The GenMark ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous detection and identification of multiple potentially pathogenic fungal organisms in positive blood culture. The ePlex BCID-FP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain fungal organism.

The following fungal organisms are identified using the ePlex BCID-FP Panel: Candida albicans, Candida auris, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus gattii, Cryptococcus neoformans, Fusarium and Rhodotorula.

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

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

Device Description

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

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

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

AI/ML Overview

This document describes the analytical and clinical performance of the GenMark ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel, an in vitro diagnostic test. The information provided is sufficient to extract the requested details about acceptance criteria and study proving the device meets them.

1. Table of acceptance criteria and reported device performance:

The document implicitly defines acceptance criteria through the reported performance characteristics. While no explicit "acceptance criteria" table is provided, the clinical performance (Sensitivity/PPA and Specificity/NPA) tables against a comparator method serve as the primary evidence of meeting performance expectations. Analytical performance characteristics also define a form of acceptance criteria (e.g., LOD, exclusivity).

Here’s a table summarizing the reported device performance for each target organism, which represents the device meeting its performance objectives. The device demonstrates high sensitivity (positive percent agreement) and specificity (negative percent agreement) across various sample types (prospective, retrospective, and contrived).

Target	Sample Type	Sensitivity/PPA % (95% CI)	Specificity/NPA % (95% CI)
Candida albicans	Overall	97.1 (89.9-99.2)	99.9 (99.3-100)
Candida auris	Overall	100 (92.7-100)	100 (99.5-100)
Candida dubliniensis	Overall	100 (93.1-100)	100 (99.5-100)
Candida famata	Overall	100 (93.0-100)	100 (99.5-100)
Candida glabrata	Overall	98.3 (91.1-99.7)	99.8 (99.1-99.9)
Candida guilliermondii	Overall	98.0 (89.5-99.6)	100 (99.5-100)
Candida kefyr	Overall	100 (93.0-100)	99.8 (99.1-99.9)
Candida krusei	Overall	100 (92.9-100)	100 (99.5-100)
Candida lusitaniae	Overall	98.0 (89.3-99.6)	99.9 (99.3-100)
Candida parapsilosis	Overall	98.3 (91.1-99.7)	99.9 (99.3-100)
Candida tropicalis	Overall	100 (92.9-100)	99.9 (99.3-100)
Cryptococcus gattii	Overall	100 (92.9-100)	100 (99.5-100)
Cryptococcus neoformans	Overall	100 (93.7-100)	100 (99.5-100)
Fusarium	Overall	98.6 (92.3-99.7)	100 (99.5-100)
Rhodotorula	Overall	96.2 (87.0-98.9)	99.9 (99.3-100)

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

The test set for evaluating clinical performance consisted of:

Prospective Samples: 21 evaluable samples (11 fresh, 10 frozen) collected at 6 clinical sites. These samples are from patients of all ages and genders. Collection dates are specified from May 2015 through July 2016 (frozen) and July through August 2018 (fresh). The country of origin is not explicitly stated but implied to be the US given the FDA submission. This data is prospective.
Retrospective Samples: 120 samples collected from 9 sites. This data is retrospective.
Contrived Samples: 725 evaluable samples prepared by spiking isolates into blood culture bottles. These are laboratory-prepared samples.

The total number of samples evaluated for clinical performance was 866 (11 fresh prospective + 10 frozen prospective + 120 retrospective + 725 contrived).

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

The document does not specify the number of experts or their qualifications (e.g., radiologists with 10 years of experience) used to establish the ground truth. Instead, it relies on standard laboratory procedures and analytically validated PCR assays followed by bi-directional sequencing as the comparator methods (ground truth). This implies that the ground truth was established through a combination of traditional microbiological methods and molecular techniques, not through expert consensus reading of images or other subjective assessments.

4. Adjudication method for the test set:

The document describes the "comparator method" as the gold standard. For specific targets like Candida auris, Fusarium, Rhodotorula, and to confirm Candida parapsilosis, PCR/sequencing was used to determine the presence or absence of the organism, effectively acting as an adjudication step for these cases. For other organisms, standard laboratory procedures (culture, MALDI-TOF IVD, etc.) defined the ground truth. There is no mention of a traditional reader adjudication process (e.g., 2+1 or 3+1) as would be common in image-based AI studies, as this is a molecular diagnostic test. For discrepant results (e.g., section "Co-detections in Clinical Samples"), PCR/sequencing was used to investigate.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done:

No, an MRMC comparative effectiveness study was not done. This type of study is typically performed for AI-assisted diagnostic tools that involve human interpretation of images. The ePlex BCID-FP Panel is an in vitro diagnostic (IVD) test that directly detects and identifies genetic material, so human readers are not involved in its direct interpretation in the way they would be in an AI imaging study. Therefore, there is no effect size of how much human readers improve with AI vs without AI assistance.

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

Yes, the primary clinical performance evaluation is a standalone performance of the algorithm (the ePlex BCID-FP Panel) against a defined ground truth (comparator methods). The reported sensitivity/PPA and specificity/NPA values are purely the device's performance.

7. The type of ground truth used:

The ground truth for the clinical performance evaluation was established using:

Standard laboratory procedures: This includes traditional and automated culture, MALDI-TOF IVD (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry), and other microbiological and biochemical techniques.
Analytically validated PCR assays followed by bi-directional sequencing: This advanced molecular method was used for specific targets (Candida auris, Fusarium, Rhodotorula) and to confirm certain identifications (Candida parapsilosis).

This represents a combination of expert consensus (through standard lab practices) and molecular outcomes data.

8. The sample size for the training set:

The document does not explicitly state the sample size for a "training set" in the context of machine learning. As this is a molecular diagnostic assay using nucleic acid hybridization and PCR, not a machine learning algorithm that learns from data in the same way, the concept of a distinct 'training set' for the device's core functionality specification might not apply directly in the conventional AI sense. The development of such assays involves analytical validation using numerous strains and concentrations (analytical reactivity, LOD, exclusivity), which implicitly serve as a form of "training" or optimization data during product development, but this is distinct from machine learning model training. The provided data focuses on the performance evaluation (test set) for regulatory approval.

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

Given that there isn't a "training set" in the typical machine learning sense, the way "ground truth" would be established for the development of such a molecular assay would involve:

Known Reference Strains: Use of well-characterized microbial strains (e.g., ATCC, CBS, CDC strains) with confirmed identities. These are used in analytical studies like Limit of Detection (LOD) and Analytical Reactivity (Inclusivity), as well as Competitive Inhibition studies. Table 21 ("Contrived Sample Summary") and Table 27 ("Analytical Reactivity (Inclusivity) Results") list numerous specific strains and their origins (e.g., ATCC, CBS, NCPF, CDC#) used in testing.
Sequencing and Phenotypic Characterization: During the assay's development, target sequences would be determined through genome sequencing, and phenotypic characteristics would be confirmed through established microbiological methods.

Therefore, the "ground truth" during device development (analogous to training/development data in AI) relies on well-characterized laboratory standards and molecular gold standards.

Summary

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December 21, 2018

GenMark Diagnostics, Incorporated Beth Stofka Sr. Regulatory Affairs Specialist 5964 La Place Court Carlsbad, California 92008

Re: K182690

Trade/Device Name: ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel 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: PEO Dated: September 26, 2018 Received: September 27, 2018

Dear Beth Stofka:

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 mav, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be avare that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's

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requirements, including, but not limited to: registration and listing (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.html; good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Uwe Scherf -S

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) K182690

Device Name

ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel

Indications for Use (Describe)

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X Prescription Use (Part 21 CFR 801 Subpart D)

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510(k) Summary

Summary of Safety and Effectiveness

Submitter Information

Submitter:	GenMark Diagnostics, Incorporated5964 La Place CourtCarlsbad, CA 92008
Manufacturer:	GenMark Diagnostics, Incorporated5964 La Place CourtCarlsbad, CA 92008
Establishment Registration Number:	3008632402
Contact:	Beth StofkaSr. Regulatory Affairs Specialist
Phone:	760-579-4778
Fax:	760-683-6961
E-mail:	Beth.Stofka@genmarkdx.com
Alternate Contact:	Alan Maderazo, Ph.D., RACVice President, Quality, Regulatory and Clinical Affairs
Phone:	760-448-4308
Fax:	760-683-6961
E-mail:	Al.Maderazo@genmarkdx.com
Date Prepared:	September 27, 2018

Name of Device and Classification

ePlex® Blood Culture Identification Fungal Pathogen (BCID-FP) Panel Product Name: Device Classification: 866.3980, Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures, Class II Product Code(s): PEO

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Predicate Device

Predicate:

FilmArray Blood Culture Identification Panel; BioFire Diagnostics; K130914

Device Description

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Intended Use/Indications for Use

The GenMark ePlex® Blood Culture Identification Fungal Pathogen (BCID-FP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous detection and identification of multiple potentially pathogenic fungal organisms in positive blood culture. The ePlex BCID-FP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain fungal organism.

