisStreptococcus spp.Streptococcus pneumoniaeStreptococcus pyogenesStreptococcus agalactiaeStreptococcus anginosus groupEnterococcus faecalisEnterococcus faeciumListeria spp.In addition, BC-GP detects the mecAresistance marker, inferring mecA -mediatedmethicillin resistance, andthe vanA and vanB resistance markers, inferringvanA/vanB -mediated vancomycin resistance. Inmixed growth, BC-GP does not specifically attributevan -mediated vancomycin resistance to either E.faecalis or E. faecium , or mecA -mediated methicillinresistance to either S. aureus or S. epidermidis .
	Bacterial Species ResistanceMarkers Staphylococcus aureusStaphylococcus epidermidisStreptococcus pneumoniaeEnterococcus faecalisEnterococcus faecium mecA - associatedwith methicillinresistancevanA - associatedwith vancomycinresistancevanB - associatedwith vancomycinresistance The iC-GPC Assay™ detects the mecA resistancemarker, inferring mecA -mediated methicillinresistance, and the vanA and vanB resistancemarkers, inferring vanA/vanB -mediatedvancomycin resistance. In mixed growth, the iC-GPC Assay™ does not specifically attribute van -mediated vancomycin resistance to either E .

Comparison of New Device with Predicate Device TABLE 1.

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Characteristic	iC-GPC Assay™ for use on the iC-System™(New Device)	VERIGENE® Gram Positive Blood Culture NucleicAcid Test (BC-GP) (K122514)(Primary Predicate Device)
	faecalis or E. faecium , or mecA-mediatedmethicillin resistance to either S. aureus or S.epidermidis .
	Sub-culturing of positive blood cultures isnecessary to recover organisms for susceptibilitytesting, identification of organisms not detectedby the iC-GPC Assay™, differentiation of mixedgrowth, association of antimicrobial resistancemarker genes to a specific organism, or forepidemiological typing.
Indication forUse	The iC-GPC Assay™ is indicated for use inconjunction with other clinical and laboratoryfindings to aid in the diagnosis of bacterialbloodstream infections; however, is not to beused to monitor these infections. Sub-culturing ofpositive blood cultures is necessary to recoverorganisms for susceptibility testing, identificationof organisms not detected by the iC-GPC Assay™,differentiation of mixed growth, association ofantimicrobial resistance marker genes to aspecific organism, or for epidemiological typing.	BC-GP is indicated for use in conjunction with otherclinical and laboratory findings to aid in the diagnosisof bacterial bloodstream infections; however, is notto be used to monitor these infections. Sub-culturingof positive blood cultures is necessary to recoverorganisms for susceptibility testing, identification oforganisms not detected by BC-GP, differentiation ofmixed 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 Principal	Arm-PCR	Gold nanoparticle probe-based PCR
CompatibleBlood CultureBottles	BACTEC™ Standard Aerobic/Anaerobic, BACTEC™Plus Aerobic/Anaerobic, BACTEC™ Lytic/10Anaerobic, BacT/ALERT Standard Aerobic,BacT/ALERT FA Aerobic FAN®, BacT/ALERT FAPlus Aerobic, VersaTREK® REDOX 1/2	BACTEC™ Standard Aerobic/Anaerobic, BACTEC™Plus Aerobic/Anaerobic, BACTEC™ Lytic/10Anaerobic, BACTEC™ Peds Plus, BacT/ALERTStandard Aerobic/Anaerobic, BacT/ALERT FA AerobicFAN®, BacT/ALERT FN Anaerobic FAN®, BacT/ALERTPF Pediatric FAN, VersaTREK® REDOX 1/2
Throughput	Four (4) samples/iC-Processor™	One (1) sample/processor

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Intended Use

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

The iC-GPC Assay™ detects organism DNA and identifies the following bacterial species and resistance markers:

Bacterial Species	Resistance Markers
Staphylococcus aureusStaphylococcus epidermidisStreptococcus pneumoniaeEnterococcus faecalisEnterococcus faecium	mecA- associated with methicillin resistancevanA- associated with vancomycin resistancevanB- associated with vancomycin resistance

The iC-GPC Assay™ detects the mecA resistance marker, inferring mecA-mediated methicillin resistance, and the vanA and vanB resistance markers, inferring vanA/vanB-mediated vancomycin resistance. In mixed growth, the iC-GPC Assay™ does not specifically attribute van-mediated vancomycin resistance to either E. faecalis or E. faecium, or mecA-mediated methicillin resistance to either S. aureus or S. epidermidis.

Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing, identification of organisms not detected by the iC-GPC 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-GPC Assay™ labeling for a more complete list of warnings, precautions and contraindications.

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Methodology

The iC-GPC Assay™ utilizes polymerase chain reaction (PCR) for the amplification of specific targets and detects the amplified target DNA with fluorescence-based microarray hybridization. The iC-GPC Assay™ uses proprietary ARM-PCR (Amplicon Rescued Multiplex PCR) technology allowing for multiple targets to be amplified in one reaction. Targets are detected directly from patient positive blood cultures, confirmed by Gram stain to contain gram positive cocci. Testing is performed within a closed, disposable cassette that contains all the reagents required to complete the iC-GPC Assay™, including a universal microarray.

To operate, the user opens the iC-GPC Cassette™ cap and pipettes an aliquot of the positive blood culture sample into the sample well of the cassette is then inserted into the iC-Processor™, which performs the processes of DNA extraction, multiplex amplification, and microarray After processing is complete, the cassette is inserted into the iC-Reader™ for hybridization. fluorescence-based detection and data analysis. Final results are generated by iC-Report, computer software for data acquisition, analysis, and display.

Extraction, amplification, and hybridization 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-GPC Cassette™ which communicates with the iC-Processor™. To access and pierce the foil-sealed reagent wells located in the bottom well plate of the cassette, the processor manipulates the cassette to move the internal 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 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 4 iC-Cassettes™ at one time. The results are interpreted via the iC-Report™ software and displayed for the user on an iMac® computer. Raw data and result interpretations are stored within the iMac®; raw data is accessible to authorized personnel only and is not available to the end user.

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Performance Data

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

TABLE 1: iC-GPC Assay Targets
Organism	Target Gene	Acronym
Staphylococcus epidermidis	gseA	SE
Staphylococcus aureus	nuc	SA
Streptococcus pneumoniae	lytA	SPN
Enterococcus faecalis	ddl	EFLS
Enterococcus faecium	fcm (ddlEFCM)	EFCM

Bottle Ring

A study was performed to establish the lowest level of each iC-GPC Assay target organism at initial bottle positivity (bottle "ring"). The nineteen organisms used to define target limits of detection were evaluated. Organisms were inoculated into BD BACTEC Plus Aerobic blood culture bottles with human Bottles were allowed to incubate on the blood culture system until initial bottle blood added. positivity. Bottles were removed from the incubator within two hours of bottle ring, and plating and subsequent colony counts were performed to confirm organism concentrations. A minimum of five bottles were evaluated for each strain. The average concentrations at bottle ring are presented in Table 2 below. These concentrations, representative of the lowest levels that may be observed in a clinical setting, are equivalent to or greater than the respective target limits of detection (see below).

TABLE 2: Target Organism Concentrations at Bottle "Ring"
Organism	Average Concentration (CFU/mL)
SE 700566	$2.68 \times 10^7$
SE 35984	$2.04 \times 10^7$
SE 12228	$2.59 \times 10^7$
SE 49134	$1.01 \times 10^7$
SA 700699	$5.00 \times 10^8$
SA BAA 1768	$5.24 \times 10^7$
SA BAA 977	$1.14 \times 10^8$
SA 25923	$6.06 \times 10^8$
SPN 6301	$5.40 \times 10^6$
SPN 700673	$5.95 \times 10^6$
EFLS 51299	$4.72 \times 10^{10}$
EFLS 700802	$2.22 \times 10^8$
EFLS JMI 12536	$2.27 \times 10^8$
EFLS 29212	$5.53 \times 10^{10}$
EFLS BAA 2128	$5.77 \times 10^7$
EFCM 700221	$1.16 \times 10^9$
EFCM 51559	$5.87 \times 10^8$
EFCM 35667	$2.86 \times 10^7$
EFCM BAA 2127	$2.70 \times 10^8$

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Reproducibility

To confirm site-to-site, operator, system-to-system, and lot-to-lot reproducibility of the iC-GPC Assay, a representative panel of target organisms and one non-target organism were tested at two concentrations: initial bottle positivity and eight hours beyond initial bottle positivity. Organisms were grown to the appropriate concentration in BD BACTEC Plus Aerobic/F 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 in-house. The six organism panel was tested in replicates of three across five, non-consecutive days. Testing was evaluated across three cassette lots and four iC-Systems. Table 3 below summarizes results by iC-GPC Assay target and concentration. Testing confirmed that performance of the iC-GPC Assay for use on the iC-System is reproducible across sites, operators, systems, and lots.

TABLE 3: iC-GPC Reproducibility Performance by Target & Concentration
Organism/Gene Target/Concentration	OverallPerformance	OverallPerformance %[95% CI]	FalseNegatives	FalsePositives	Positive ControlsCheck Failures	SystemFailures
S. epidermidis (gseA)Bottle Ring	89/89	100.0[95.86-100.0]	0/89(0.00%)	1/976(0.10%)	0/90(0.00%)	1/90(1.11%)
S. epidermidis (gseA)Bottle Ring + 8 hours	90/90	100.0[95.91-100.0]	0/90(0.00%)	0/975(0.00%)	0/90(0.00%)	0/90(0.00%)
S. aureus (nuc)Bottle Ring	89/89	100.0[95.86-100.0]	0/89(0.00%)	0/976(0.00%)	1/90(1.11%)	0/90(0.00%)
S. aureus (nuc)Bottle Ring + 8 hours	89/90	98.9[93.97-99.80]	1/90(1.12%)	0/975(0.00%)	0/90(0.00%)	0/90(0.00%)
S. pneumoniae (lytA)Bottle Ring	88/88	100.0[95.82-100.0]	0/88(0.00%)	0/977(0.00%)	1/90(1.11%)	1/90(1.11%)
S. pneumoniae (lytA)Bottle Ring + 8 hours	89/89	100.0[95.86-100.0]	0/89(0.00%)	0/976(0.00%)	0/90(0.00%)	1/90(1.11%)
E. faecalis (ddl)Bottle Ring	89/89	100.0[95.86-100.0]	0/89(0.00%)	2/976(0.20%)	0/90(0.00%)	1/90(1.11%)
E. faecalis (ddl)Bottle Ring + 8 hours	89/89	100.0[95.86, 100.0]	0/89(0.00%)	3/976(0.31%)	1/90(1.11%)	0/90(0.00%)
E. faecium (fcm)Bottle Ring	90/90	100.0[95.91-100.0]	0/90(0.00%)	0/975(0.00%)	0/90(0.00%)	0/90(0.00%)
E. faecium (fcm)Bottle Ring + 8 hours	89/89	100.0[95.86-100.0]	0/89(0.00%)	0/976(0.00%)	1/90(1.11%)	0/90(0.00%)
mecABottle Ring	177/178	99.4[96.89-99.90]	1/178(0.56%)	1/887(0.11%)	1/180(0.56%)	1/180(0.56%)
mecABottle Ring + 8 hours	180/180	100.0[97.91-100.0]	0/180(0.00%)	0/885(0.00%)	0/180(0.00%)	0/180(0.00%)
vanABottle Ring	90/90	100.0[95.91-100.0]	0/90(0.00%)	0/975(0.00%)	0/90(0.00%)	0/90(0.00%)
vanABottle Ring + 8 hours	89/89	100.0[95.86-100.0]	0/89(0.00%)	0/976(0.00%)	1/90(1.11%)	0/90(0.00%)
vanBBottle Ring	89/89	100.0[95.86-100.0]	0/89(0.00%)	0/976(0.00%)	0/90(0.00%)	1/90(1.11%)
vanBBottle Ring + 8 hours	89/89	100.0[95.86, 100.0]	0/89(0.00%)	0/976(0.00%)	1/90(1.11%)	0/90(0.00%)

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Limit of Detection (LoD)

A study was conducted to determine the limit of detection, defined as the lowest concentration (CFU/mL) of analyte that can be detected approximately 95% of the time, for the eight targets detected by the iC-GPC Assay. A panel of nineteen organisms was evaluated, including a minimum of two bacterial strains per target analyte. Three concentrations were tested for each LoD panel member in replicates of twenty on three unique cassette lots. The LoD of each target is considered the lowest concentration with an approximately 95% detection rate, presented in Table 4 below.

