Predicate Devices

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Reference Devices

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AI/ML (Artificial Intelligence / Machine Learning)

No
The device description details a molecular assay based on nucleic acid detection and microarray analysis. The decision-making process for results is based on light scatter intensity thresholds, not complex pattern recognition or learning algorithms. There is no mention of AI, ML, or related concepts in the document.

Therapeutic

No.
The device is an in vitro diagnostic test for detecting and identifying specific bacteria and resistance markers, which aids in the diagnosis of bacterial bloodstream infections. It does not provide treatment or therapy.

Diagnostic

Yes

The text explicitly states: "The Verigene® Gram Positive Blood Culture Nucleic Acid Test (BC-GP) ... is a qualitative, multiplexed in vitro diagnostic test for the simultaneous detection and identification of potentially pathogenic gram-positive bacteria..." and "...is indicated for use in conjunction with other clinical and laboratory findings, such as culture, to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor bloodstream infections." These phrases directly classify the device as diagnostic.

SaMD (Software as a Medical Device)

No

The device description clearly states that the Verigene System consists of both a Reader and a Processor SP, which are hardware components. The test itself also utilizes physical consumables and a test cartridge. While software is involved in the user interface and results generation, the device is not solely software.

IVD (In Vitro Diagnostic)

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

The "Intended Use / Indications for Use" section explicitly states: "The Verigene® Gram Positive Blood Culture Nucleic Acid Test (BC-GP) performed using the sample-to-result Verigene System is a qualitative, multiplexed in vitro diagnostic test for the simultaneous detection and identification of potentially pathogenic gram-positive bacteria which may cause bloodstream infection (BSI)."

This statement directly identifies the device as an in vitro diagnostic test.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Verigene® Gram Positive Blood Culture Nucleic Acid Test (BC-GP) performed using the sample-to-result Verigene System is a qualitative, multiplexed in vitro diagnostic test for the simultaneous detection and identification of potentially pathogenic gram-positive bacteria which may cause bloodstream infection (BSI). BC-GP is performed directly on positive blood culture using BACTEC™ Plus Aerobic/F and BacT/ALERT FA FAN® Aerobic blood culture bottles, which contain gram positive bacteria. BC-GP is indicated for use in conjunction with other clinical and laboratory findings, such as culture, to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor bloodstream infections.

BC-GP detects and identifies the following bacterial genera and species:

Staphylococcus spp.	Streptococcus spp.	Enterococcus
Staphylococcus aureus	Streptococcus	faecalis
Staphylococcus	pneumoniae	Enterococcus
epidermidis	Streptococcus pyogenes	faecium
Staphylococcus	Streptococcus agalactiae
lugdunensis	Streptococcus anginosus	Listeria spp.
	group

In addition, BC-GP 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, BC-GP does not specifically attribute van-mediated vancomycin resistance to either E. faecium, or mecA-mediated methicillin resistance to either S. aureus or S. epidermidis.

BC-GP is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial bloodstream infections; however, is not to be used to monitor these infections. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing. identification of organisms not detected by BC-GP, differentiation of mixed growth, association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing.

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

PAM

Device Description

The Verigene® Gram Positive Blood Culture Nucleic Acid Test (BC-GP) is a molecular assav which relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial nucleic acid sequences detected by the BC-GP test, Capture and Mediator oligonucleotides are utilized for gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides bind to a specific portion of the nucleic acid target and are themselves bound onto a substrate in the microarray. The Mediator oligonucleotides bind to a different portion of the same nucleic acid target and allow binding of a gold nanoparticle probe to a portion complementary to a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency.

The BC-GP test is performed on the Verigene® System, a 'sample-to-result', fully automated, bench-top molecular diagnostics workstation. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP. The BC-GP test utilizes single-use disposable test consumables and a self-contained Verigene Test Cartridge for each sample tested. For the BC-GP test, the Verigene System allows automated nucleic acid extraction from Gram-positive bacteria-containing blood culture specimens and target detection of bacteria-specific DNA.

The Reader is the Verigene System's user interface, which serves as the central control unit for all aspects of test processing and results generation. The Reader's graphical user interface guides the user through test processing and test results using a barcode scanner. The user inserts the Test Cartridge into the Verigene Processor SP, which executes the test procedure. automating the steps of (1) Sample Preparation - Cell lysis and magnetic bead-based bacterial DNA isolation from blood culture samples and (2) Verigene Hybridization Test - Detection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology.

After test processing is complete, to obtain the test results the user removes the Test Cartridge from the Processor SP, removes the reagent pack from the substrate holder, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make decisions regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte.

Mentions image processing

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Mentions AI, DNN, or ML

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Input Imaging Modality

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Anatomical Site

Blood culture

Indicated Patient Age Range

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Intended User / Care Setting

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

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

A method comparison study (n=1767) was conducted at five external, geographically-diverse clinical study sites to evaluate the comparative performance of BC-GP to applicable conventional biochemical, culture, and bidirectional sequencing reference methods. The total evaluable specimens tested in the studies included; 1251 fresh, 175 frozen, and 216 contrived blood culture specimens. Eligible study subjects included individuals receiving routine care requiring blood culture testing. Blood culture specimens were collected from the patients and incubated on either the BACTEC or the BacT Alert continuous monitoring blood culture systems. Bottles that were flagged positive by the instrument were gram stained and then bottles confirmed to contain gram positive organisms were then tested with the BC-GP Test.