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Summary of Technological Characteristics of the Device Compared to the Predicate Device

The GenMark ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel ("Subject Device") and the legally marketed device, FilmArray Blood Culture Identification Panel, K130914, ("Predicate Device") are described below:

Characteristic	Subject Device	Predicate Device
Product Name	ePlex BCID-FP Panel	FilmArray BCID Panel
Manufacturer	GenMark Diagnostics, Inc.	BioFire Diagnostics, Inc.
OrganismsDetected	• Candida albicans• Candida auris• Candida dubliniensis,• Candida famata• Candida glabrata• Candida guilliermondii• Candida kefyr• Candida krusei• Candida lusitaniae• Candida parapsilosis• Candida tropicalis• Cryptococcus gattii• Cryptococcus neoformans• Fusarium• Rhodotorula	Enterococci, Listeria monocytogenes,commonly encountered Staphylococci(including specific differentiation ofStaphylococcus aureus), commonlyencountered Streptococci (withspecific differentiation ofStreptococcus agalactiae,Streptococcus pneumoniae, andStreptococcus pyogenes),Acinetobacter baumannii, commonlyencountered Enterobacteriaceae(including specific differentiation ofthe Enterobacter cloacae complex,Escherichia coli, Klebsiella oxytoca,Klebsiella pneumoniae, Proteus, andSerratia marcescens), Haemophilusinfluenzae, Neisseria meningitidis(encapsulated), Pseudomonasaeruginosa, Candida albicans,Candida glabrata, Candida krusei,Candida parapsilosis, and Candidatropicalis.
Indication forUse	The ePlex BCID-FP Panel is indicatedas an aid in the diagnosis ofbloodstream infections. The use ofadditional laboratory testing (e.g. sub-culturing of positive blood cultures foridentification of organisms not detectedby the ePlex BCID-FP Panel and forsusceptibility testing, differentiation ofmixed growth) and clinical presentationmust be taken into consideration in thefinal diagnosis of blood streaminfection.	FilmArray BCID is indicated as an aidin the diagnosis of specific agents ofbacteremia and fungemia and resultsshould be used in conjunction withother clinical and laboratory findings.
Characteristic	Subject Device	Predicate Device
Specimen Type	Blood culture samples identified aspositive by a continuous monitoringblood culture system and whichcontain fungal organism.	Blood culture samples identified aspositive by a continuous monitoringblood culture system that demonstratesthe presence of organisms asconfirmed by Gram stain.
Chemistry	Reagents on cartridge include: samplelysis and nucleic acid extraction, PCRamplification and hybridization-basedelectrochemical detection reagents.	The FilmArray BCID pouch containsfreeze-dried reagents to performnucleic acid purification and nested,multiplex PCR with DNA meltanalysis.
Hardware	GenMark ePlex Instrument & SingleUse Cartridge	FilmArray Instrument and assay pouch
SoftwareInterfaceResult Reporting	GenMark ePlex System Software GenMark ePlex BCID-FP PanelSoftware	The FilmArray Software automaticallyinterprets the results of each DNAmelt curve analysis and combines thedata with the results of the internalpouch controls to provide a test resultfor each organism and antimicrobialresistance gene on the panel.

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Summary of Performance Data

EXPECTED VALUES

A prospective, multicenter clinical study was conducted to evaluate the clinical performance of the ePlex BCID-FP Panel in positive blood culture samples. A total of 447 positive blood culture samples were collected at 6 clinical sites in 2 phases from patients of all ages and genders. Samples were collected and frozen for future testing from May 2015 through July 2016. Samples were collected from July through August 2018 and tested fresh (never frozen). Of these 447 samples, 21 had a Gram stain result indicating fungal organism. The expected values of individual analytes based on the ePlex BCID-FP Panel results in the 21 prospective samples are summarized by age group and by site in Tables 1 and 2 below.

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Target	All Ages(N=21)n (%)	Age <1(N=1)n (%)	Age 1-17(N=2)n (%)	Age 18-44(N=4)n (%)	Age 45-64(N=11)n (%)	Age 65-84(N=2)n (%)
Candida albicans	4 (19.0)	1 (100)	0 (0.0)	0 (0.0)	2 (18.2)	1 (50.0)
Candida auris	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida dubliniensis	1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	1 (9.1)	0 (0.0)
Candida famata	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida glabrata	6 (28.6)	0 (0.0)	1 (50.0)	1 (25.0)	3 (27.3)	1 (50.0)
Candida guilliermondii	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida kefyr	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida krusei	2 (9.5)	0 (0.0)	0 (0.0)	0 (0.0)	2 (18.2)	0 (0.0)
Candida lusitaniae	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida parapsilosis	2 (9.5)	0 (0.0)	1 (50.0)	0 (0.0)	1 (9.1)	0 (0.0)
Candida tropicalis	2 (9.5)	0 (0.0)	0 (0.0)	1 (25.0)	1 (9.1)	0 (0.0)
Cryptococcus gattii	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Cryptococcus neoformans	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Fusarium	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Rhodotorula	1 (4.8)	0 (0.0)	0 (0.0)	1 (25.0)	0 (0.0)	0 (0.0)

Table 1: Expected Value by Age Group (Prospective Samples)

Table 2: Expected Value by Collection Site (Prospective Samples)

	All Sites(N=21)	Site 1(N=1)	Site 2(N=8)	Site 3(N=2)	Site 4(N=4)	Site 5(N=4)	Site 6(N=2)
Target	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
Candida albicans	4 (19.0)	1 (100)	0 (0.0)	2 (100)	1 (25.0)	0 (0.0)	0 (0.0)
Candida auris	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida dubliniensis	1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	1 (25.0)	0 (0.0)	0 (0.0)
Candida famata	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida glabrata	6 (28.6)	0 (0.0)	2 (25.0)	0 (0.0)	1 (25.0)	2 (50.0)	1 (50.0)
Candida guilliermondii	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida kefyr	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida krusei	2 (9.5)	0 (0.0)	2 (25.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida lusitaniae	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Candida parapsilosis	2 (9.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (25.0)	1 (50.0)
Candida tropicalis	2 (9.5)	0 (0.0)	2 (25.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Cryptococcus gattii	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Cryptococcus neoformans	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Fusarium	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Rhodotorula	1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (25.0)	0 (0.0)

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PERFORMANCE CHARACTERISTICS

Clinical Performance

Samples with a Gram stain result indicating fungal organism, a final, valid investigational test result, and a valid comparator result were evaluable and included in summaries and analyses of demographics, expected values (positivity rate), and performance characteristics. Evaluable samples included 11 prospective fresh and 10 prospective frozen samples as well as 120 retrospective samples and 725 contrived samples.

Comparator Method

The performance of the ePlex BCID-FP Panel was compared to standard laboratory procedures, including traditional and automated culture, MALDI-TOF IVD, and microbiological and biochemical techniques. In addition, all prospective samples were tested with analytically validated PCR assays followed by bi-directional sequencing to determine the presence or absence of Candida auris, Fusarium, and Rhodotorula. Identification for samples with Candida parapsilosis identified by standard laboratory procedures was confirmed using analytically validated PCR assays followed by bi-directional sequencing.

The comparator method(s) results were used to determine the Detected / Not Detected status for each target organism on the ePlex BCID-FP Panel. The comparator methods for each target are summarized in Table 3.

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Target	Comparator Method
Candida albicans
Candida dubliniensis
Candida famata
Candida glabrata
Candida guilliermondii
Candida kefyr	Standard laboratory procedures for organism identification.
Candida krusei
Candida lusitaniae
Candida tropicalis
Cryptococcus gattii
Cryptococcus neoformans
Candida parapsilosis	Standard laboratory procedures for organism ID. PCR/sequencing toconfirm C. parapsilosis or identify C. metapsilosis, C. orthopsilosis.
Candida auris, Fusarium, andRhodotorula	Standard laboratory procedures for organism identification.PCR/sequencing in prospective samples.

Table 3: Comparator Method(s) by ePlex BCID-FP Panel Target

Demographics of Clinical Samples

Clinical performance was evaluated in samples prospectively and retrospectively collected. Prospective samples were collected at 6 clinical sites, with 21 evaluable samples. Sample with final, valid, ePlex BCID-FP Panel results and valid comparator results were considered evaluable. Demographic information for prospectively-collected samples is described in Table 4. Subjects enrolled in this study were from a diverse demographic distribution and represent the intended patient population.

To supplement the results of the prospective collection, 120 samples were collected retrospectively from a total of 9 sites, and 725 evaluable samples were contrived for organisms with low prevalence. Demographic information for retrospectively-collected samples is described in Table 5.