TABLE 4: iC-GPC Assay Target LoD
iC-GPC Assay Target	Phenotype Identification	Defined LoD (CFU/mL)
gseA	Staphylococcus epidermidis	$1.6 \times 10^6 - 1.7 \times 10^7$
nuc	Staphylococcus aureus	$1.7 \times 10^6 - 4.4 \times 10^6$
mecA	Associated with methicillin resistance	$7.4 \times 10^5 - 9.5 \times 10^6$
lytA	Streptococcus pneumoniae	$1.3 \times 10^6 - 6.0 \times 10^6$
ddl	Enterococcus faecalis	$3.0 \times 10^5 - 5.8 \times 10^6$
fcm	Enterococcus faecium	$4.9 \times 10^6 - 7.9 \times 10^6$
vanA	Associated with vancomycin resistance	$7.2 \times 10^5 - 1.1 \times 10^7$
vanB	Associated with vancomycin resistance	$3.9 \times 10^6 - 5.8 \times 10^6$

Analytical Reactivity (Inclusivity)

The analytical reactivity (inclusivity) for each iC-GPC Assay target was evaluated using multiple wellcharacterized and clinically relevant strains chosen to represent temporal, geographic, and genetic diversity. Inclusivity panel organisms included the following:

5 Staphylococcus epidermidis, mecA negative strains O
6 Staphylococcus epidermidis, mecA positive strains O
O 5 Staphylococcus aureus, mecA negative strains
58 Staphylococcus aureus, mecA positive strains, representing various pulse-O
- field gel electrophoresis types and including the following:
  - . Vancomycin-intermediate SA (VISA)
  - . Panton-Valentine Leukocidin (PVL)-producing SA
  - . ATCC 43300 (hetero-resistant, mecA positive)
2 borderline oxacillin-resistant Staphylococcus aureus strains (BORSA) O
O 10 Streptococcus pneumoniae strains
5 Enterococcus faecalis, vanA/vanB negative strains O
5 Enterococcus faecalis, vanA or vanB positive strains O
5 Enterococcus faecium, vanA/vanB negative strains O
5 Enterococcus faecium, vanA or vanB positive strains O

A total of 106 organisms were evaluated for iC-GPC Assay Inclusivity testing. Strains were tested near the target LoD (2-3x LoD) or at the lowest level of bottle positivity. Testing was performed in the event of a false negative result, the stain was retested near the target LoD in replicates of ten. All expected targets were detected by the iC-GPC Assay. Results of inclusivity testing are summarized in Table 5 below.

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TABLE 5: iC-GPC Inclusivity Results
Species	Strain ID	Other IDs/Notes	Targets Detected/Total Replicates
S. epidermidis	Z318	N/A	3/3
S. epidermidis	Z0801689	HER 1292	3/3
S. epidermidis	ATCC 700583	N/A	3/3
S. epidermidis	Z291	ATCC 14990	3/3
S. epidermidis	Z049	N/A	3/3
S. epidermidis, mecA (+)	Z0801651	RP62A	3/3
S. epidermidis, mecA (+)	Z256	N/A	3/3
S. epidermidis, mecA (+)	Z257	N/A	3/3
S. epidermidis, mecA (+)	Z258	N/A	12/13
S. epidermidis, mecA (+)	Z259	N/A	3/3
S. epidermidis, mecA (+)	ATCC 29887	N/A	3/3
S. aureus	Z021	ATCC 6538P	3/3
S. aureus	ATCC 12600	N/A	3/3
S. aureus	Z0801675	N/A	3/3
S. aureus	Z057	N/A	3/3
S. aureus	Z153	N/A	3/3
S. aureus, mecA (+)	HM-466	131	3/3
S. aureus, mecA (+)	HM-467	177	3/3
S. aureus, mecA (+)	NR-10129	TCH60	3/3
S. aureus, mecA (+)	NR-10189	HFH-30364, USA400, PVL+	3/3
S. aureus, mecA (+)	NR-10192	HFH-30106, non USA100-1100	3/3
S. aureus, mecA (+)	NR-13524	H342087	3/3
S. aureus, mecA (+)	NR-28983	S0385	3/3
S. aureus, mecA (+)	NR-45872	HIP07930, USA600	3/3
S. aureus, mecA (+)	NR-45880	LIM1	3/3
S. aureus, mecA (+)	NR-45890	BR 5, VISA	3/3
S. aureus, mecA (+)	NR-46062	H2138 (isolate 10)	3/3
S. aureus, mecA (+)	NR-46063	P1V44, VISA	3/3
S. aureus, mecA (+)	NR-46072	1078, USA700	3/3
S. aureus, mecA (+)	NR-46080	AIS 2006061, USA1000, PVL+	3/3
S. aureus, mecA (+)	NR-46081	HIP12899, USA1100, PVL+	3/3
S. aureus, mecA (+)	NR-46218	GA-442, USA700	3/3
S. aureus, mecA (+)	NR-46070	USA300-0114	3/3
S. aureus, mecA (+)	NR-46171	CA-126, USA100	3/3
S. aureus, mecA (+)	NR-46172	CA-127, USA300, PVL+	3/3
S. aureus, mecA (+)	NR-46177	CA-347, USA600	3/3
S. aureus, mecA (+)	NR-46180	CA-409, USA200	3/3
S. aureus, mecA (+)	NR-46182	CA-513, USA800	3/3
S. aureus, mecA (+)	NR-46191	CO-34, USA300, PVL+	3/3
S. aureus, mecA (+)	NR-46197	CO-72, USA800	3/3
TABLE 5: iC-GPC Inclusivity Results
Species	Strain ID	Other IDs/Notes	Targets Detected/Total Replicates
S. aureus, mecA (+)	NR-46199	CT-110, USA100	3/3
S. aureus, mecA (+)	NR-46207	CT-58, USA500	3/3
S. aureus, mecA (+)	NR-46215	GA-356	3/3
S. aureus, mecA (+)	NR-46221	GA-656, USA800	3/3
S. aureus, mecA (+)	NR-46223	GA-92, USA300, PVL+	3/3
S. aureus, mecA (+)	NR-46224	NY-315, USA600	3/3
S. aureus, mecA (+)	NR-46250	OR-130, USA100	3/3
S. aureus, mecA (+)	NR-46251	OR-131, USA200	3/3
S. aureus, mecA (+)	NR-46261	TN-112, USA300	3/3
S. aureus, mecA (+)	NR-46269	TN-82, USA200	3/3
S. aureus, mecA (+)	NR-41875	M0001	3/3
S. aureus, mecA (+)	NR-41876	M0006	3/3
S. aureus, mecA (+)	NR-41877	M0055	3/3
S. aureus, mecA (+)	NR-41878	M0102	3/3
S. aureus, mecA (+)	NR-41879	M0108	3/3
S. aureus, mecA (+)	NR-41880	M0197	3/3
S. aureus, mecA (+)	NR-41881	M0200	3/3
S. aureus, mecA (+)	NR-41882	M0288	3/3
S. aureus, mecA (+)	NR-41883	M0334	3/3
S. aureus, mecA (+)	NR-41887	M0663	3/3
S. aureus, mecA (+)	NR-41889	M0934	3/3
S. aureus, mecA (+)	NR-41890	M0943	3/3
S. aureus, mecA (+)	NR-41895	M1510	12/13
S. aureus, mecA (+)	NR-41896	M1565	3/3
S. aureus, mecA (+)	NR-10187	HFH-29994, USA100	3/3
S. aureus, mecA (+)	NR-13525	F338081	3/3
S. aureus, mecA (+)	NR-13526	W342179	3/3
S. aureus, mecA (+)	NR-13533	S247312	3/3
S. aureus, mecA (+)	NR-13546	SU-1	3/3
S. aureus, mecA (+)	NR-30544	HI049, USA300, PVL+	3/3
S. aureus, mecA (+)	NR-45924	LinR #12	3/3
S. aureus, mecA (+)	ATCC 700698	N/A	3/3
S. aureus, mecA (+)	ATCC BAA44	N/A	3/3
S. aureus, mecA (+)	ATCC 43300	Hetero-resistant	3/3
BORSA	MCW 109	N/A	3/3
BORSA	MCW 141	N/A	3/3
S. pneumoniae	Z022	Clinical isolate, serotype 19F	3/3
S. pneumoniae	Z073	Clinical isolate, serotype 19F	3/3
S. pneumoniae	Z319	Clinical isolate, serotype 12F	3/3
S. pneumoniae	Z278	ATCC BAA-255, R6 (non-virulent)	3/3
TABLE 5: iC-GPC Inclusivity Results
Species	Strain ID	Other IDs/Notes	Targets Detected/Total Replicates
S. pneumoniae	Z279	PHE NCTC 11910, serotype 23F	3/3
S. pneumoniae	Z280	PHE NCTC 11897, serotype 9V	3/3
S. pneumoniae	Z282	ATCC 33400, NCTC 7465, serotype 1	3/3
S. pneumoniae	Z295	ATCC 49619, serotype 19F	3/3
S. pneumoniae	Z261	Clinical isolate	3/3
S. pneumoniae	Z262	Clinical isolate	3/3
E. faecalis	Z0801637	N/A	3/3
E. faecalis	ATCC 33186	N/A	3/3
E. faecalis	ATCC 49532	N/A	3/3
E. faecalis	Z289	ATCC 19433	3/3
E. faecalis	Z266	ATCC 6055	3/3
E. faecalis, vanB (+)	Z0801693	vanB	3/3
E. faecalis, vanA (+)	Z324	vanA	3/3
E. faecalis, vanA (+)	Z267	PHE NCTC 12201- vanA	3/3
E. faecalis, vanA (+)	Z269	PHE NCTC 12203- vanA	3/3
E. faecalis, vanB (+)	ATCC 51575	vanB	3/3
E. faecium	Z322	N/A	3/3
E. faecium	ATCC 8459	N/A	3/3
E. faecium	Z265	ATCC 9756	3/3
E. faecium	Z290	ATCC 19434	3/3
E. faecium	Z320	ATCC 19634	3/3
E. faecium, vanA (+)	Z0801892	vanA	3/3
E. faecium, vanB (+)	Z323	vanB	3/3
E. faecium, vanA (+)	Z270	PHE NCTC 12202- vanA	3/3
E. faecium, vanA (+)	Z271	PHE NCTC 12204- vanA	3/3
E. faecium, vanA (+)	Z260	vanA	3/3

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{14}------------------------------------------------

Analytical Exclusivity

Analytical specificity of the iC-GPC Assay was evaluated by testing a comprehensive panel of non-target microorganisms that may be encountered in positive blood cultures. Exclusivity panel members included organisms phylogenetically related to iC-GPC target organisms as well as common blood culture contaminants. A total of ninety-four (94) exclusivity organisms were tested. Potential cross-reactivity was evaluated by testing the exclusivity panel organisms at high concentrations in blood culture bottle/blood media. Exclusivity results are presented in Table 6 below; performance is based on the observation of all expected negative results. In the event of a false positive result, the organism was retested in replicates of three (3) or ten (10). One organism, Streptococcus bovis, demonstrated reproducible cross-reactivity with the iC-GPC target Enterococcus faecalis.