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	All Sites(N=21)	Site 1(N=1)	Site 2(N=8)	Site 3(N=2)	Site 4(N=4	Site 5(N=4)	Site 6(N=2)
	n (%)	n (%)	n (%)	n (%)	n (%))	n (%)	n (%)
Sex, n (%)
Male	14(66.7)	1(100)	7(87.5)	1(50.0)	3(75.0)	1(25.0)	1(50.0)
Female	7(33.3)	0(0.0)	1(12.5)	1(50.0)	1(25.0)	3(75.0)	1(50.0)
Age (years)
<1 yr	1(4.8)	0(0.0)	0(0.0)	0(0.0)	1(25.0)	0(0.0)	0(0.0)
1-17 yrs	2(9.5)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	2(50.0)	0(0.0)
18-44 yrs	4(19.0)	0(0.0)	2(25.0)	0(0.0)	1(25.0)	1(25.0)	0(0.0)
45-64 yrs	11(52.4)	1(100)	4(50.0)	1(50.0)	2(50.0)	1(25.0)	2(100)
65-84 yrs	2(9.5)	0(0.0)	1(12.5)	1(50.0)	0(0.0)	0(0.0)	0(0.0)
85+ yrs	1(4.8)	0(0.0)	1(12.5)	0(0.0)	0(0.0)	0(0.0)	0(0.0)

Table 4: Demographic Data for Clinical Samples by Collection Site (Prospective Collection)

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

	All Sites(N=120)	Site 1(N=13)	Site 2(N=14)	Site 3(N=17)	Site 4(N=4)	Site 5(N=3)	Site 6(N=13)	Site 7(N=16)	Site 8(N=5)	Site 9(N=35)
	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
Sex, n (%)
Male	68(56.7)	10(76.9)	8(57.1)	8(47.1)	1(25.0)	2(66.7)	8(61.5)	9(56.3)	3(60.0)	19(54.3)
Female	52(43.3)	3(23.1)	6(42.9)	9(52.9)	3(75.0)	1(33.3)	5(38.5)	7(43.8)	2(40.0)	16(45.7)
Age (years)
<1 yr	2(1.7)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	2(5.7)
1-17 yrs	8(6.7)	1(7.7)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	5(38.5)	0(0.0)	0(0.0)	2(5.7)
18-44 yrs	27(22.5)	4(30.8)	2(14.3)	2(11.8)	1(25.0)	0(0.0)	3(23.1)	3(18.8)	1(20.0)	11(31.4)
45-64 yrs	39(32.5)	2(15.4)	6(42.9)	6(35.3)	1(25.0)	2(66.7)	2(15.4)	7(43.8)	1(20.0)	12(34.3)
65-84 yrs	39(32.5)	6(46.2)	6(42.9)	8(47.1)	2(50.0)	0(0.0)	2(15.4)	5(31.3)	2(40.0)	8(22.9)
85+ yrs	5(4.2)	0(0.0)	0(0.0)	1(5.9)	0(0.0)	1(33.3)	1(7.7)	1(6.3)	1(20.0)	0(0.0)

Table 5: Demographic Data for Clinical Samples by Collection Site (Retrospective Collection)

{15}------------------------------------------------

Clinical Performance

Sensitivity or positive percent agreement (PPA) was calculated by dividing the number of true positive (TP) results by the sum of TP and false negative (FN) results, while specificity or negative percent agreement (NPA) was calculated by dividing the number of true negative (TN) results by the sum of TN and false positive (FP) result being defined as a sample where the detected ePlex BCID-FP Panel result matched the detected comparator method result, while a TN result was one where a negative ePlex BCID-FP Panel result matched a negative comparator method result. The two-sided 95% confidence interval was also calculated.

A total of 866 positive blood culture samples with a Gram stain result indicating fungal organism consisting of 11 fresh prospective, 10 frozen prospective, 120 retrospective, and 725 contrived samples were evaluated for the ePlex BCID-FP Panel targets. Contrived samples were prepared by spiking an isolate into a blood culture bottle and growing until flagged positive by a continuously monitoring blood culture system. Samples were removed from the system within 8 hours of positivity and stored frozen until the time of testing. PPA and NPA results are summarized by target in Tables 6-20 below and the strains used to contrive samples are summarized in Table 21.

{16}------------------------------------------------

Target	Sample Type	TP/TP+FN	Sensitivity/PPA% (95% CI)	TN/TN+FP	Specificity/NPA% (95% CI)
Candida albicans	Prospective (Fresh)	2/2	100 (34.2-100)	9/9	100 (70.1-100)
	Prospective (Frozen)	2/2	100 (34.2-100)	8/8	100 (67.6-100)
	Prospective (All)	4/4	100 (51.0-100)	17/17	100 (81.6-100)
	Retrospective	49/50	98.0 (89.5-99.6)	70/70	100 (94.8-100)
	Prospective/Retrospective	53/54	98.1 (90.2-99.7)	87/87	100 (95.8-100)
	Contrived	13/14	92.9 (68.5-98.7)	710/711	99.9 (99.2-100)
	Overall	66/68	97.1 (89.9-99.2)	797/798	99.9 (99.3-100)

Table 6: Clinical Performance for Candida albicans

CI= Confidence Interval

			Table 7: Clinical Performance for Candida auris
--	--	--	-------------------------------------------------	--	--	--

Target	Sample Type	TP/TP+FN	Sensitivity/PPA% (95% CI)	TN/TN+FP	Specificity/NPA% (95% CI)
Candida auris	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	0/0	---	120/120	100 (96.9-100)
	Prospective/Retrospective	0/0	---	141/141	100 (97.3-100)
	Contrived	49/49	100 (92.7-100)	676/676	100 (99.4-100)
	Overall	49/49	100 (92.7-100)	817/817	100 (99.5-100)

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

Target	Sample Type	TP/TP+FN	Sensitivity/PPA% (95% CI)	TN/TN+FP	Specificity/NPA% (95% CI)
Candidadubliniensis	Prospective (Fresh)	1/1	100 (20.7-100)	10/10	100 (72.2-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	1/1	100 (20.7-100)	20/20	100 (83.9-100)
	Retrospective	3/3	100 (43.9-100)	117/117	100 (96.8-100)
	Prospective/Retrospective	4/4	100 (51.0-100)	137/137	100 (97.3-100)
	Contrived	48/48	100 (92.6-100)	677/677	100 (99.4-100)
	Overall	52/52	100 (93.1-100)	814/814	100 (99.5-100)

Table 8: Clinical Performance for Candida dubliniensis

Table 9: Clinical Performance for Candida famata

Target	Sample Type		Sensitivity/PPA	Specificity/NPA
		TP/TP+FN	% (95% CI)	TN/TN+FP	% (95% CI)
Candida famata	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	0/0	---	120/120	100 (96.9-100)
	Prospective/Retrospective	0/0	---	141/141	100 (97.3-100)
	Contrived	51/51	100 (93.0-100)	674/674	100 (99.4-100)
	Overall	51/51	100 (93.0-100)	815/815	100 (99.5-100)

{18}------------------------------------------------

Target	Sample Type	Sensitivity/PPA		Specificity/NPA
		TP/TP+FN	% (95% CI)	TN/TN+FP	% (95% CI)
Candida glabrata	Prospective (Fresh)	4/4	100 (51.0-100)	7/7	100 (64.6-100)
	Prospective (Frozen)	2/2	100 (34.2-100)	8/8	100 (67.6-100)
	Prospective (All)	6/6	100 (61.0-100)	15/15	100 (79.6-100)
	Retrospective	37/38	97.4 (86.5-99.5)	80/82	97.6 (91.5-99.3)
	Prospective/Retrospective	43/44	97.7 (88.2-99.6)	95/97A	97.9 (92.8-99.4)
	Contrived	16/16	100 (80.6-100)	709/709	100 (99.5-100)
	Overall	59/60	98.3 (91.1-99.7)	804/806	99.8 (99.1-99.9)

Table 10: Clinical Performance for Candida glabrata

A. C. glabrata was detected in 2/2 false positive clinical samples using PCR/sequencing.

					Table 11: Clinical Performance for Candida guilliermondii
--	--	--	--	--	-----------------------------------------------------------	--

Target	Sample Type	TP/TP+FN	Sensitivity/PPA% (95% CI)	TN/TN+FP	Specificity/NPA% (95% CI)
Candidaguilliermondii	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
Candidaguilliermondii	Retrospective	0/0	---	120/120	100 (96.9-100)
Candidaguilliermondii	Prospective/Retrospective	0/0	---	141/141	100 (97.3-100)
Candidaguilliermondii	Contrived	49/50	98.0 (89.5-99.6)	675/675	100 (99.4-100)
Candidaguilliermondii	Overall	49/50	98.0 (89.5-99.6)	816/816	100 (99.5-100)

{19}------------------------------------------------

Target	Sample Type	Sensitivity/PPA		Specificity/NPA
		TP/TP+FN	% (95% CI)	TN/TN+F P	% (95% CI)
Candida kefyr	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	0/0	---	120/120	100 (96.9-100)
	Prospective/Retrospective	0/0	---	141/141	100 (97.3-100)
	Contrived	51/51	100 (93.0-100)	672/674	99.7 (98.9-99.9)
	Overall	51/51	100 (93.0-100)	813/815	99.8 (99.1-99.9)

Table 12: Clinical Performance for Candida kefyr

Table 13: Clinical Performance for Candida krusei

Target	Sample Type		Sensitivity/PPA	Specificity/NPA
		TP/TP+FN	% (95% CI)	TN/TN+FP	% (95% CI)
Candida krusei	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	2/2	100 (34.2-100)	8/8	100 (67.6-100)
	Prospective (All)	2/2	100 (34.2-100)	19/19	100 (83.2-100)
	Retrospective	2/2	100 (34.2-100)	118/118	100 (96.8-100)
	Prospective/Retrospective	4/4	100 (51.0-100)	137/137	100 (97.3-100)
	Contrived	46/46	100 (92.3-100)	679/679	100 (99.4-100)
	Overall	50/50	100 (92.9-100)	816/816	100 (99.5-100)

{20}------------------------------------------------

Target	Sample Type	Sensitivity/PPA		Specificity/NPA
		TP/TP+FN	% (95% CI)	TN/TN+F P	% (95% CI)
Candida lusitaniae	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	3/4	75.0 (30.1-95.4)	116/116	100 (96.8-100)
	Prospective/Retrospective	3/4	75.0 (30.1-95.4)	137/137	100 (97.3-100)
	Contrived	45/45	100 (92.1-100)	679/680	99.9 (99.2-100)
	Overall	48/49	98.0 (89.3-99.6)	816/817	99.9 (99.3-100)

Table 14: Clinical Performance for Candida lusitaniae

Table 15: Clinical Performance for Candida parapsilosis

Target	Sample Type	TP/TP+FN	% (95% CI)	TN/TN+FP	% (95% CI)
Candidaparapsilosis	Prospective (Fresh)	2/2	100 (34.2-100)	9/9	100 (70.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	2/2	100 (34.2-100)	19/19	100 (83.2-100)
	Retrospective	16/17	94.1 (73.0-99.0)	102/103	99.0 (94.7-99.8)
	Prospective/Retrospective	18/19	94.7 (75.4-99.1)	121/122A	99.2 (95.5-99.9)
	Contrived	41/41	100 (91.4-100)	684/684	100 (99.4-100)
	Overall	59/60	98.3 (91.1-99.7)	805/806	99.9 (99.3-100)

C. parapsilosis was detected in 1/1 false positive clinical sample using PCR/sequencing. A.

{21}------------------------------------------------

Target	Sample Type	TP/TP+FN	Sensitivity/PPA% (95% CI)	Specificity/NPATN/TN+FP	% (95% CI)
Candida tropicalis	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	2/2	100 (34.2-100)	8/8	100 (67.6-100)
	Prospective (All)	2/2	100 (34.2-100)	19/19	100 (83.2-100)
	Retrospective	3/3	100 (43.9-100)	116/117	99.1 (95.3-99.8)
	Prospective/Retrospective	5/5	100 (56.6-100)	135/136A	99.3 (96.0-99.9)
	Contrived	45/45	100 (92.1-100)	680/680	100 (99.4-100)
	Overall	50/50	100 (92.9-100)	815/816	99.9 (99.3-100)

Table 16: Clinical Performance for Candida tropicalis

A. C. tropicalis was detected in 1/1 false positive clinical sample using PCR/sequencing.

		Table 17: Clinical Performance for Cryptococcus gattii
--	--	--------------------------------------------------------	--	--	--

Target	Sample Type	TP/TP+FN	% (95% CI)	TN/TN+F P	% (95% CI)
Cryptococcus gattii	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	0/0	---	120/120	100 (96.9-100)
	Prospective/Retrospective	0/0	---	141/141	100 (97.3-100)
	Contrived	50/50	100 (92.9-100)	675/675	100 (99.4-100)
	Overall	50/50	100 (92.9-100)	816/816	100 (99.5-100)

{22}------------------------------------------------

Target	Sample Type	TP/TP+FN	Sensitivity/PPA% (95% CI)	TN/TN+FP	Specificity/NPA% (95% CI)
Cryptococcusneoformans	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	5/5	100 (56.6-100)	115/115	100 (96.8-100)
	Prospective/Retrospective	5/5	100 (56.6-100)	136/136	100 (97.3-100)
	Contrived	52/52	100 (93.1-100)	673/673	100 (99.4-100)
	Overall	57/57	100 (93.7-100)	809/809	100 (99.5-100)

Table 18: Clinical Performance for Cryptococcus neoformans

Table 19: Clinical Performance for Fusarium

		Sensitivity/PPA		Specificity/NPA
Target	Sample Type	TP/TP+FN	% (95% CI)	TN/TN+FP	% (95% CI)
Fusarium	Prospective (Fresh)	0/0	---	11/11	100 (74.1-100)
	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	0/0	---	21/21	100 (84.5-100)
	Retrospective	0/0	---	120/120	100 (96.9-100)
	Prospective/Retrospective	0/0	---	141/141	100 (97.3-100)
	Contrived	69/70	98.6 (92.3-99.7)	655/655	100 (99.4-100)
	Overall	69/70	98.6 (92.3-99.7)	796/796	100 (99.5-100)

{23}------------------------------------------------

Target	Sample Type	Sensitivity/PPA		Specificity/NPA
		TP/TP+FN	% (95% CI)	TN/TN+FP	% (95% CI)
	Prospective (Fresh)	1/1	100 (20.7-100)	10/10	100 (72.2-100)
Rhodotorula	Prospective (Frozen)	0/0	---	10/10	100 (72.2-100)
	Prospective (All)	1/1	100 (20.7-100)	20/20	100 (83.9-100)
	Retrospective	1/1	100 (20.7-100)	119/119	100 (96.9-100)
	Prospective/Retrospective	2/2	100 (34.2-100)	139/139	100 (97.3-100)
	Contrived	48/50	96.0 (86.5-98.9)	674/675	99.9 (99.2-100)
	Overall	50/52	96.2 (87.0-98.9)	813/814	99.9 (99.3-100)

Table 20: Clinical Performance for Rhodotorula

Table 21: Contrived Sample Summary

Target	Organism	Strain	IndependentContrived SamplesTested
Candida albicans	Candida albicans	ATCC 10231	2
		ATCC 14053	2
		ATCC 24433	2
		ATCC 90028	5
		ATCC MYA-4441	3
	Candida albicans total		14
Candida auris	Candida auris	ATCC 10913	1
		ATCC 12372	1
		ATCC 12766	1
		CBS 10913	3
		CBS 12372	3
		CBS 12373	2
		CBS 12766	3
		CBS 12767	3
		CBS 12768	2
		CDC#0385	5
		CDC#0386	5
		CDC#0387	5
		CDC#0388	5
		CDC#0389	5
		CDC#0390	5
		Candida auris total
		ATCC MYA-577	6
Target	Organism	Strain	IndependentContrived SamplesTested
Candidadubliniensis	Candida dubliniensis	ATCC MYA-578	12
	Candida dubliniensis	ATCC MYA-579	12
	Candida dubliniensis	ATCC MYA-582	13
	Candida dubliniensis	NCPF3949	5
	Candida dubliniensis total		48
Candida famata	Debaryomyces fabryi	CBS 789	21
	Debaryomyces hansenii	CBS 1961	3
	Debaryomyces subglobosus	CBS 1796	27
	Candida famata total		51
Candida glabrata	Candida glabrata	128-4058	1
		128-4072	1
		ATCC 15126	2
		ATCC 15545	2
		ATCC 2001	1
		ATCC 66032	4
		ATCC 90030	2
	Candida glabrata	ATCC MYA-2950	3
	Candida glabrata total		16
Candidaguilliermondii	Candida guilliermondii	ATCC 22017	13
		ATCC 6260	12
	Meyerozyma guilliermondii	ATCC 90197	10
		ATCC 90198	9
		ATCC 90199	6
	Candida guilliermondii total		50
Candida kefyr	Candida kefyr	ATCC 204093	4
		ATCC 2512	10
		ATCC 4135	13
		ATCC 66028	12
		ATCC 8553	12
	Candida kefyr total		51
Candida krusei	Candida krusei	ATCC 14243	8
		ATCC 22985	9
		ATCC 28870	9
		ATCC 32196	10
		ATCC 34135	10
	Candida krusei total		46
Target	Organism	Strain	IndependentContrived SamplesTested
Candidalusitaniae	Candida lusitaniae	ATCC 26287	5
		ATCC 34449	10
		ATCC 42720	9
		ATCC 66035	11
		ATCC MYA-766	10
	Candida lusitaniae total		45
Candidaparapsilosis	Candida parapsilosis	ATCC 22019	11
		ATCC 28474	5
		ATCC 28475	10
		ATCC 58895	7
		ATCC 90018	8
	Candida parapsilosis total		41