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TABLE 6: iC-GPC Exclusivity Results	Test Concentration (CFU/mL, oras designated, bottle ring orTCID50/mL)	Exclusivity
Abiotrophia defectiva	3.09 × 108	3/3
Acinetobacter baumannii	6.55 × 108	3/3
Acinetobacter lwoffi	5.70 × 108	3/3
Aerococcus viridans	5.05 × 107	3/3
Aeromonas hydrophila	3.50 × 108	3/3
Alcaligenes faecalis	1.01 × 1010	3/3
Anaerococcus tetradius	3.55 × 108	3/3
Aspergillus niger	8.10 × 107	3/3
Bacillus cereus	7.30 × 106	3/3
Bacteroides fragilis	4.20 × 109	3/3
Campylobacter coli	2.55 × 108	3/3
Campylobacter jejuni	2.24 × 108	3/3
Candida albicans	8.00 × 107	3/3
Candida catenulata	1.14 × 109	3/3
Candida dubliniensis	7.05 × 106	3/3
Candida glabrata	1.15 × 108	3/3
Candida guilliermondii	1.03 × 107	3/3
Candida krusei	2.82 × 107	5/61
Candida parapsilosis	3.15 × 107	3/3
Candida tropicalis	7.65 × 107	2/2
Citrobacter amalonaticus	2.35 × 109	3/3
Citrobacter freundii	6.40 × 108	3/3
Citrobacter koseri	3.29 × 109	3/3
Citrobacter sedlakii	1.70 × 109	3/3
Clostridium difficile (NAP-1 toxigenic)	2.44 × 107	3/3
Clostridium difficile (non-toxigenic)	2.97 × 107	3/3
Clostridium oedematiens (novyi)	5.70 × 106	3/3
Collinsella aerofaciens	5.95 × 107	3/3
Corynebacterium amycolatum	3.79 × 108	5/62
Corynebacterium genitalium	2.50 × 108	3/3
Corynebacterium jeikeium	4.19 × 108	3/3
Coxsackie virus	1 × 106.34 (TCID50)	3/3
Cryptococcus neoformans	1.08 × 108	3/3
Cytomegalovirus	1 × 105.07 (TCID50)	3/3
Echovirus	1 × 107.77 (TCID50)	3/3
Edwardsiella tarda	5.05 × 109	3/3
Eggerthella lenta	1.42 × 108	3/3
Enterobacter aerogenes	6.50 × 109	3/3
TABLE 6: iC-GPC Exclusivity Results
Exclusivity Organism	Test Concentration (CFU/mL, oras designated, bottle ring orTCID50/mL)	Exclusivity
Enterococcus avium	$5.85 \times 10^7$	3/3
Enterococcus casseliflavus	$9.60 \times 10^6$	3/3
Enterococcus cecorum	$3.80 \times 10^8$	3/3
Enterococcus dispar	$6.10 \times 10^8$	3/3
Enterococcus gallinarum	$3.20 \times 10^8$	3/3
Enterococcus hirae	$1.04 \times 10^9$	3/3
Enterococcus raffinosus	$4.67 \times 10^8$	3/3
Enterovirus Type 71	$1 \times 10^{6.10}$ (TCID50)	3/3
Escherichia coli	$1.27 \times 10^9$	3/3
Escherichia hermannii	$3.09 \times 10^9$	3/3
Fusobacterium varium	$1.25 \times 10^9$	3/3
Klebsiella oxytoca	$7.25 \times 10^9$	3/3
Klebsiella pneumoniae	$2.27 \times 10^9$	3/3
Kocuria kristinae	$3.45 \times 10^8$	3/3
Kytococcus schroeteri	$1.85 \times 10^8$	3/3
Lactobacillus acidophilus	$5.30 \times 10^7$	3/3
Lactobacillus plantarum subsp.plantarum	$1.53 \times 10^9$	3/3
Lactobacillus reuteri	$1.24 \times 10^8$	5/63
Lactococcus lactis	$2.57 \times 10^9$	3/3
Leminorella grimontii	$1.22 \times 10^9$	3/3
Leuconostoc mesenteroides	$3.17 \times 10^7$	3/3
Micrococcus luteus	$1.13 \times 10^7$	3/3
Oerskovia enterophila	$1.33 \times 10^{10}$	3/3
Pediococcus pentosaceus	$2.82 \times 10^9$	3/3
Planococcus citreus	$1.14 \times 10^9$	3/3
Propionibacterium acnes	$9.75 \times 10^7$	3/3
Proteus mirabilis	$9.25 \times 10^8$	3/3
Proteus penneri	$1.11 \times 10^9$	3/3
Proteus vulgaris	$1.13 \times 10^9$	3/3
Providencia alcalifaciens	$1.62 \times 10^9$	10/114
Providencia rettgeri	$1.05 \times 10^9$	3/3
Providencia stuartii	$1.48 \times 10^9$	3/3
Pseudomonas aeruginosa	$2.14 \times 10^8$	3/3
Pseudomonas putida	$4.95 \times 10^8$	3/3
Rothia mucilaginosus	$1.60 \times 10^8$	3/3
Salmonella spp (typhimurium)	$4.45 \times 10^9$	3/3
Staphylococcus capitis	$4.05 \times 10^7$	3/3
Staphylococcus delphini	$2.12 \times 10^9$	3/3
TABLE 6: iC-GPC Exclusivity Results
Exclusivity Organism	Test Concentration (CFU/mL, oras designated, bottle ring orTCID50/mL)	Exclusivity
Staphylococcus hominis	$1.87 \times 10^8$	3/3
Staphylococcus intermedius	$8.55 \times 10^7$	5/65
Staphylococcus lugdunensis	$3.05 \times 10^9$	3/3
Staphylococcus lutrae	$6.00 \times 10^9$	3/3
Staphylococcus pettenkoferi	$1.36 \times 10^9$	3/3
Staphylococcus schleiferi	$2.67 \times 10^9$	3/3
Staphylococcus schleiferi subsp. coagulans	$2.67 \times 10^8$	5/66
Staphylococcus warneri	$6.48 \times 10^8$	3/3
Streptococcus agalactiae	$6.05 \times 10^8$	13/147
Streptococcus anginosus	$4.65 \times 10^8$	13/148
Streptococcus bovis	$5.50 \times 10^8$	3/149
Streptococcus dysgalactiae	$8.35 \times 10^6$	3/3
Streptococcus intermedius	Bottle ring + 8 hours	11/1210
Streptococcus mitis	Bottle ring + 8 hours	3/3
Streptococcus pseudopneumoniae	Bottle ring + 8 hours	3/3
Streptococcus pyogenes	$8.20 \times 10^8$	3/3
Streptococcus salivarus	$8.85 \times 10^7$	3/3
Streptococcus uberis	$7.45 \times 10^7$	3/3

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{17}------------------------------------------------

Candida krusei: 1/3 false positive E. faecalis in initial testing, 3/3 repeats negative. 1)

Corynebacterium amycolatum: 1/3 false positive S. epidermidis in initial testing, 3/3 repeats negative.
Lactobacillus reuteri: 1/3 false positive mecA in initial testing, 3/3 repeats negative. Note that mecA would not be reported as the S. aureus and S. epidermidis targets were negative as expected.

1. Providencia alcalifaciens: 1/3 false positive S. aureus in initial testing, 8/8 repeats negative.
  Staphylococus intermedius: 1/3 false positive mecA in initial testing, 3/3 repeats negative. Note that mecA would not be 5) reported as the S. aureus and S. epidermidis targets were negative as expected.
୧) Staphylococus schlejferi subsp. coagulans: 1/3 false positive mecA in initial testing, 3/3 repeats negative. Note that mecA would not be reported as the S. aureus and S. epidermidis targets were negative as expected.
1. Streptococus agalactiae: 1/3 false positive mecA in initial testing, 11/11 repeats negative. Note that mecA would not be reported as the S. aureus and S. epidermidis targets were negative as expected.
Streptococcus anginosus: 1/2 false positive E. faecalis in initial testing, 12/12 repeats negative. 8)
1. Streptococcus bovis: 1/2 false positive E. faecalis in initial testing, 10/12 false positive E. faecalis in repeat testing
1. Streptococcus intermedius: 1/3 false positive E. faecalis in initial testing, 9/9 repeats negative

Microbial Interference

Potential microbial interference was evaluated by testing high concentrations of 85 exclusivity organisms in combination with iC-GPC target organisms present near LoD concentrations. Microbial interference results are presented in Table 7 below; performance is based on all expected targets detected. In the event of a false negative result, the combination was repeated with the target organism near target LoD concentrations in replicates of 10. If additional false negative results were observed, testing was repeated with the target organism at bottle ring concentrations. Microbial interference was not observed for the majority of organism combinations. For those combinations with unexpected initial false negative results, additional replicate testing at the same concentration or at a higher "bottle ring" concentration generated 100% iC-GPC Assay target detection as expected.