{24}------------------------------------------------

{25}------------------------------------------------

Candida tropicalis	Candida tropicalis	ATCC 1369	9
		ATCC 13803	12
		ATCC 201380	9
		ATCC 201381	7
		ATCC 750	8
		Candida tropicalis total	45
Cryptococcusgattii	Cryptococcus gattii	ATCC 14248	11
			ATCC 76108
			ATCC MYA-4138
			ATCC MYA-4560
		ATCC MYA-4877	9
		Cryptococcus gattii total	50
Cryptococcusneoformans	Cryptococcus neoformans var. grubii	ATCC 14116	9
			ATCC208821(H99)
			ATCC 90112
			NCPF8195
			NCPF8299
			NCPF8357
	Cryptococcus neoformans var. neoformans(Filobasidiella bacillispora Kwon-Chung,teleomorph (serotype D))	ATCC 34873	3
	Cryptococcus neoformans var. neoformans(serotype D)	ATCC 36556	3
		ATCC MYA-565	9
		Cryptococcus neoformans total	52
Fusarium	Bisifusarium dimerum	CBS 108944	3
			CBS 110317
			CBS 116520

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

Target	Organism	Strain	IndependentContrived SamplesTested
	Fusarium moniliforme	ATCC 38159	11
	Fusarium oxysporum	CBS 116611	1
	Fusarium sacchari	ATCC 24379	10
		CBS 119828	11
	Fusarium solani	ATCC 36031	11
	Fusarium verticillioides	CBS 100312	11
Fusarium total			70
Rhodotorula	Rhodotorula glutinis	ATCC 32765	3
	Rhodotorula glutinis	ATCC 32766	3
	Rhodotorula glutinis (Fresenius) Harrison	ATCC 96365	4
	Rhodotorula mucilaginosa	ATCC 66034	21
		ATCC 9449	19
	Rhodotorula total		50

{27}------------------------------------------------

Genus and Group Assay Species Stratification

The ePlex BCID-FP Panel reports genus level results for Fusarium and Rhodotorula targets. Sensitivity/PPA of these genus and group level targets from all clinical and contrived samples tested are summarized in Table 22 below.

Target Species Detected	All Samples
	TP/TP+FN	% (95% CI)
Fusarium	69/70	98.6 (92.3-99.7)
Bisifusarium dimerum	14/15	93.3 (70.2-98.8)
Fusarium moniliforme	11/11	100 (74.1-100)
Fusarium oxysporum	1/1	100 (20.7-100)
Fusarium sacchari	21/21	100 (84.5-100)
Fusarium solani	11/11	100 (74.1-100)
Fusarium verticillioides	11/11	100 (74.1-100)
Rhodotorula	50/52	96.2 (87.0-98.9)
Rhodotorula*	2/2	100 (34.2-100)
Rhodotorula glutinis	6/6	100 (61.0-100)
Rhodotorula glutinis (Fresenius) Harrison	4/4	100 (51.0-100)
Rhodotorula mucilaginosa	38/40	95.0 (83.5-98.6)

Table 22: Species Detected by Genus Assays

*Results are from 2 clinical samples (1 retrospective). All other Fusarium and Rhodotorula samples were contrived.

{28}------------------------------------------------

Co-detections in Clinical Samples

The ePlex BCID-FP Panel did not identify any fungal co-detections in prospective samples, and 6 fungal co-detections were identified in the retrospective samples. Of the 120 retrospective samples, 114 (95.0%) had single detections and 6 (5.0%) had double detections. In the 6 codetections, 4 included 1 organism that comparator method(s) did not detect. See Table 27 below for a summary of co-detections in retrospective samples.

Distinct Organism Combinations Detected bythe ePlex Panel in Retrospective Samples		Number of Samples(Number Discrepant)	DiscrepantOrganism(s)A,B
Target 1	Target 2
Candida albicans	Candida dubliniensis	1 (0)
Candida albicans	Candida parapsilosis	1 (0)
Candida dubliniensis	Candida parapsilosis	1 (1)	C. parapsilosis (1)
Candida dubliniensis	Candida tropicalis	1 (1)	C. tropicalis (1)
Candida glabrata	Candida lusitaniae	2 (2)	C. glabrata (2)

Table 23 Co-Detections Identified by the ePlex BCID-FP Panel (Retrospective Samples)

A discrepant organism is defined as one that was detected by the BCID-FP Panel but not by the comparator method(s). A. B.

4/4 organisms were investigated using PCR/sequencing and the discrepant organism was detected in 4/4 cases.

i. In 1/1 sample, C. parapsilosis was detected.

ii. In 1/1 sample, C. tropicalis was detected.

iii. In 2/2 samples, C. glabrata was detected.

Summaries of additional distinct fungal co-detections detected by the comparator method in prospective and retrospective samples are provided in Table 29. These tables include additional distinct fungal co-detections not included in the co-detections identified by the BCID-FP Panel; they have 1 or more organism not detected by the BCID-FP Panel or an offpanel fungal organism.

Table 24: Co-Detections Identified by the Comparator Method (Prospective Samples
Distinct Organism Combinations Detected by

Distinct Organism Combinations Detected bythe Comparator Method in ProspectiveSamples		Number of Samples(Number Discrepant)	DiscrepantOrganism(s)A
Target 1	Target 2
Candida metapsilosis*	Trichosporon asahii*	1 (0)

A. A discrepant organism is defined as one that was detected by the comparator method(s) but not by the BCID-FP Panel (excludes organisms not targeted by the BCID-FP Panel).

Off-panel organism not targeted by the BCID-FP Panel.

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

Distinct Organism Combinations Detected by theComparator Method in Retrospective Samples			Number of Samples(NumberDiscrepant)	DiscrepantOrganism(s)A
Target 1	Target 2	Target 3
Candidaalbicans	Candidadubliniensis	Candida glabrata	1 (1)	C. glabrata (1)
Candidaalbicans	Candidaparapsilosis		1 (1)	C. parapsilosis (1)

Table 25: Co-Detections Identified by the Comparator Method (Retrospective Samples)

A discrepant organism is defined as one that was detected by the comparator method(s) but not by the BCID-FP Panel A. (excludes organisms not targeted by the BCID-FP Panel).

{30}------------------------------------------------

Clinical Study ePlex Instrument Performance

A total of 867 samples (including prospective, retrospective, and contrived samples) were initially tested in the clinical evaluations. Of these, 7/867 (0.8%) did not complete the run and the sample was retested. After repeat testing, all 867 sampleted testing and 839/867 (96.8%, 95% CI: 95.4%-97.8%) generated valid results and 28/867 (3.2%, 95% CI: 2.2%-4.6%) generated invalid results on the first completed attempt.

Upon repeat testing of the 28 samples with initially invalid results, all completed the run and 27/28 (96.4%) generated valid results. Overall, after final testing, 1/867 (0.1%, 95% CI: 0.0%-0.7%) had final, invalid results, resulting in a final validity rate of 866/867 (99.9%, 95% CI: 99.3%-100%).

Analytical Performance Characteristics

Limit of Detection (Analytical Sensitivity)

The Limit of Detection (LOD), or analytical sensitivity, was identified and verified for each assay on the BCID-FP Panel using at least two quantified reference strains in simulated blood culture sample matrix, which is defined as whole blood with EDTA added to a blood culture bottle in the same ratio as the manufacturer recommends and incubated for 8 hours. At least 20 replicates per target were tested for each condition. The limit of detection was defined as the lowest concentration of each target that is detected in ≥95% of tested replicates. The confirmed LOD for each ePlex BCID-FP Panel organism is shown in Table 26.

{31}------------------------------------------------

Table 26: LOD Results Summary

Target	Organism	Strain	LODConcentration(CFU/mL)
Candida albicans	Candida albicans	ATCC 14053	$1 x 10^6$
	Candida albicans	ATCC 24433	$1 x 10^5$
Candida auris	Candida auris	CBS 10913	$1 x 10^5$
	Candida auris	CBS 12766	$1 x 10^5$
Candida dubliniensis	Candida dubliniensis	ATCC MYA-577	$1 x 10^4$
	Candida dubliniensis	NCPF 3949	$1 x 10^5$
Candida famata	Candida famata	CBS 767	$1 x 10^3$
	Candida famata	CBS 766	$1 x 10^4$
Candida glabrata	Candida glabrata	ATCC 2001	$1 x 10^6$
	Candida glabrata	ATCC 15545	$1 x 10^6$
Candida guilliermondii	Candida guilliermondii	ATCC 22017	$1 x 10^5$
	Candida guilliermondii	ATCC 6260	$1 x 10^5$
Candida kefyr	Candida kefyr	ATCC 4135	$1 x 10^3$
	Candida kefyr	ATCC 8553	$1 x 10^4$
Candida krusei	Candida krusei	ATCC 22985	$1 x 10^5$
	Candida krusei	ATCC 28870	$1 x 10^6$
Candida lusitaniae	Candida lusitaniae	ATCC 34449	$1 x 10^6$
	Candida lusitaniae	ATCC 66035	$1 x 10^5$
Candida parapsilosis	Candida parapsilosis	ATCC 28474	$1 x 10^4$
	Candida parapsilosis	ATCC 28475	$1 x 10^5$
Candida tropicalis	Candida tropicalis	ATCC 13803	$1 x 10^5$
	Candida tropicalis	ATCC 1369	$1 x 10^6$
Cryptococcus gattii	Cryptococcus gattii	ATCC MYA-4877	$1 x 10^3$
Target	Organism	Strain	LODConcentration(CFU/mL)
	Cryptococcus gattii	ATCC MYA-4138	1 x 103
Cryptococcus neoformans	Cryptococcus neoformans	ATCC 208821	1 x 105
	Cryptococcus neoformans	ATCC MYA-565	1 x 105
Fusarium	Fusarium oxysporum	CBS 116611	1 x 106spores/mL
	Fusarium solani	ATCC 36301	1 x 106spores/mL
Rhodotorula	Rhodotorula mucilaginosa	ATCC 4058	1 x 105
	Rhodotorula glutinis	ATCC 32765	1 x 105
Target	Organism	Strain
Candida albicans	Candida albicans	ATCC 10231
	Candida albicans	ATCC 90028
	Candida albicans	ATCC MYA-4441
Candida auris	Candida auris	CDC#385
	Candida auris	CDC#386
	Candida auris	CDC#387
	Candida auris	CDC#388
	Candida auris	CDC#389
	Candida auris	CDC#390
	Candida auris	CBS 12766
Candida dubliniensis	Candida dubliniensis	ATCC MYA-578
	Candida dubliniensis	ATCC MYA-579
	Candida dubliniensis	ATCC MYA-582
Candida famata	Candida famata	ATCC 20850
	Candida famata	CBA 1961
	Candida famata	CBS 789
Candida glabrata	Candida glabrata	ATCC 15126
	Candida glabrata	ATCC 66032
	Candida glabrata	ATCC MYA-2950
Candida guilliermondii	Candida guilliermondii	ATCC 90197
	Candida guilliermondii	ATCC 90198
	Candida guilliermondii	ATCC 90199
Candida kefyr	Candida kefyr	ATCC 204093
	Candida kefyr	ATCC 2512
	Candida kefyr	ATCC 66028
Candida krusei	Candida krusei	ATCC 14243
	Candida krusei	ATCC 32196
	Candida krusei	ATCC 34135
Candida lusitaniae	Candida lusitaniae	ATCC 42720
	Candida lusitaniae	ATCC MYA-766
	Candida lusitaniae	Z010
Candida parapsilosis	Candida parapsilosis	ATCC 22019
	Candida parapsilosis	ATCC 58895
	Candida parapsilosis	ATCC 90018
Candida tropicalis	Candida tropicalis	ATCC 201380
	Candida tropicalis	ATCC 201381
Target	Organism	Strain
	Candida tropicalis	ATCC 750
Cryptococcus gattii	Cryptococcus gattii	ATCC 14248
Cryptococcus gattii	Cryptococcus gattii	ATCC 4560
Cryptococcus gattii	Cryptococcus gattii	ATCC 76108
Cryptococcus neoformans	Cryptococcus neoformans	ATCC 14116
Cryptococcus neoformans	Cryptococcus neoformans	ATCC 90112
Cryptococcus neoformans	Cryptococcus neoformans	NCPF 8299
Fusarium	Bisifusarium dimerum	CBS 110317
Fusarium	Fusarium moniliforme	ATCC 38159
Fusarium	Fusarium proliferatum	CBS 131570
Fusarium	Fusarium sacchari	CBS 119828
Fusarium	Fusarium verticillioides	CBS 100312
Rhodotorula	Rhodotorula glutinis	ATCC 96365
Rhodotorula	Rhodotorula mucilaginosa	ATCC 66034
Rhodotorula	Rhodotorula mucilaginosa	ATCC 9449

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

Analytical Reactivity (Inclusivity)

A panel of 51 strains/isolates, representing the genetic, temporal and geographic diversity of each target on the ePlex BCID-FP Panel was evaluated to demonstrate analytical reactivity. Each strain was tested in triplicate at concentrations approximating bottle positivity (1 x 10° CFU/mL for Candida and Rhodotorula, 1 x 107 CFU/mL for Cryptococcus, and 1 x 108 spores/mL for Fusarium). All organisms tested were detected at bottle positive concentrations. Results of the analytical reactivity are summarized in Table 27. An additional 29 unique strains were detected as a part of the Limit of Detection (Analytical Sensitivity) study and are summarized in Table 26.

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

Table 27: Analytical Reactivity (Inclusivity) Results

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

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

Predicted (in silico) Reactivity for Genus and Group Assays

Note: the performance of the ePlex BCID-FP Panel has not been established for all of the organisms listed in the tables below. See the Analytical Reactivity (Inclusivity) and Limit of Detection (Analytical Sensitivity) sections of the package insert for data on organisms for which performance characteristics have been established (indicated with an asterisk in Tables 32 and 33). Some species were not assessed in silico due to lack of sequence data, though they may appear in the analytical sensitivity or specificity studies.

In addition to species-specific assays, the ePlex BCID-FP Panel contains two broader genuslevel assays: Fusarium and Rhodotorula. Table 28 and Table 29 highlight the predicted (in silico) reactivity (inclusivity) for these assay targets.

Detection Predicted for ≥95% of target sequences
Fusarium acaciae-mearnsii	Fusarium cortaderiae	Fusarium napiforme
Fusarium acuminatum	Fusarium culmorum	Fusarium nisikadoi
Fusarium acutatum	Fusarium denticulatum	Fusarium nygamai
Fusarium aethiopicum	Bisifusarium dimerum*	Fusarium oxysporum*
Fusarium ananatum	Fusarium dlaminii	Fusarium palustre
Fusarium andiyazi	Fusarium equiseti	Fusarium phyllophilum
Fusarium anthophilum	Fusarium falciforme	Fusarium poae
Fusarium armeniacum	Fusarium foetens	Fusarium polyphialidicum
Fusarium asiaticum	Fusarium fujikuroi	Fusarium proliferatum*
Fusarium austroamericanum	Fusarium gaditjirri	Fusarium pseudoanthophilum
Fusarium avenaceum	Fusarium globosum	Fusarium pseudocircinatum
Fusarium aywerte	Fusarium guttiforme	Fusarium pseudograminearum
Fusarium bactridioides	Fusarium hostae	Fusarium pseudonygamai
Fusarium begoniae	Fusarium incarnatum	Fusarium ramigenum
Fusarium beomiforme	Fusarium inflexum	Fusarium sacchari*
Fusarium boothii	Fusarium konzum	Fusarium secorum
Fusarium brachygibbosum	Fusarium lacertarum	Fusarium sinensis
Fusarium brasilicum	Fusarium lactis	Fusarium solani*
Fusarium brevicatenulatum	Fusarium langsethiae	Fusarium sporotrichioides
Fusarium bulbicola	Fusarium lichenicola	Fusarium sterilihyphosum

Table 28: Predicted (in silico) Reactivity (Inclusivity) Results for Fusarium

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

	(Cylindrocarpon lichenicola)
Fusarium bullatum	Fusarium louisianense	Fusarium subglutinans
Fusarium camptoceras	Fusarium lunulosporum	Fusarium temperatum
Fusarium cerealis	Fusarium mangiferae	Fusarium thapsinum
Fusarium circinatum	Fusarium meridionale	Fusarium udum
Fusarium commune	Fusarium mesoamericanum	Fusarium verticillioides*
Fusarium concentricum	Fusarium mexicanum
Fusarium concolor	Fusarium musae
Detection Predicted for 85%-94% of target sequences
Fusarium torulosum	Fusarium xylarioides
Detection Predicted for <85% of target sequences
Fusarium chlamydosporum (66.7%)	Fusarium graminearum (59.4%)	Fusarium longipes (25.0%)
Fusarium coeruleum (50.0%)	Fusarium lateritium (50.0%)	Fusarium nelsonii (16.7%)
Detection Not Predicted
Fusarium kyushuense	Fusarium sambucinum	Fusarium venenatum
Fusarium miscanthi	Fusarium stilboides
Fusarium redolens	Fusarium succisae

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

Detection Predicted for ≥95% of target sequences
Rhodotorula araucariae	Rhodotorula graminis	Rhodotorula taiwanensis
Rhodotorula glutinis*	Rhodotorula mucilaginosa*
Detection Predicted for 85%-94% of target sequences
None Identified
Detection Predicted for <85% of target sequences
None Identified
Detection Not Predicted
Rhodotorula acheniorum	Rhodotorula fragariae	Rhodotorula marina
Rhodotorula acuta	Rhodotorula fujisanensis	Rhodotorula minuta
Rhodotorula armeniaca	Rhodotorula hinnulea	Rhodotorula muscorum
Rhodotorula aurantiaca	Rhodotorula hordea	Rhodotorula nothofagi
Rhodotorula auriculariae	Rhodotorula hylophila	Rhodotorula philyla
Rhodotorula bacarum	Rhodotorula ingeniosa	Rhodotorula phylloplana
Rhodotorula bogoriensis	Rhodotorula javanica	Rhodotorula pilati
Rhodotorula buffonii	Rhodotorula lactosa	Rhodotorula pustula
Rhodotorula ferulica	Rhodotorula lignophila	Rhodotorula sonckii

Table 29: Predicted (in silico) Reactivity (Inclusivity) Results for Rhodotorula

{38}------------------------------------------------

Analytical Specificity (Cross-Reactivity and Exclusivity)

Cross-reactivity of on-panel and off-panel analytes was evaluated with the BCID-FP Panel. Onpanel organisms were tested in triplicate at concentrations approximating bottle positivity (see Analytical Reactivity (Inclusivity) section of the package insert for additional details). Offpanel organisms were tested at concentrations of ≥1x10° CFU/mL for bacteria and ≥1x107 CFU/mL or spores/mL for fungi unless otherwise noted in Table 30. If the target concentration could not be reached, the organism was diluted 2-fold from stock for use.