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TABLE 7: iC-GPC Microbial Interference Results
		SE	SA	SPN	EFLS	EFCM
Exclusivity Organism	Test Concentration(CFU/mL)	Test Concentration for initial three replicates (CFU/mL)
		1.4 x 107	1.4 x 107	BottleRing	1.2 x 107	1.2 x 107
Abiotrophia defective	1.54 x 108	3/3	3/3	3/3	3/3	3/3
Acinetobacter baumannii	3.28 x 108	3/3	3/3	3/3	3/3	3/3
Acinetobacter lwoffi	2.85 x 108	3/3	3/3	3/3	3/3	3/3
Aerococcus viridans	2.53 x 107	3/3	3/3	3/3	12/131	3/3
Aeromonas hydrophila	1.75 x 108	3/3	3/3	3/3	3/3	3/3
Anaerococcus tetradius	1.78 x 108	3/3	3/3	3/3	3/3	3/3
Bacillus cereus	3.65 x 106	3/3	3/3	3/3	3/3	3/3
Bacteroides fragilis	2.10 × 109	3/3	3/3	2/2	3/3	3/3
Campylobacter coli	1.28 x 108	3/3	18/222	3/3	3/3	3/3
Campylobacter jejuni	1.12 x 108	3/3	3/3	3/3	3/3	3/3
Candida albicans	4.00 × 107	3/3	3/3	3/3	3/3	3/3
Candida catenulate	5.68 x 108	3/3	3/3	3/3	3/3	3/3
Candida dubliniensis	3.53 × 106	3/3	3/3	3/3	3/3	3/3
Candida glabrata	5.75 x 107	3/3	3/3	3/3	3/3	3/3
Candida guilliermondii	5.15 x 106	3/3	3/3	3/3	3/3	3/3
Candida krusei	1.41 x 107	3/3	3/3	3/3	20/223	3/3
Candida parapsilosis	1.58 x 107	3/3	3/3	3/3	3/3	3/3
Candida tropicalis	3.83 x 107	3/3	3/3	3/3	3/3	3/3
Citrobacter amalonaticus	1.18 x 108	3/3	3/3	3/3	3/3	5/64
Citrobacter freundii	3.20 x 108	3/3	3/3	3/3	3/3	3/3
Citrobacter koseri	1.64 x 109	3/3	3/3	3/3	3/3	3/3
Citrobacter sedlakii	8.50 × 108	3/3	3/3	3/3	3/3	3/3
Clostridium difficile (NAP-1toxigenic)	1.22 x 107	3/3	5/65	3/3	3/3	3/3
Clostridium difficile (non-toxigenic)	1.48 x 107	5/66	3/3	3/3	3/3	3/3
Clostridium oedematiens (novyi)	2.85 x 106	3/3	3/3	3/3	3/3	3/3
Collinsella aerofaciens	2.98 x 107	3/3	3/3	3/3	3/3	3/3
Corynebacterium amycolatum	1.89 x 108	3/3	3/3	3/3	3/3	3/3
Corynebacterium genitalium	1.25 x 108	3/3	3/3	3/3	3/3	3/3
Corynebacterium jeikeium	2.09 × 108	3/3	3/3	3/3	2/2	3/3
Edwardsiella tarda	2.53 × 109	3/3	3/3	3/3	3/3	3/3
Enterobacter aerogenes	3.25 × 109	3/3	3/3	3/3	3/3	3/3
Enterobacter cloacae	1.36 × 109	3/3	3/3	3/3	3/3	3/3
Enterococcus avium	2.93 x 107	3/3	3/3	3/3	3/3	3/3
Enterococcus casseliflavus	4.80 x 106	3/3	3/3	3/3	3/3	3/3
Enterococcus cecorum	1.90 x 108	3/3	3/3	3/3	3/3	3/3
Enterococcus dispar	3.05 × 108	3/3	3/3	3/3	3/3	3/3
TABLE 7: iC-GPC Microbial Interference Results
		SE	SA	SPN	EFLS	EFCM
Exclusivity Organism	Test Concentration(CFU/mL)	Test Concentration for initial three replicates (CFU/mL)
		1.4 x 107	1.4 x 107	BottleRing	1.2 x 107	1.2 x 107
Enterococcus gallinarum	1.60 x 108	3/3	3/3	3/3	3/3	3/3
Enterococcus hirae	5.18 x 108	3/3	3/3	3/3	3/3	3/3
Enterococcus raffinosus	2.33 x 108	3/3	3/3	3/3	3/3	3/3
Escherichia coli	6.35 × 108	3/3	3/3	3/3	3/3	3/3
Escherichia hermannii	1.54 × 109	3/3	3/3	3/3	11/127	3/3
Fusobacterium varium	6.23 x 108	3/3	3/3	3/3	3/3	3/3
Klebsiella oxytoca	3.63 x 109	3/3	3/3	3/3	3/3	3/3
Klebsiella pneumoniae	1.13 x 109	3/3	3/3	3/3	3/3	3/3
Kocuria kristinae	1.73 x 108	3/3	12/138	3/3	3/3	3/3
Kytococcus schroeteri	9.25 x 107	3/3	3/3	3/3	3/3	3/3
Lactobacillus acidophilus	2.65 x 107	3/3	3/3	3/3	3/3	3/3
Lactobacillus plantarumsubsp.plantarum	7.63 x 108	3/3	3/3	3/3	3/3	3/3
Lactobacillus reuteri	6.18 x 107	3/3	3/3	3/3	3/3	3/3
Lactococcus lactis	1.28 × 109	3/3	3/3	3/3	3/3	3/3
Leminorella grimontii	6.08 x 108	3/3	3/3	3/3	3/3	3/3
Leuconostoc mesenteroides	1.58 x 107	3/3	3/3	3/3	3/3	3/3
Micrococcus luteus	5.63 x 106	3/3	11/129	3/3	3/3	3/3
Oerskovia enterophila	6.63 x 109	3/3	3/3	3/3	3/3	3/3
Pediococcus pentosaceus	1.41 × 109	3/3	3/3	3/3	3/3	3/3
Propionibacterium acnes	4.88 x 107	3/3	3/3	3/3	3/3	3/3
Proteus mirabilis	4.63 x 108	3/3	12/1310	3/3	11/1211	3/3
Proteus penneri	5.55 x 108	3/3	11/1312	3/3	3/3	3/3
Proteus vulgaris	5.63 x 108	3/3	3/3	3/3	3/3	3/3
Providencia alcalifaciens	8.08 × 108	3/3	3/3	3/3	3/3	3/3
Providencia rettgeri	5.25 x 108	3/3	3/3	3/3	3/3	3/3
Providencia stuartii	7.40 x 108	3/3	5/613	3/3	3/3	3/3
Pseudomonas aeruginosa	1.07 x 108	3/3	3/3	3/3	3/3	3/3
Pseudomonas putida	2.48 x 108	3/3	3/3	3/3	3/3	3/3
Rothia mucilaginosus	8.00 × 107	3/3	3/3	3/3	3/3	3/3
Salmonella spp (typhimurium)	2.23 x 109	3/3	3/3	3/3	3/3	3/3
Staphylococcus capitis	2.03 x 107	3/3	21/2314	3/3	3/3	3/3
Staphylococcus delphini	1.06 x 109	3/3	12/1315	3/3	3/3	3/3
Staphylococcus haemolyticus	9.75 x 106	3/3	3/3	11/1216	3/3	3/3
Staphylococcus hominis	9.33 x 107	3/3	3/3	3/3	3/3	3/3
Staphylococcus intermedius	4.28 x 107	3/3	3/3	3/3	3/3	3/3
Staphylococcus lugdunensis	1.53 × 109	3/3	3/3	3/3	3/3	3/3
Staphylococcus lutrae	3.00 x 109	3/3	3/3	3/3	3/3	3/3
TABLE 7: iC-GPC Microbial Interference Results
Exclusivity Organism	Test Concentration(CFU/mL)	SE	SA	SPN	EFLS	EFCM
		Test Concentration for initial three replicates (CFU/mL)
		1.4 × 107	1.4 × 107	BottleRing	1.2 × 107	1.2 × 107
Staphylococcus pettenkoferi	6.78 × 108	12/1317	3/3	3/3	3/3	3/3
Staphylococcus schleiferi	1.33 × 109	3/3	3/3	3/3	3/3	3/3
Staphylococcus schleiferi subsp.coagulans	1.22 × 109	3/3	3/3	3/3	3/3	3/3
Staphylococcus warneri	3.20 × 108	3/3	3/3	3/3	3/3	3/3
Streptococcus agalactiae	3.03 × 108	3/3	3/3	3/3	3/3	3/3
Streptococcus anginosus	2.33 × 108	3/3	3/3	3/3	3/3	12/1318
Streptococcus bovis	2.75 × 108	5/619	3/3	3/3	3/3	3/3
Streptococcus dysgalactiae	4.18 × 107	3/3	3/3	3/3	3/3	3/3
Streptococcus intermedius	8.25 × 107	3/3	3/3	3/3	3/3	3/3
Streptococcus mitis	Bottle ring + 8 hours	3/3	3/3	3/3	3/3	3/3
Streptococcus pseudopneumoniae	Bottle ring + 8 hours	3/3	3/3	3/3	3/3	3/3
Streptococcus pyogenes	4.10 × 108	3/3	3/3	3/3	3/3	3/3
Streptococcus salivarus	4.43 × 107	3/3	12/1320	3/3	3/3	2/2
Streptococcus uberis	3.73 × 107	3/3	3/3	3/3	3/3	3/3

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1 false negative vanB in initial testing (2/3), 10/10 repeats passed 1)

2 false negative S. aureus and 1 false negative mec4 in initial testing (1/3), 2 false negative mecA 2) in repeat testing near LoD (8/10). No false negatives in repeat testing at bottle ring concentration (9/9).

1. 1 false negative vanB in initial testing (2/3), 1 false negative vanB in repeat testing near LoD (9/10). No false negatives in repeat testing at bottle ring concentration (9/9).
1 false positive mec4 in initial testing (2/3), 3/3 repeats passed. Note that mecA would not be reported as the S aureus and S. 4) epidermidis targets were negative as expected.
1. 1 false positive S. epidermidis in initial testing (2/3), 3/3 repeats passed
1. 1 false positive S. aureus in initial testing (2/3), 3/3 repeats passed
1. 1 false negative vanB in initial testing (2/3), 9/9 repeats passed
1. 1 false negative S. aureus and 1 false negative mecA in initial testing (2/3), 10/10 repeats passed
1 false negative S. aureus and 1 false negative mecA in initial testing (2/3), 9/9 repeats passed 9)
1. 1 false negative S. aureus in initial testing (2/3), 10/10 repeats passed
1. 1 false negative vanB in initial testing (2/3), 9/9 repeats passed
1. 2 false negative mecA in initial testing (1/3), 10/10 repeats passed
1. 1 false positive S. epidermidis in initial testing (2/3), 3/3 repeats passed
1. 1 false negative mecA in initial testing (2/3), 1 false negative mecA in repeat testing near LoD (9/10). No false negatives in repeat testing at bottle ring concentration (9/9).
1. 1 false negative S. aureus and 1 false negative mecA in initial testing (2/3), 10/10 repeats passed
1. 1 false negative S. pneumoniae in initial testing (1/2), 10/10 repeats passed at bottle ring concentration.
1. 1 false negative mecA in initial testing (2/3), 10/10 repeats passed
1. 1 false negative E. faecium in initial testing (2/3), 10/10 repeats passed
1. 1 false positive E. faecalis in initial testing (2/3), 3/3 repeats passed
1. 1 false negative S. aureus in initial testing (2/3), 10/10 repeats passed

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Competitive Inhibition

iC-GPC Assay performance was evaluated with combinations of target analytes that may be found in mixed positive blood cultures. One target organism was tested at a concentration near LoD ("low organism") in combination with a second target organism at a concentration of bottle ring + 8 hours ("high organism"). Combinations of five organisms representing each of the iC-GPC Assay targets were tested, for a total of 20 combinations. Competitive inhibition results are summarized in Table 8 below. Results represent the number of detected targets out of the total number of replicates tested. All high concentration iC-GPC targeted organisms were detected in all organism combinations. Due to competitive inhibition, low concentration organism targets were only detected in 3/20 (15%) of organism combinations. Target organisms present near LoD concentrations may not be detected by the iC-GPC Assay when a second target organism is present in the blood culture specimen.

TABLE 8: iC-GPC Competitive Inhibition Performance		Target Performance
LoD Strain	High Strain	Low Organism(~2-3x LoD)	Low ResistanceMarker(~2-3x LoD)	High Organism(Bottle ring+ 8 hours)	High ResistanceMarker (Bottlering + 8 hours)
S. epidermidis(SE/mecA)	SA/mecA	0/3	3/3*	3/3	3/3*
	SPN	3/3	3/3	3/3	NA
	EFLS/vanB	0/3	0/3	3/3	3/3
	EFCM/vanA	0/3	0/3	3/3	3/3
S. aureus(SA/mecA)	SE/mecA	0/3	3/3*	3/3	3/3*
	SPN	3/3	3/3	3/3	NA
	EFLS/vanB	0/3	0/3	3/3	3/3
	EFCM/vanA	0/3	0/3	3/3	3/3
S. pneumoniae(SPN)	SE/mecA	0/3	NA	3/3	3/3
	SA/mecA	0/3	NA	3/3	3/3
	EFLS/vanB	0/3	NA	3/3	3/3
	EFCM/vanA	0/3	NA	3/3	3/3
E. faecalis(EFLS/vanB)	SE/mecA	0/3	0/3	3/3	3/3
	SA/mecA	1/3	0/3	3/3	3/3
	SPN	3/3	2/3	3/3	NA
	EFCM/vanA	0/3	0/3	3/3	3/3
E. faecium(EFCM/vanA)	SE/mecA	0/3	3/3**	3/3	3/3
	SA/mecA	0/3	3/3**	3/3	3/3
	SPN	3/3	3/3	3/3	NA
	EFLS/vanB	0/3	2/3	3/3	3/3
Total		13/60 (21.7%)	25/48 (52.1%)	60/60 (100%)	48/48 (100%)

*The source of the resistance marker cannot be distinguished between high concentration organism and low concentration organism.