No cross reactivity was observed for any of the organisms tested. See Table 30 for a summary of the on-panel strains tested and Table 31 for a summary of off-panel strains tested.

{39}------------------------------------------------

On-Panel Exclusivity

Organism	Strain	Organism
Candida albicans	ATCC 10231	Candida krusei
Candida albicans	ATCC 90028	Candida krusei
Candida albicans	ATCC MYA-4441	Candida lusitaniae
Candida auris	CBS 12766	Candida lusitaniae
Candida auris	CDC#385	Candida lusitaniae
Candida auris	CDC#386	Candida parapsilosis
Candida auris	CDC#387	Candida parapsilosis
Candida auris	CDC#388	Candida parapsilosis
Candida auris	CDC#389	Candida tropicalis
Candida auris	CDC#390	Candida tropicalis
Candida dubliniensis	ATCC MYA-578	Candida tropicalis
Candida dubliniensis	ATCC MYA-579	Cryptococcus gattii
Candida dubliniensis	ATCC MYA-582	Cryptococcus gattii
Candida famata	ATCC 20850	Cryptococcus gattii
Candida famata	CBA 1961	Cryptococcus neoformans
Candida famata	CBS 789	Cryptococcus neoformans
Candida glabrata	ATCC 15126	Cryptococcus neoformans
Candida glabrata	ATCC 66032	Bisifusarium dimerum
Candida glabrata	ATCC MYA-2950	Fusarium lichenicola(Cylindrocarpon lichenicola)
Candida guilliermondii	ATCC 90197	Fusarium moniliforme
Candida guilliermondii	ATCC 90198	Fusarium proliferatum
Candida guilliermondii	ATCC 90199	Fusarium sacchari
Candida kefyr	ATCC 204093	Fusarium verticillioides
Candida kefyr	ATCC 2512	Rhodotorula glutinis
Candida kefyr	ATCC 66028	Rhodotorula mucilaginosa
Candida krusei	ATCC 14243	Rhodotorula mucilaginosa
Organism	Strain
Acinetobacter Iwoffii	ATCC 15309
Acremonium kiliense	ATCC 4301
Aspergillus fumigatus	ATCC 204305A
Bacteroides fragilis	ATCC 25285
Bordetella pertussis	ATCC 9340
Candida bracarensis	CBS 10154
Candida carpophila	CBS 5256
Candida duobushaemulonii	CDC#394
Candida haemulonii	CDC#393
Candida inconspicua	ATCC 16783
Candida lambica	ATCC 24750
Candida lipolytica	ATCC 20177
Candida metapsilosis	ATCC 96144
Candida nivariensis	CBS 9984
Candida norvegensis	ATCC 22977
Candida orthopsilosis	ATCC 96139
Candida pelliculosa	ATCC 10262
Candida rugosa	CBS 96275
Candida sake	ATCC 22021
Candida utilis	ATCC 9256
Citrobacter freundii	ATCC 6879
Clostridium perfringens	ATCC 13124
Corynebacterium striatum	ATCC 7094
Enterobacter aerogenes	ATCC 29751
Enterobacter cloacae	ATCC 23373
Enterococcus faecium	ATCC 31282
Exophiala jeanselmei	ATCC 12734
Filobasidium elegans	CBS 7637
Filobasidium globisporum	CBS 7642
Klebsiella oxytoca	ATCC 43165
Kluyveromyces lactis	ATCC 10689
Organism	Strain
Kodamaea ohmeri	CDC#0396
Lactobacillus rhamnosus	ATCC 53103
Malassezia furfur	ATCC 12078
Malassezia furfur	ATCC 14521
Malassezia furfur	CBS 7710
Malassezia globosa	ATCC MYA-4612
Malassezia restricta	ATCC MYA-4611
Malassezia sympodialis	ATCC 44031
Meyerozyma caribbica(Candida fermentati)	ATCC 20296
Micrococcus luteus	ATCC 19212
Morganella morganii	ATCC 25830
Mucor velutinosus	ATCC MYA-4766
Penicillium marneffei	ATCC 200050
Proteus mirabilis	ATCC 35659
Rhodotorula minuta	ATCC 36236
Saccharomyces cerevisiae	ATCC 18824
Salmonella enterica (Typhi)	ATCC 19430
Scedosporium prolificans	ATCC 200543
Schizosaccharomyces pombe	LPY 02387
Serratia marcescens	ATCC 43861
Sporidiobolus salmonicolor	ATCC 24217
Sporothrix schenckii	ATCC 18616
Staphylococcus hominis	ATCC 27844
Staphylococcus intermedius	ATCC 29663
Staphylococcus saprophyticus	ATCC 15305
Streptococcus agalactiae	ATCC 12401
Streptococcus anginosus	ATCC 9895
Streptococcus pyogenes	ATCC 12384
Trichosporon asahii	ATCC 201110
Trichosporon asteroides	ATCC 90043
Trichosporon dermatis	ATCC 204094

Table 30: On-Panel Organisms Assessed for Cross-reactivity with the ePlex BCID-FP Panel (Exclusivity)

{40}------------------------------------------------

Off-Panel Exclusivity

Table 31: Off-Panel Organisms Assessed for Cross-Reactivity

with the ePlex BCID-FP Panel (Exclusivity)

A. Tested at 1 x 106 spores/mL

{41}------------------------------------------------

Bottle Positivity

Several representative fungal organisms were spiked into blood culture bottles along with the manufacturer's recommended volume of human whole blood and grown to positivity in a commercially-available continuously monitoring blood culture system. Bottles were removed from the incubator within two hours of being identified as positive as well as eight hours after bottle positivity. At least two independent positive blood culture replicates were quantified for each organism on culture plates. Organisms tested and approximate bottle positivity concentrations are summarized in Table 32. Concentrations shown below represent approximate levels that may be observed in a clinical setting. All estimated bottle positivity concentrations are equivalent or greater than the established Limit of Detection (LOD) for each of the assays of the ePlex BCID-FP Panel.

Organism	Strain ID	Mean Bottle PositivityConcentration	Mean Bottle Positivity+8 hours Concentration
Candida albicans	ATCC 90082	1.6 x 106 CFU/mL	1.4 x 106 CFU/mL
Cryptococcus neoformans var. grubii	ATCC 14116	1.3 x 107 CFU/mL	6.5 x 107 CFU/mL
Fusarium solani	ATCC 36031	9.6 x 106 spores/mL	7.7 x 106 spores/mL
Rhodotorula mucilaginosa	ATCC 66034	1.6 x 106 CFU/mL	4.2 x 106 CFU/mL

Table 32: Bottle Positivity Concentrations

{42}------------------------------------------------

Reproducibility

Two positive mixes including 5 on-panel organisms at two concentrations and one negative mix including an off-panel organism were tested. Concentrations in the positive mixes reflected those observed at time of bottle positivity (BP) and time of bottle positivity plus 8 hours or one log higher than that expected at bottle positivity (BP+8) and one mix containing an off-panel organism grown to bottle positivity, which is expected to yield a negative result. Bottle concentrations used in this study are summarized in Table 32. Testing occurred at three sites, with two operators testing the mixes over six days using three cartridge lots.

Organism	Bottle PositivityConcentration	Bottle Positivity +8Hours Concentration
Candida albicans	1 x 106 CFU/mL	1 x 107 CFU/mL
Candida kefyr	1 x 106 CFU/mL	1 x 107 CFU/mL
Cryptococcus neoformans	1 x 107 CFU/mL	1 x 108 CFU/mL
Fusarium sacchari	6.5 x 106 spores/mL	6.1 x106 spores/mL
Rhodotorula mucilaginosa	1 x 106 CFU/mL	1 x 107 CFU/mL

Table 32: Bottle Positivity Concentrations

The percent agreement of each target with the expected result is summarized in Tables 33-37.

{43}------------------------------------------------

Concentration ofCandida albicans	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Bottle Positive+ 8 Hours(1x107 CFU/mL)	1	34/35	97.1	(85.5-99.5)
	2	35/36	97.2	(85.8-99.5)
	3	36/36	100	(90.4-100)
	All	105/107	98.1	(93.4-99.5)
Bottle Positive(1x106 CFU/mL)	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Negative	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	108/108	100	(96.6-100)
	All	324/324	100	(98.8-100)

Table 33: Percent Agreement for Candida albicans

Table 34: Percent Agreement for Candida kefyr

Concentration ofCandida kefyr	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Bottle Positive+ 8 Hours(1x107 CFU/mL)	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Bottle Positive(1x106 CFU/mL)	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Negative	1	107/107	100	(96.5-100)
	2	108/108	100	(96.6-100)
	3	108/108	100	(96.6-100)
	All	323/323	100	(98.8-100)

{44}------------------------------------------------

Concentration of	Site	Agreement with Expected Results
Cryptococcus neoformans		Agreed / N	%	95% CI
Bottle Positive+ 8 Hours(1x108 CFU/mL)	1	35/35	100	(90.1-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	107/107	100	(96.5-100)
Bottle Positive(1x107 CFU/mL)	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Negative	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	108/108	100	(96.6-100)
	All	324/324	100	(98.8-100)

Table 35: Percent Agreement for Cryptococcus neoformans

Table 36: Percent Agreement for Fusarium

Concentration ofFusarium sacchari	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Bottle Positive+ 8 Hours(6.1x106 spores/mL)	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Bottle Positive(6.5x106 spores/mL)	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Negative	1	107/107	100	(96.5-100)
	2	108/108	100	(96.6-100)
	3	108/108	100	(96.6-100)
	All	323/323	100	(98.8-100)

{45}------------------------------------------------

Concentration of	Site	Agreement with Expected Results
Rhodotorula mucilaginosa		Agreed / N	%	95% CI
Bottle Positive+ 8 Hours(1x107 CFU/mL)	1	36/36	100	(90.4-100)
	2	34/36	94.4	(81.9-98.5)
	3	35/36	97.2	(85.8-99.5)
	All	105/108	97.2	(92.1-99.1)
Bottle Positive(1x106 CFU/mL)	1	35/36	97.2	(85.8-99.5)
	2	36/36	100	(90.4-100)
	3	35/36	97.2	(85.8-99.5)
	All	106/108	98.1	(93.5-99.5)
Negative	1	107/107	100	(96.5-100)
	2	108/108	100	(96.6-100)
	3	108/108	100	(96.6-100)
	All	323/323	100	(98.8-100)

Table 37: Percent Agreement for Rhodotorula

{46}------------------------------------------------

Interfering Substances and Sample Matrix Equivalency (Bottle Evaluation)

Two organism mixes consisting of 5 on-panel organisms and negative blood matrix were used to assess potentially interfering substances and bottle types for interference. The concentration of each organism tested is summarized in Table 38.

Organism	Concentration
Candida albicans	1 x 106 CFU/mL
Candida kefyr	1 x 106 CFU/mL
Cryptococcus neoformans	1 x 107 CFU/mL
Fusarium sacchari	6.5 x 106 spores/mL
Rhodotorula mucilaginosa	1 x 106 CFU/mL

Table 38: Interfering Substance and Bottle Equivalency Concentrations

Interfering Substances

Eighteen substances were used to assess the ePlex BCID-FP Panel for potential interference. The organisms in Table 38 were spiked into negative blood matrix and tested in triplicate with and without each potentially interfering substance. Negative blood matrix was tested to control for potential positive interference. Potentially interfering substances are summarized in Table 39. None of the eighteen substances commonly found in blood culture specimens or as medications commonly used to treat skin or blood infections were found to inhibit the ePlex BCID-FP Panel at the clinically relevant concentrations. The effect of interfering substances has only been evaluated for the organisms listed in Table 38. Interference due to substances other than those described in this section can lead to erroneous results.

{47}------------------------------------------------

Endogenous Substances	Testing Concentration
Bilirubin	60 µg/mL
Hemoglobin	0.6 g/L
Human Genomic DNA	6 x 105 copies/mL
Triglycerides	1000 mg/dL
γ-globulin	0.85 g/dL
Exogenous Substances	Testing Concentration
Amoxicillin/Clavulanate	3.5 µg/mL
Amphotericin B	2 µg/mL
Caspofungin	5 µg/mL
Ceftriaxone	0.23 mg/mL
Ciprofloxacin	3 mg/L
Fluconazole	25 mg/L
Flucytosine	90 µg/mL
Gentamicin sulfate	3 µg/mL
Heparin	0.9 U/mL
Imipenem	83 µg/mL
Sodium Polyanethol Sulfonate(SPS)	0.25% w/v
Tetracycline	5 mg/L
Vancomycin	30 mg/L

Table 39: Potentially Interfering Substances: Substance List

{48}------------------------------------------------

Sample Matrix Equivalency (Bottle Evaluation)

Fifteen bottle types were tested for interference with each of the organisms listed in Table 38. Five replicates of each organism were tested in each of two bottle lots. Negative blood matrix was run as a negative control. Thirteen of the bottle types tested showed no interference for any of the targets tested. One lot of the BACT/Alert® PF Plus bottle type showed lower sensitivity for Rhodotorula and one lot BACT/Alert® FA Plus bottle type showed lower sensitivity for Candida albicans. A summary of the bottle types assessed and the study outcomes is found in Table 40.

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Manufacturer	Bottle Brand	Bottle Type	Study Outcome
BD	BACTEC™	Plus Aerobic	No interference observed
BD	BACTEC	Plus Anaerobic	No interference observed
BD	BACTEC	Standard Aerobic	No interference observed
BD	BACTEC	Standard Anaerobic	No interference observed
BD	BACTEC	Peds Plus™	No interference observed
BD	BACTEC	Lytic Anaerobic	No interference observed
BD	BACTEC	Myco	No interference observed
bioMérieux	BACT/ALERT®	SA Standard Aerobic	No interference observed
bioMérieux	BACT/ALERT	SN Standard Anaerobic	No interference observed
bioMérieux	BACT/ALERT	FA Plus	A false negative result wasobserved for the Candidaalbicans target in one lot.
bioMérieux	BACT/ALERT	FN Plus	No interference observed
bioMérieux	BACT/ALERT	PF Plus	False negative results wereobserved for the Rhodotorulatarget in one lot.
bioMérieux	BACT/ALERT	MP Mycobacteria	No interference observed
Thermo Scientific™	VersaTREK™	REDOX™ 1 EZ DrawAerobic	No interference observed
		REDOX 2 EZ DrawAnaerobic	No interference observed

Table 40: Sample Matrix Equivalency (Bottle Evaluation) Bottle Types

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Carryover and Cross-Contamination

Carryover and cross-contamination were evaluated for the ePlex BCID-FP Panel within and between runs by alternating high positive and negative samples across multiple runs over 5 rounds of testing. Fusarium sacchari was grown to bottle positivity +8 hours and spiked with 1 x 107 CFU/mL Candida albicans to simulate clinically relevant high positive samples for positive testing. Negative blood culture matrix was used to represent negative samples. No false positives were detected, indicating that no carryover or cross-contamination was observed between bays or within bays with the ePlex BCID-FP Panel when testing consecutively or in adjacent bays.

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Competitive Inhibition Study

Competitive inhibition was evaluated for the ePlex BCID-FP Panel by pairing twelve clinically relevant organisms (including 9 off-panel organisms) in thirteen simulated dual infection sample mixes. Candida albicans was tested at low titer (concentration expected at bottle positivity) while in the presence of other organisms at higher titer (concentrations expected at 8 hours beyond bottle positivity, or one log higher than that expected at bottle positivity). Candida glabrata and Candida parapsilosis were also tested at the concentration expected at bottle positivity in the presence of Candida albicans at a higher titer concentration. No competitive inhibition was observed in any of the sample mixes evaluated at the concentrations listed in Table 41.

On-panel Organisms	HighConcentration	LowConcentration
Candida albicans	1 x 107 CFU/mL	1 x 106 CFU/mL
Candida glabrata	1 x 107 CFU/mL	1 x 106 CFU/mL
Candida parapsilosis	1 x 107 CFU/mL	1 x 106 CFU/mL
Off-panel Organisms	High Concentration
Acinetobacter baumannii	1 x 109 CFU/mL
Cutibacterium acnes	1 x 109 CFU/mL
Enterococcus faecalis	1 x 108 CFU/mL
Escherichia coli	1 x 109 CFU/mL
Klebsiella pneumoniae	1 x 109 CFU/mL
Staphylococcus aureus	1 x 108 CFU/mL
Staphylococcus epidermidis	1 x 108 CFU/mL
Streptococcus pneumoniae	4 x 108 CFU/mL
Pseudomonas aeruginosa	4 x 108 CFU/mL

Table 41: Competitive Inhibition Organisms and Concentrations Tested

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).