** Results include all positive resistance marker detections, regardless of associated organism detection. In the absence of a positive Enterococcus detection, vanA would not be reported.

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-GPC Assay performance. Nineteen representative iC-GPC target

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organisms plus one non-target organism were tested in 10 BCB media types (see Table 9 below). Target organisms were tested near LoD concentrations (2-3x LoD). Target performance is based on all expected targets identified and no false positive targets detected. Non-target performance is based on expected negative results. Performance in all BACTEC™ and VersaTREK® bottle types and BacT/ALERT® SA Standard Aerobic media met acceptance criteria of ≥ 95%; these bottles are validated for use with the iC-GPC Assay.

False positive Enterococcus faecium results were observed in 15/59 (25.4%) of BacT/ALERT® FAN and 13/60 (21.7%) of BacT/ALERT® FA Plus Aerobic media. Testing in these bottle types was repeated at higher concentrations, approximately 5-10x LoD, with false positive E. faecium results observed in 8/59 (13.6%) and 5/60 (8.3%) of BacT/ALERT FA Aerobic FAN and BacT/ALERT FA Plus Aerobic media, respectively. Overall performance results are presented in the table below.

TABLE 9: iC-GPC Assay BCB Equivalency Results
Blood Culture Bottle Type	OverallPerformance (%)	TargetPerformance (%)	FalseNegatives (%)	Non-TargetPerformance (%)	False Positives(%)
BACTEC™ Plus Aerobic	69/70 (98.6%)	66/67 (98.5%)	1/67 (1.5%)1	3/3 (100.0%)	0/70 (0.0%)
BACTEC™ Plus Anaerobic	70/72 (97.2%)	67/69 (97.1%)	1/69 (1.4%)2	3/3 (100.0%)	1/72 (1.4%)3
BACTEC™ Standard Aerobic	62/63 (98.4%)	59/60 (98.3%)	1/60 (1.7%)4	3/3 (100.0%)	0/63 (0.0%)
BACTEC™ StandardAnaerobic	77/80 (96.3%)	74/77 (96.1%)	2/77 (2.6%)5	3/3 (100.0%)	1/80 (1.3%)6
BACTEC™ Lytic/10Anaerobic	78/81 (96.3%)	75/78 (96.2%)	2/78 (2.6%)7	3/3 (100.0%)	1/81 (1.2%)8
VersaTREK® REDOX 1	60/60 (100.0%)	57/57 (100.0%)	0/57 (0.0%)	3/3 (100.0%)	0/60 (0.0%)
VersaTREK® REDOX 2	59/59 (100.0%)	56/56 (100.0%)	0/56 (0.0%)	3/3 (100.0%)	0/59 (0.0%)
BacT/ALERT® SA StandardAerobic	60/60 (100.0%)	57/57 (100.0%)	0/57 (0.0%)	3/3 (100.0%)	0/60 (0.0%)
BacT/ALERT® FA AerobicFAN (2-3x LoD)	44/59 (74.6%)	44/56 (78.6%)	0/56 (0.0%)	0/3 (0.0%)	15/59 (25.4%)9
BacT/ALERT® FA AerobicFAN (5-10x LoD)	51/59 (86.4%)	51/57 (89.5%)	0/57 (0.0%)	0/2 (0.0%)	8/59 (13.6%)10
BacT/ALERT® FA PlusAerobic (2-3x LoD)	47/60 (78.3%)	46/57 (80.7%)	0/57 (0.0%)	1/3 (33.3%)	13/60 (21.7%)11
BacT/ALERT® FA PlusAerobic (5-10x LoD)	55/60 (91.7%)	54/57 (94.7%)	0/57 (0.0%)	1/3 (33.3%)	5/60 (8.3%)12

1 false negative mecA 1)
1 false negative S. epidermidis 2)
1 false positive E. faecalis 3)
1 false negative S. epidermidis 4)
1. 1 false negative S. epidermidis, 1 false negative vanA
1 false positive S. epidermidis ର)
1. 1 false negative S. epidermidis, 1 false negative S. pneumonige
1. 1 false positive E. faecalis/vanB
15 false positive E. faecium 9)
1. 8 false positive E. faecium
1. 12 false positive E. faecium, 1 false positive S. aureus
1. 5 false positive E. faecium

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Further investigation suggests that the increased percentage of false positive E. faecium results observed in BacT/Alert® FA Aerobic FAN and BacT/Alert® FA Plus Aerobic media may be due to the presence of remnant nucleic acids and/or non-viable organisms in the culture media. FA Aerobic FAN media was further evaluated in clinical method comparison studies. In 96 clinical FA Aerobic FAN blood culture specimens tested, no false positive E. faecium results were observed, suggesting the risk of false positive results for E. faecium may be minimized when other organisms are present at bottle positivity concentrations in this bottle type.

Positive E. faecium results should be confirmed using alternative methods when using BacT/ALERT® FA Aerobic FAN or FA Plus Aerobic blood culture bottles.

The iC-GPC Assay is not recommended for use with BacT/ALERT® SN Standard Anaerobic, FN Anaerobic FAN, or FN Plus Anaerobic blood culture bottles.

Interfering Substances

iC-GPC Assay performance was evaluated in the presence of potentially inhibiting substances encountered in blood and blood culture media. Five representative target organisms plus one gram positive, non-target organism (Group B Streptococus) were evaluated. Target organisms were tested near LoD concentrations in combination with potential interferents at "worst-case" concentrations. Target performance is based on all expected targets detected, while non-target performance is based on all negative results. In the event of a false negative result, the organism/interferent was retested in replicates of ten. In the event of a false positive result or other system failure, the organism/interferent was retested in replicates of three. If the discordant result was not observed upon repeat testing, the compound was not considered an iC-GPC Assay interferent (Table 10). If the discordant result was observed upon repeat testing, the combination was retested at a decreased inhibitor concentration until interference was no longer observed (Table 11). Interference testing was performed in BD BACTEC Plus Aerobic blood culture bottle media with a sodium polyanetholesulfonate (SPS) concentration of 0.05% w/v. Additional SPS at concentrations greater than 0.03% w/v was found to interfere with iC-GPC Assay performance. Total protein (gamma-globulin and albumin) in concentrations greater than 3g/dL was also found to interfere with iC-GPC Assay performance. Both interferents may result in false negative results or positive controls check failures.

TABLE 10: Non-Interfering Substances Performance
		Target Performance
Potential Interferent	Test Concentration	SE	SA	SPN	EFLS	EFCM	Group B Strep
Hemoglobin	14 g/L	3/3	3/3	3/3	3/3	3/3	5/6¹
Triglyceride	1000 mg/dL	3/3	3/3	3/3	3/3	3/3	3/3
Conjugated bilirubin	20 mg/dL	3/3	3/3	12/13²	12/13³	3/3	3/3
Unconjugated bilirubin	20 mg/dL	3/3	3/3	3/3	3/3	3/3	3/3
Human genomic DNA	1 x 10⁶ cells/mL	3/3	3/3	3/3	12/13⁴	3/3	3/3
Vancomycin	100 mg/mL	3/3	3/3	12/13⁵	3/3	3/3	3/3
Piperacillin	160 mg/mL	3/3	3/3	3/3	3/3	3/3	3/3
Meropenem	80 mg/mL	3/3	3/3	3/3	3/3	3/3	3/3
Cefepime	80 mg/mL	3/3	12/13⁶	11/12⁷	3/3	3/3	3/3
Ceftriaxone	80 mg/mL	3/3	3/3	3/3	3/3	3/3	3/3
Fluconazole	125 mg/mL	3/3	3/3	3/3	3/3	3/3	3/3
Gentamicin	100 mg/mL	3/3	3/3	11/12⁸	12/14⁹	3/3	3/3

1 false positive E. faecalis, 3/3 repeats passed 1)

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1 false negative S. pneumoniae, 10/10 repeats passed 기
1. 1 false negative vanB, 10/10 repeats passed
1. 1 false negative vanB, 10/10 repeats passed
1. 1 false negative S. pneumoniae, 10/10 repeats passed
1 false negative mecA, 10/10 repeats passed ୧)
1 false negative S. pneumoniae, 9/9 repeats passed 7)
1. 1 false negative S. pneumoniae, 9/9 repeats passed
1 false negative vanB, 1 false positive S. epidermidis, 11/11 repeats passed 9)

TABLE 11: Interfering Substances Performance
		Target Performance
Interferent	Test Concentration	SE	SA	SPN	EFLS	EFCM	Group B Strep
Total Protein(γ-globulin + albumin)	3 g/dL	2/2	3/3	3/3	2/31	3/3	3/3
Total Protein(γ-globulin + albumin)	6g/dL*	0/3	0/3	2/3	0/3	0/3	2/3
SPS	0.03% w/v	3/3	12/132	3/3	3/3	3/3	5/63
SPS	0.04% w/v*	2/3	2/3	2/2	1/3	3/3	3/3

*Concentration at which interference observed; concentration decreased for subsequent testing.

1. 1 false negative S. aureus/mecA, 10/10 repeats passed
1. 1 false positive mecA, 3/3 repeats passed

Carryover and Cross-Contamination

The iC-System is a closed system, designed to eliminate the potential for carryover or cross-contamination between samples. Testing was performed to confirm that no false positive results from carryover or crosscontamination will occur from a high positive sample into a negative sample. Two representative iC-GPC target organisms, Staphylococcus aureus ATCC BAA977 (nuc positive) and Enterococcus faecium ATCC 700221 (fcm positive, vanA positive), were tested as representative positive samples. A gram positive nontarget organism, Corynebacterium striatum, was used as the negative sample. All organisms were tested at high concentrations, eight hours beyond initial bottle positivity. Positive, target organism samples were tested in an alternating pattern with negative, non-target organism samples across eight blades of an iC-Processor. Performance was based on accurate iC-GPC Assay target detection. No false positives indicative of sample carryover or cross-contamination were observed.

Method Comparison

A method comparison study was performed at four geographically dispersed clinical sites to evaluate the performance of the iC-GPC Assay™ for use on the iC-System™. Sites tested 966 leftover, de-identified specimens from aerobic and 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: BD BACTEC™, bioMérieux BacT/ALERT®, and Thermo Fisher VersaTREK®. Positive blood cultures were confirmed by Gram stain to contain gram positive cocci prior to being entered into the study. Specimens demonstrating mixed Gram stain results were excluded from the study.

Performance of the iC-GPC Assay was compared to an FDA-cleared multiplex assay that detects gram positive organisms and associated resistance markers directly from positive blood culture specimens. PCR and bidirectional sequencing were also performed for all specimens with positive results for resistance markers by either the iC-GPC Assay or the FDA-cleared multiplex assay. These PCR and bi-directional sequencing results were used to analyze discordant results between the iC-GPC Assay and the FDA-cleared multiplex assay.

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Additionally, performance of the iC-GPC Assay was compared to conventional reference methods including subculture, identification of isolates using biochemical methods or MALDI-TOF, and phenotypic antimicrobial susceptibility testing (AST). PCR and bi-directional sequencing were not used for discordant analysis between iC-GPC and conventional reference method results.

A total of 966 positive blood culture specimens, confirmed by Gram stain to contain gram positive cocci, were enrolled in the iC-GPC Assay Method Comparison Study. A total of 53 samples were withdrawn from the study (see below). Of the 913 samples in the study after identified samples were withdrawn, 879 were fresh prospective samples and 34 were frozen prospective samples.

An additional 168 contrived samples were evaluated for low prevalence organisms and resistance markers. Contrived specimens were prepared by inoculating low concentrations of organisms into BD BACTEC Plus Aerobic blood culture bottles with human blood added and incubating the blood culture system until bottle positivity. Multiple strains of S. pneumoniae, E. faecium were used to prepare contrived specimens.

Performance vs. FDA-Cleared Multiplex Assay:

When performance of the iC-GPC Assay was compared to the FDA-cleared multiplex assay, there was no statistical difference in performance noted between the four study sites or between the three blood culture systems. The following blood culture bottle types were evaluated: BD BACTEC Standard Aerobic, VersaTREK REDOX 1/2, BacT/ALERT SA Standard Aerobic, and BacT/ALERT FA Aerobic FAN.

Performance of the iC-GPC Assay in comparison to the FDA-cleared multiplex assay is provided in the tables Performance is stratified by prospectively tested fresh specimens, prospectively collected and below. retrospectively tested frozen specimens, and contrived specimens.

iC-GPC Assay Performance: Staphylococcus aureus (nuc)				iC-GPC Assay Performance: Staphylococcus epidermidis (gseA)
	Specimen Type	N=	Percent AgreementPositive (95% CI)	Percent AgreementNegative (95% CI)	Comparator Method		Specimen Type	N=	Percent AgreementPositive (95% CI)	Percent AgreementNegative (95% CI)	Comparator Method
Prospective	Fresh	879	96.9%249/257(94.0-98.4)	99.7%620/622(98.8-99.9)	FDA-ClearedMultiplex Assay		Fresh	879	98.3%227/231(95.6-99.3)	98.0%635/648(96.6-98.8)	FDA-ClearedMultiplex Assay
	Frozen	34	100%9/9(70.1-100)	100%25/25(86.7-100)			Frozen	34	100%4/4(51.0-100)	96.7%29/30(83.3-99.4)
	TOTAL	913	97.0%258/266*(94.2-98.5)	99.7%645/647**(98.9-99.9)			TOTAL	913	98.3%231/235*(95.7-99.3)	97.9%664/678**(96.6-98.8)
Contrived	168	-0/0-	100%168/168(97.8-100)			Contrived	168	-0/0-	100%168/168(97.8-100)

*Of the 8 observed false negative S. aureus results by the iC-GPC Assay, 7/8 were positive for S. aureus by PCR/bi-directional sequencing.

** Of the 2 observed false positive S. aureus results by the iC-GPC Assay, 1/2 was positive for S. aureus by PCR/bi-directional sequencing. One sample was not available for discordant analysis.

*Of the 4 observed false negative S. epidermidis results by the iC-GPC Assay, 3/4 were positive for S. epidermidis by PCR/bi-directional sequencing.

**Of the 14 observed false positive S. epidermidis results by the iC-GPC Assay, 9/14 were positive for S. epidermidis by PCR/bi-directional sequencing.

{26}------------------------------------------------

iC-GPC Assay Performance: Streptococcus pneumoniae (lytA)
SpecimenType	N=	Percent Agreement		ComparatorMethod
		Positive(95% CI)	Negative(95% CI)
Prospective	Fresh	82.6%*19/23(62.9-93.0)	99.9%855/856(99.3-100)	FDA-ClearedMultiplexAssay
	Frozen	100%4/4(51.0-100)	100%30/30(88.6-100)
	TOTAL	85.2%23/27*(67.5-94.1)	99.9%885/886**(99.4-100)
Contrived	168	100%36/36(90.4-100)	100%132/132(97.2-100)

*Of the 4 observed false negative S. pneumoniae results by the iC-GPC Assay, 0/4 were positive for S. pneumoniae by PCR/bi-directional sequencing and 0/4 culture isolates obtained from these specimens were positive for S. pneumoniae by MALDI ID.

**Of the 1 observed false positive S. pneumoniae result by the iC-GPC Assay, 1/1 was positive for S. pneumoniae by PCR/bi-directional sequencing.

SpecimenType	N=	Percent Agreement	ComparatorMethod
		Positive(95% CI)	Negative(95% CI)
Prospective	Fresh	879	96.6%56/58(88.3-99.0)	99.9%820/821(99.3-100)	FDA-ClearedMultiplexAssay
	Frozen	34	100%3/3(43.8-100)	100%31/31(89.0-100)
	TOTAL	913	96.7%59/61*(88.8-99.1)	99.9%851/852**(99.3-100)
	Contrived	168	100%90/90(95.9-100)	100%78/78(95.3-100)

*Of the 2 observed false negative E. faecalis results by the iC-GPC Assay, 1/2 was positive for E. faecalis by PCR/bi-directional sequencing. **Of the 1 observed false positive E. faecalis result by the iC-GPC Assay, 0/1 was positive for E. faecalis by PCR/bi-directional sequencing.

iC-GPC Assay Performance: Enterococcus faecium (fcm)				iC-GPC Assay Performance: mecA (methicillin resistance)
Specimen Type	N=	Percent Agreement Positive (95% CI)	Percent Agreement Negative (95% CI)	Specimen Type	N=	Percent Agreement Positive (95% CI)	Percent Agreement Negative (95% CI)
Fresh	879	96.4%27/28(82.3-99.4)	99.8%849/851(99.1-99.9)	Fresh	879	96.1%269/280(93.1-97.8)	98.2%588/599(96.7-99.0)
Frozen	34	100%1/1(20.7-100)	100%33/33(89.6-100)	Frozen	34	100%5/5(56.6-100)	100%29/29(88.3-100)
TOTAL	913	96.6%28/29*(82.8-99.4)	99.8%882/884**(99.2-99.9)	TOTAL	913	96.1%274/285†(93.2-97.8)	98.2%617/628**(96.9-99.0)
Contrived	168	100%42/42(91.6-100)	100%126/126(97.0-100)	Contrived	168	-0/0-	100%168/168(97.8-100)

Comparator Percent Agreement Method N= Positive Negative (95% CI) (95% CI) 96.1% 98.2% sh 879 269/280 588/299 FDA-(93.1-97.8) (96.7-99.0) Cleared 100% 100% Multiplex 34 5/5 29/29 en Assay (56.6-100) (88.3-100) 96.1% 98.2% 274/285** AL ਰੇਹਤ 617/628** (96.9-99.0) (93.2-97.8) 100% -0/0 ed 168 168/168 (97.8-100) -

*Of the 1 observed false negative E. faecium result by the iC-GPC Assay, 1/1 was positive for E. faecium by PCR/bi-directional sequencing.

**Of the 2 observed false positive E. faecium results by the iC-GPC Assay, 1/2 were positive for E. faecium by PCR/bi-directional sequencing.

TOf the 274 observed true positive mecA results by the iC-GPC Assay, 269/274 were positive for mecA by PCR/bi-directional sequencing. *Of the 11 observed false negative mecA results by the iC-GPC Assay, 10/11 were positive for mecA by PCR/bi-directional sequencing.

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8/11 were positive for mecA by PCR/bi-directional sequencing.

iC-GPC Assay Performance: vanA (vancomycin resistance)				iC-GPC Assay Performance: vanB (vancomycin resistance)
Specimen Type	N=	Percent Agreement Positive (95% CI)	Percent Agreement Negative (95% CI)	Specimen Type	N=	Percent Agreement Positive (95% CI)	Percent Agreement Negative (95% CI)	Comparator Method
Fresh	879	94.7%18/19(75.4-99.1)	99.4%855/860(98.6-99.8)	Fresh	879	100%2/2(34.2-100)	99.9%876/877(99.4-100)	FDA-Cleared Multiplex Assay
Prospective	Frozen	34	100%1/1(20.7-100)	100%33/33(89.6-100)	Frozen	34	-0/0-
	TOTAL	913	95.0%19/20†*(76.4-99.1)	99.4%888/893**(98.7-99.8)	TOTAL	913	100%2/2†(34.2-100)
Contrived	168	100%40/40(91.2-100)	100%128/128(97.1-100)	Contrived	168	100%56/56(93.6-100)	100%112/112(96.7-100)

Of the 19 observed true positive vanA results by the iC-GPC Assay, 19/19 were positive for vanA by PCR/bi-directional sequencing. *Of the 1 observed false negative vanA result by the iC-GPC Assay, 1/1 was positive for vanA by PCR/bi-directional sequencing. **Of the 5 observed false positive vanA results by the iC-GPC Assay, 2/5 were positive for vanA by PCR/bi-directional sequencing.

'Of the 2 observed true positive vanB results by the iC-GPC Assay, were positive for vanB by PCR/bi-directional sequencing. *Of the 1 observed false positive vanB result by the iC-GPC Assay, 0/1 was positive for vanB by PCR/bi-directional sequencing.

The following tables provide the performance of prospective clinical samples in comparison to the FDA-cleared multiplex assay for mecA detection with S. aureus/S. epidermidis, vanA detection with E. faecium, and vanB detection with E. faecalis/E. faecium.

Detection of mecA with S. aureus: Prospective
	FDA-Cleared Comparator Assay
Staphylococcus aureus/mecA	SA+/mecA+	SA+/mecA-	SA-	TOTAL
iCubateiC-GPC™	SA+/mecA+	111	3	0	114
	SA+/mecA-	0	144	2	146
	SA-	4	4	645	653
	TOTAL	115	151	647	913
Staphylococcus aureus/mecA		95% C.I.
% Agreement (SA+/mecA+)		96.5% (111/115)	91.4%	98.6%
% Agreement (SA+/mecA-)		95.4% (144/151)	90.7%	97.7%
% Agreement (SA-)		99.7% (645/647)	98.9%	99.2%

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Detection of mecA with S. epidermidis: Prospective
Staphylococcusepidermidis/mecA	FDA-Cleared Comparator Assay
	SE+/mecA+	SE+/mecA-	SE-	TOTAL
SE+/mecA+	163	2	6	171
iCubateiC-GPC™	SE+/mecA-	4	62	8	74
	SE-	3	1	664	668
	TOTAL	170	65	678	913
	Staphylococcus epidermidis/mecA			95% C.I.
% Agreement (SE+/mecA+)		95.9% (163/170)	91.8%	98.0%
% Agreement (SE+/mecA-)		95.4% (62/65)	87.3%	98.4%
% Agreement (SE-)		97.9% (664/678)	96.6%	98.8%

Detection of vanA with E. faecalis: Prospective
	Enterococcus faecalis/vanA	FDA-Cleared Comparator Assay
		Efs+/vanA+	Efs+/vanA-	Efs-	TOTAL
iCubateiC-GPC™	Efs+/vanA+	0	2	1	3
	Efs+/vanA-	0	57	0	57
	Efs-	0	2	851	853
	TOTAL	0	61	852	913
		Enterococcus faecalis/vanA			95% C.I.
% Agreement (Efs+/vanA+)		-	-	-
% Agreement (Efs+/vanA-)		93.4% (57/61)		84.3%	97.4%
% Agreement (Efs-)			99.9% (851/852)	99.3%	99.9%

Detection of vanB with E. faecalis: Prospective
	Enterococcus faecalis/vanB	FDA-Cleared Comparator Assay
		Efs+/vanB+	Efs+/vanB-	Efs-	TOTAL
iCubateiC-GPC™	Efs+/vanB+	2	0	1	3
	Efs+/vanB-	0	57	0	57
	Efs-	0	2	851	853
	TOTAL	2	59	852	913
Enterococcus faecalis/vanB					95% C.I.
% Agreement (Efs+/vanB+)		100.0% (2/2)	34.2%		100.0%
% Agreement (Efs+/vanB-)		96.6% (57/59)	88.5%		99.1%
% Agreement (Efs-)		99.9% (851/852)	99.3%		99.9%

{29}------------------------------------------------

Detection of vanA with E. faecium: Prospective
	FDA-Cleared Comparator Assay
Enterococcus faecium/vanA	Efm+/vanA+	Efm+/vanA-	Efm-	TOTAL
iCubateiC-GPC™	Efm+/vanA+	19	2	2	23
	Efm+/vanA-	0	7	0	7
	Efm-	1	0	882	883
	TOTAL	20	9	884	913
Enterococcus faecium/vanA		95% C.I.
% Agreement (Efm+/vanA+)		95.0% (19/20)	76.4%	99.1%
% Agreement (Efm+/vanA-)		77.8% (7/9)	45.3%	93.7%
% Agreement (Efm-)		99.8% (882/884)	99.2%	99.9%

Detection of vanB with E. faecium: Prospective
	FDA-Cleared Comparator Assay
Enterococcus faecium/vanB	Efm+/vanB+	Efm+/vanB-	Efm-	TOTAL
iCubateiC-GPC™	Efm+/vanB+	0	0	1	1
	Efm+/vanB-	0	28	1	29
	Efm-	0	1	882	883
	TOTAL	0	29	884	913
Enterococcus faecium/vanB		95% C.I.
% Agreement (Efm+/vanB+)		-	-	-
% Agreement (Efm+/vanB-)		96.6% (28/29)	82.8%	99.4%
% Agreement (Efm-)		99.8% (882/884)	99.2%	99.9%

The following tables provide the performance of contrived samples for vanA/vanB detection with E. faecalis and E. faecium.

Detection of vanA with E. faecalis: Contrived
	vanA/E. faecalis	Expected Result
		POS	NEG	TOTAL
iCubateiC-GPC™	POS	16	0	16
	NEG	0	152	152
	TOTAL	16	152	168
	vanA/E. faecalis	95% C.I.
	Positive % Agreement	100.0%	80.6%	100.0%
	Negative % Agreement	100.0%	97.5%	100.0%
Detection of vanA with E. faecium: Contrived
vanA/E. faecium		Expected Result
		POS	NEG	TOTAL
iCubateiC-GPC™	POS	24	0	24
	NEG	0	144	144
	TOTAL	24	144	168
	vanA/E. faecium	95% C.I.
	Positive % Agreement	100.0%	86.2%	100.0%
	Negative % Agreement	100.0%	97.4%	100.0%

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Detection of vanB with E. faecalis: Contrived
	vanB/E. faecalis	Expected Result
		POS	NEG	TOTAL
iCubateiC-GPC™	POS	56	0	56
	NEG	0	112	112
	TOTAL	56	112	168
	vanB/E. faecalis	95% C.I.
	Positive % Agreement	100.0%	93.6%	100.0%
	Negative % Agreement	100.0%	96.7%	100.0%
Detection of vanB with E. faecium : Contrived
	vanB/E. faecium	Expected Result
		POS	NEG	TOTAL
iCubateiC-GPC™	POS	0	0	0
	NEG	0	168	168
	TOTAL	0	168	168
	vanB/E. faecium	95% C.I.
	Positive % Agreement	-	-	-
	Negative % Agreement	100.0%	97.8%	100.0%

Performance vs. Traditional Culture and Susceptibility:

Performance of the iC-GPC Assay was compared to traditional laboratory reference methods: culture followed by testing blood culture isolates with conventional biochemicals/MALDI-TOF and AST testing (cefoxitin disc for mecA and vancomycin Etest for vanA/vanB).

Performance of the iC-GPC Assay in comparison to traditional laboratory reference methods is provided in the tables below. Performance is stratified by prospectively tested fresh specimens and prospectively collected, retrospectively tested frozen specimens.

iC-GPC Assay Performance: Staphylococcus aureus (nuc)				iC-GPC Assay Performance: Staphylococcus epidermidis (gseA)
Specimen Type	N=	Percent Agreement Positive (95% CI)	Percent Agreement Negative (95% CI)	Reference Method	Specimen Type	N=	Percent Agreement Positive (95% CI)	Percent Agreement Negative (95% CI)	Reference Method
Fresh	849	95.8%250/261(92.6-97.6)	99.8%587/588(99.0-100)	Culture & Biochemicals	Fresh	849	95.9%213/222(92.5-97.9)	96.5%605/627(94.7-97.7)	Culture & Biochemicals
Frozen	32	100%9/9(70.1-100)	100%23/23(85.7-100)		Frozen	32	100%3/3(43.8-100)	96.6%28/29(82.8-99.4)
TOTAL	881	95.9%259/270(92.9-97.7)	99.8%610/611(99.1-100)		TOTAL	881	96.0%216/225(92.6-97.9)	96.5%633/656(94.8-97.7)

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iC-GPC Assay Performance: Streptococcus pneumoniae (lytA)				iC-GPC Assay Performance: Enterococcus faecalis (ddl)
Specimen Type	N=	Percent AgreementPositive (95% CI)	Percent AgreementNegative (95% CI)	Specimen Type	N=	Percent AgreementPositive (95% CI)	Percent AgreementNegative (95% CI)
Prospective	Fresh	856	100%18/18(82.4-100)	99.9%837/838(99.3-100)	Prospective	Fresh	858	96.6%57/59(88.5-99.1)	99.9%798/799(99.3-100)
	Frozen	33	100%3/3(43.8-100)	96.7%29/30(83.3-99.4)		Frozen	33	100%3/3(43.8-100)	100%30/30(88.6-100)
	TOTAL	889	100%21/21(84.5-100)	99.8%866/868(99.2-99.9)		TOTAL	891	96.8%60/62(89.0-99.1)	99.9%828/829(99.3-100)
Culture & Biochemicals				Culture & Biochemicals

iC-GPC Assay Performance: Enterococcus faecium (fcm)					iC-GPC Assay Performance: mecA (methicillin resistance)
SpecimenType	N=	Percent Agreement		ComparatorMethod	SpecimenType	N=	Percent Agreement		ComparatorMethod
		Positive(95% CI)	Negative(95% CI)					Positive(95% CI)
Fresh	858	96.4%27/28(82.3-99.4)	99.6%827/830(98.9-99.9)	Culture &Biochemicals	Fresh	848	90.0%252/280(85.9-93.0)	97.0%551/568(95.3-98.1)	Culture,Biochemicals& CefoxitinDisc
Prospective	Frozen	33	100%1/1(20.7-100)		100%32/32(89.3-100)	Frozen	32	100%5/5(56.6-100)
	TOTAL	891	96.6%28/29(82.8-99.4)		99.7%859/862(99.0-99.9)	TOTAL	880	90.2%257/285(86.2-93.1)

Of the 28 observed false negative mecA results by the iC-GPC Assay,
14/28 were positive for mecA by multiple FDA-cleared multiplex assays.
Repeat AST testing also confirmed See note below.

{32}------------------------------------------------

iC-GPC Assay Performance: vanA/B (vancomycin resistance)
SpecimenType	N=	Percent Agreement	ComparatorMethod
		Positive(95% CI)
		Negative(95% CI)
Prospective	Fresh	855	87.5%21/24(69.0-95.7)	99.8%829/831(99.1-99.9)	Culture,Biochemicals&VancomycinEtest
	Frozen	33	100%1/1(20.7-100)	100%32/32(89.3-100)
	TOTAL	888	88.0%*22/25(70.0-95.8)	99.8%861/863(99.2-99.9)

*Of the 3 observed false negative results by the iC-GPC Assay, 1/3 was positive for vanA/vanB by the FDA-cleared multiplex assay.

The following tables provide the performance of prospective clinical samples in comparison to traditional laboratory reference methods for mecA detection with S. oureus and S. epidermidis and vanA/vanB detection with E. faecalis and E. faecium.

NOTE: Of the 21 observed false negative mecA results by the iC-GPC Assay for specimens identified to contain methicillin-resistant S. aureus by conventional identification methods and cefoxitin disc testing, 16 were also mecA negative by the FDA-cleared multiplex assay used for method comparison testing. To further investigate, isolates from 14 of the specimens identified at a single site were tested with an alternate real-time PCR assay that detects mecA and mecC. This assay confirmed the isolates to be negative for both mecA and mecC. Cefoxitin disc testing was also repeated on the 14 sample isolates. All isolates were found to be susceptible to cefoxitin with a zone size of 22mm. These tests confirm the iC-GPC result of mecA negative for 14 of the 21 discrepant results.

Detection of mecA with S. aureus : Prospective
		Culture, Biochemicals & Cefoxitin Disc Testing
	Staphylococcus aureus/mecA	SA+/ mecA +	SA+/ mecA -	SA-	TOTAL
iCubateiC-GPC™	SA+/ mecA +	113	1	0	114
	SA+/ mecA -	16	128	1	145
	SA-	5	6	610	621
TOTAL		134	135	611	880
Staphylococcus aureus/mecA		95% C.I.
% Agreement (SA+/ mecA +)		84.3%* (113/134)	77.2%	89.5%
% Agreement (SA+/ mecA -)		94.8% (128/135)	89.7%	97.5%
% Agreement (SA-)		99.8% (610/611)	99.1%	100%
*Corrected based on reviewer comments

See note above

{33}------------------------------------------------

Detection of mecA with S. epidermidis : Prospective
		Culture, Biochemicals & Cefoxitin Disc Testing
	Staphylococcus epidermidis/mecA	SE+/ mecA +	SE+/ mecA -	SE-	TOTAL
iCubateiC-GPC™	SE+/ mecA +	144	10	15	169
	SE+/ mecA -	2	60	8	70
	SE-	5	4	632	641
	TOTAL	151	74	655	880
Staphylococcus epidermidis/mecA		95% C.I.
% Agreement (SE+/ mecA +)		95.4% (144/151)	90.7%	97.7%
% Agreement (SE+/ mecA -)		81.2% (60/74)	70.7%	88.4%
% Agreement (SE-)		96.5% (632/655)	94.8%	97.7%

Detection of vanA/vanB with E. faecalis : Prospective
	Culture, Biochemicals & Vancomycin AST
	E. faecalis/vanA/vanB	Efs+/van+	Efs+/van-	Efs-	TOTAL
iCubateiC-GPC™	Efs+/van+	1	1	1	3
	Efs+/van-	2	53	0	55
	Efs-	0	2	828	830
	TOTAL	3	56	829	888
E. faecalis/vanA/vanB		95% C.I.
% Agreement (Efs+/van+)		33.3% (1/3)	6.2%	79.2%
% Agreement (Efs+/van-)		94.6% (53/56)	85.4%	98.2%
% Agreement (Efs-)		99.9% (828/829)	99.3%	100%

Detection of vanA/vanB with E. faecium: Prospective
	E. faecalis/vanA/vanB	Culture, Biochemicals & Vancomycin AST
		Efm+/van+	Efm+/van-	Efm-	TOTAL
iCubateiC-GPC™	Efm+/van+	20	1	3	24
	Efm+/van-	0	7	0	7
	Efm-	1	0	856	857
	TOTAL	21	8	859	888
E. faecium/vanA/vanB		95% C.I.
% Agreement (Efm+/van+)		95.2% (20/21)	77.3%	99.2%
% Agreement (Efm+/van-)		87.5% (7/8)	52.9%	97.8%
% Agreement (Efm-)		99.7% (856/859)	99.0%	99.9%

{34}------------------------------------------------

Mixed Culture Results:

In total, there were seven (7) mixed culture specimens that were detected by the iC-GPC Assay, the FDAcleared multiplex assay, or both. The tables below lists the mixed target combinations detected by iC-GPC and the comparator assay in the clinical study. There was one (1) discrepant mixed sample for which iC-GPC detected a target that was not detected by the comparator assay. There was one (1) discrepant mixed sample for which the comparator assay detected targets that were not detected by iC-GPC. Due to competitive inhibition, target organisms present near LoD concentrations may not be detected by the iC-GPC Assay when a second target organism is present at higher concentrations.

Multiple Detections by iC-GPC						TotalTargetsDetected	DiscrepantTargets	Discrepant Results(Targets Not Detected byFDA-Cleared MultiplexAssay)
Site	ID	Target 1	Target 2	Target 3	Target 4
LAC	1081	Staphylococcus aureus	Enterococcus faecalis			2	0	NA
LAC	1149	Staphylococcus epidermidis	mecA	Enterococcus faecalis		3	0	NA
LAC	1150	Enterococcus faecalis	Enterococcus faecium	vanA		3	1	vanA
LAC	1289	Staphylococcus aureus	mecA	Enterococcus faecalis	vanB	4	0	NA
LAC	1403	Staphylococcus epidermidis	mecA	Enterococcus faecalis		3	0	NA
TGH	4029	Staphylococcus aureus	Enterococcus faecalis			2	0	NA

Multiple Detections by FDA-Cleared Multiplex Assay						Total TargetsDetected	DiscrepantTargets	Discrepant Results(Targets Not Detected byiC-GPC)
Site	ID	Target 1	Target 2	Target 3	Target 4
LAC	1081	Staphylococcus aureus	Enterococcus faecalis			2	0	NA
LAC	1149	Staphylococcus epidermidis	mecA	Enterococcus faecalis		3	0	NA
LAC	1150	Enterococcus faecalis	Enterococcus faecium			2	0	NA
LAC	1289	Staphylococcus aureus	mecA	Enterococcus faecalis	vanB	4	0	NA
LAC	1403	Staphylococcus epidermidis	mecA	Enterococcus faecalis		3	0	NA
MCW	2134	Staphylococcus aureus	mecA	Enterococcus faecium	vanA	4	2	Staphylococcus aureus,mecA
TGH	4029	Staphylococcus aureus	Enterococcus faecalis			2	0	NA

Of the six (6) mixed culture specimens detected by the iC-GPC Assay, there were two (2) discrepant samples for which iC-GPC detected a target that was not detected by culture, biochemicals, and AST testing.

Multiple Detections by iC-GPC					Total Targets Detected	Discrepant Targets	Discrepant Results (Targets Not Detected by Culture/AST)
Site	ID	Target 1	Target 2	Target 3				Target 4
LAC	1081	Staphylococcus aureus	Enterococcus faecalis			2	0	NA
LAC	1149	Staphylococcus epidermidis	mecA	Enterococcus faecalis		3	0	NA
LAC	1150	Enterococcus faecalis	Enterococcus faecium	vanA		3	3	Enterococcus faecium, vanA
LAC	1289	Staphylococcus aureus	mecA	Enterococcus faecalis	vanB	4	0	vanB (E-Test Intermediate)
LAC	1403	Staphylococcus epidermidis	mecA	Enterococcus faecalis		3	0	NA
TGH	4029	Staphylococcus aureus	Enterococcus faecalis			2	0	NA

There were fifty-six (56) mixed culture specimens detected by culture and biochemicals, including organisms not detected by the iC-GPC Assay. The table below lists the mixed target combinations detected by traditional reference methods. There were twelve (12) discrepant samples for which the reference methods detected targets that were not detected by iC-GPC (false negatives). There were nine (9) discrepant samples for which iC-GPC detected targets were not detected by the reference

{35}------------------------------------------------

methods (false positives). Of these, two (2) were positive mecA results associated with an iC-GPC target organism where reference testing associated the mecA with a non-target organism.

Multiple Detections by Culture, Biochemicals & AST
Target 1	Target 2	Target 3	Frequency	No. w/ Discrepant Targets	Discrepant Target
Staphylococcus aureus	Enterococcus faecalis		3	1	Staphylococcus aureus
Staphylococcus aureus, mecA	Enterococcus faecalis		1	0
Staphylococcus aureus, mecA	Enterococcus faecalis	Streptococcus agalactiae	1	1	Staphylococcus aureus, mecA,Enterococcus faecalis,Staphylococcus epidermidis*
Staphylococcus aureus, mecA	Enterococcus faecium, vanA	Candida glabrata	1	1	Staphylococcus aureus, mecA
	Stenotrophomonas rhizophila	Pseudomonas fulva	1	1
Staphylococcus epidermidis, mecA	Enterococcus faecalis		4	2	Staphylococcus epidermidis (2),mecA (2)
Staphylococcus epidermidis, mecA	Staphylococcus epidermidis		1	0	NA
Staphylococcus epidermidis, mecA	Staphylococcus hominis		4	0	NA
Staphylococcus epidermidis	Staphylococcus hominis		2	1	Staphylococcus epidermidis
Staphylococcus epidermidis	Staphylococcus hominis	Staphylococcus capitis	1	1	Staphylococcus epidermidis
Staphylococcus epidermidis	Staphylococcus hominis, mecA		1	1	mecA*
Staphylococcus epidermidis, mecA	Staphylococcus haemolyticus		1	0	NA
Staphylococcus epidermidis	Staphylococcus haemolyticus		1	0	NA
Staphylococcus epidermidis, mecA	Staphylococcus haemolyticus, mecA		1	0	NA
Staphylococcus epidermidis	Staphylococcus haemolyticus, mecA		1	1	mecA*
Staphylococcus epidermidis, mecA	Staphylococcus capitus		1	0	Staphylococcus epidermidis, mecA
Staphylococcus epidermidis	Staphylococcus capitus		1	1	NA
Staphylococcus epidermidis, mecA	Staphylococcus capitus, mecA		1	1	Staphylococcus epidermidis, mecA
Staphylococcus epidermidis, mecA	Staphylococcus lugdunensis		1	0	NA
Staphylococcus epidermidis, mecA	Staphylococcus warneri, mecA		1	0	NA
Staphylococcus epidermidis, mecA	Staphylococcus species, mecA		1	0	NA
Staphylococcus epidermidis, mecA	Aerococcus viridans		1	0	NA
Staphylococcus epidermidis	Bacillus circulans		1	0	NA
Staphylococcus epidermidis	Streptococcus agalactiae		1	0	NA
Staphylococcus epidermidis	Streptococcus oralis		1	0	NA
Staphylococcus epidermidis	Low Disc: Strep. mitis/oralis		1	1	Staphylococcus epidermidis
Staphylococcus aureus, mecA	Streptococcus mitis		1	0	NA
Staphylococcus aureus, mecA	Unidentified Organism		1	1	Staphylococcus aureus, mecA
Enterococcus faecalis	Staphylococcus simulans	Aerococcus viridans	1	1	Enterococcus faecalis
Enterococcus faecalis	Staphylococcus haemolyticus, mecA		1	0	NA
Enterococcus faecalis	Klebsiella pneumoniae		1	0	NA
Staphylococcus hominis, mecA	Staphylococcus hominis		4	3	Staphylococcus epidermidis (2)*,Enterococcus faecalis
Staphylococcus hominis, mecA	Staphylococcus capitis		2	1	Staphylococcus epidermidis*
Staphylococcus hominis	Staphylococcus lentus		1	0	NA
Staphylococcus hominis	Staphylococcus species		2	0	NA
Staphylococcus hominis	Aerococcus viridans		1	0	NA
Multiple Detections by Culture, Biochemicals & AST
Target 1	Target 2	Target 3	Frequency	No. w/ Discrepant Targets	Discrepant Target
Staphylococcus haemolyticus	Staphylococcus capitis		1	1	Staphylococcus epidermidis*
Staphylococcus capitis	Low Disc: Streptococcus mitis/oralis		1	0	NA
Staphylococcus capitis	Staphylococcus warneri		1	1	Staphylococcus epidermidis*
Staphylococcus lentus, mecA	Low Disc: Strep. mitis/oralis		1	0	NA
Staphylococcus cohnii ssp urealyticus, mecA	Staphylococcus cohnii ssp cohnii, mecA		1	0	NA
Streptococcus sanguinis	Streptococcus gordonii		1	0	NA

{36}------------------------------------------------

*false positive result

Expected Values:

913 prospectively collected fresh and frozen blood culture specimens were obtained from four geographically dispersed clinical sites. The number and percentage of positive cases (positivity rate) determined by the iC-GPC Assay stratified by U.S. state for each of the organisms and resistance markers detected by the assay are presented below. Overall, the iC-GPC Assay detected at least one organism in 66.9% (611/913) prospectively collected specimens and at least one resistance marker in 33.4% (305/913) prospectively collected specimens.

	U.S. State	NY	WI	NM	FL	TOTAL
Organism	TOTAL n	300	269	248	96	913
	POSITIVE n	86	57	92	31	266
S. aureus	% Positivity	28.7%	21.2%	37.1%	32.3%	29.1%
S. epidermidis	POSITIVE n	63	85	66	21	235
	% Positivity	21.0%	31.6%	26.6%	21.9%	25.7%
	POSITIVE n	12	7	7	1	27
S. pneumoniae	% Positivity	4.0%	2.6%	2.8%	1.0%	3.0%
	POSITIVE n	22	26	6	7	61
E. faecalis	% Positivity	7.3%	9.7%	2.4%	7.3%	6.7%
	POSITIVE n	5	22	1	1	29
E. faecium	% Positivity	1.7%	8.2%	0.4%	1.0%	3.2%
Resistance Marker	TOTAL n	300	269	248	96	913
	POSITIVE n	77	91	81	36	285
mecA	% Positivity	25.7%	33.8%	32.7%	37.5%	31.2%
	POSITIVE n	4	15	1	0	20
vanA	% Positivity	1.3%	5.6%	0.4%	0.0%	2.2%
	POSITIVE n	1	1	0	0	2
vanB	% Positivity	0.3%	0.4%	0.0%	0.0%	0.2%

Error Rates and Sample Withdrawals:

Throughout the course of the study, an initial error rate of 7.6% (83/1098) was observed. Errors included positive controls check failures, array registration errors, and processor or other system errors. When a failed control or system error was observed, the sample was retested on the iC-GPC Assay. Upon repeat testing, the error rate was reduced to 1.6% (17/1098). Samples generating reproducible iC-GPC errors were withdrawn from the study.

{37}------------------------------------------------

No-Calls
Internal Positive Control Failure		Instrument Errors		Total Non-Reportable Rate
Initial#fail/#totalPercent (95% CI)	Final#fail/#totalPercent (95% CI)	Initial#fail/#totalPercent (95% CI)	Final#fail/#totalPercent (95% CI)	Initial#fail/#totalPercent (95% CI)	Final#fail/#totalPercent (95% CI)
5.9%	1.5%	1.6%	0.1%	7.6%	1.5%
65/1098	16/1098	18/1098	1/1098	83/1098	17/1098
[4.7-7.5%]	[0.9-2.4%]	[1.0-2.6%]	[0.0-0.5%]	[6.1-9.3%]	[1.0-2.5%]

The total specimens excluded from the iC-GPC Assay Method Comparison Study (n=53) are listed by site and reason for exclusion in the following table. The most common reasons for exclusion included repeat iC-GPC error, repeat comparator assay error, or specimens demonstrating mixed Gram stain results.

Withdrawn Samples
	Reason for Withdrawal
Site	Mixed GramStain	RepeatiC-GPC Error	RepeatComparatorAssay Error	NoComparatorAssay ID/Other	TOTAL
1	6	4	2	0	12
2	13	7	2	0	22
3	3	8	1	4	16
4	1	0	0	2	3
TOTAL	23	19	5	6	53

Additional specimens were excluded when iC-GPC Assay performance was compared to conventional reference methods for the following reasons: no culture identification, indeterminate culture identification, or non-specific AST results (i.e., intermediate susceptibility).

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).