Additionally, simulated specimens were utilized for very rare organisms such as S. lugdunensis, S. agalactiae, S. anginosus group, S. pyogenes, and Listeria species. These specimens were prepared by spiking blood culture bottles containing whole blood with bacterial suspensions of bacterial isolates. Prepared blood culture bottles were then grown to positivity on the continuous monitoring blood culture system, until flagged positive. Gram stains were performed to verify the presence of gram positive bacteria, and then testing was performed with the BC-GP Test.

BC-GP Test results were compared with results from traditional laboratory reference methods (i.e. culture followed by testing blood culture isolates with conventional biochemicals. Vitek2, and cefoxitin disc testing. Cefoxitin discs were used as the reference method for confirming mecA mediated resistance in S. aureus and S. epidermidis. Vancomycin resistance and the presence of vanA or vanB in E. faecium and E. faecalis was performed using vancomycin E-tests followed by bidirectional sequencing on resistant organisms.

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

A method comparison study (n=1767) was conducted at five external, geographically-diverse clinical study sites to evaluate the comparative performance of BC-GP to applicable conventional biochemical, culture, and bidirectional sequencing reference methods. The total evaluable specimens tested in the studies included; 1251 fresh, 175 frozen, and 216 contrived blood culture specimens.

The initial No-call rate in the clinical study was 4.7% (77/1642) , and the final No-call rate was 1.1% (18/1642). The total rate of initial No-Calls and Pre-analysis (pre-AE) Errors was 6.2% (101/1642). The final No-Call and Pre-AE rate was 1.2% (20/1642).

Key results:

Staphylococcus aureus (SA):
- Fresh: 99.1% Positive Agreement (322/325), 100% Negative Agreement (926/926)
- Frozen: 100% Positive Agreement (10/10), 100% Negative Agreement (165/165)
- Total: 99.1% Positive Agreement (332/335), 100% Negative Agreement (1091/1091)
- Simulated: 100% Negative Agreement (216/216)
Staphylococcus species:
- Fresh: 97.9% Positive Agreement (895/914), 99.4% Negative Agreement (335/337)
- Frozen: 100% Positive Agreement (30/30), 99.3% Negative Agreement (144/145)
- Total: 98.0% Positive Agreement (925/944), 99.4% Negative Agreement (479/482)
- Simulated: 100% Positive Agreement (25/25), 100% Negative Agreement (191/191)
Staphylococcus epidermidis (SE):
- Fresh: 93.0% Positive Agreement (294/316), 98.7% Negative Agreement (923/935)
- Frozen: 100% Positive Agreement (2/2), 100% Negative Agreement (173/173)
- Total: 93.1% Positive Agreement (296/318), 98.9% Negative Agreement (1096/1108)
- Simulated: 100% Positive Agreement (2/2), 100% Negative Agreement (214/214)
Staphylococcus lugdunensis:
- Fresh: 87.5% Positive Agreement (7/8), 100% Negative Agreement (1243/1243)
- Frozen: 100% Positive Agreement (12/12), 100% Negative Agreement (163/163)
- Total: 95.0% Positive Agreement (19/20), 100% Negative Agreement (1406/1406)
- Simulated: 100% Positive Agreement (20/20), 99.5% Negative Agreement (195/196)
mecA:
- Fresh: 94.1% Positive Agreement (366/389), 97.8% Negative Agreement (843/862)
- Frozen: 100% Positive Agreement (9/9), 100% Negative Agreement (166/166)
- Total: 94.2% Positive Agreement (375/398), 98.2% Negative Agreement (1009/1028)
- Simulated: 100% Negative Agreement (216/216)
Listeria species:
- Fresh: 100% Positive Agreement (3/3), 100% Negative Agreement (1248/1248)
- Frozen: 100% Negative Agreement (175/175)
- Total: 100% Positive Agreement (3/3), 100% Negative Agreement (1423/1423)
- Simulated: 100% Positive Agreement (34/34), 100% Negative Agreement (182/182)
Enterococcus faecalis (EFL):
- Fresh: 96.0% Positive Agreement (72/75), 99.9% Negative Agreement (1175/1176)
- Frozen: 100% Positive Agreement (21/21), 100% Negative Agreement (154/154)
- Total: 96.9% Positive Agreement (93/96), 99.9% Negative Agreement (1329/1330)
- Simulated: 92.3% Positive Agreement (12/13), 100% Negative Agreement (203/203)
vanA:
- Fresh: 91.9% Positive Agreement (34/37), 100% Negative Agreement (1214/1214)
- Frozen: 96.9% Positive Agreement (31/32), 97.9% Negative Agreement (140/143)
- Total: 94.2% Positive Agreement (65/69), 99.8% Negative Agreement (1354/1357)
- Simulated: 100% Positive Agreement (15/15), 100% Negative Agreement (201/201)
Enterococcus faecium (EFC):
- Fresh: 94.4% Positive Agreement (34/36), 100% Negative Agreement (1215/1215)
- Frozen: 100% Positive Agreement (32/32), 100% Negative Agreement (143/143)
- Total: 97.1% Positive Agreement (66/68), 100% Negative Agreement (1358/1358)
- Simulated: 100% Positive Agreement (46/46), 99.4% Negative Agreement (169/170)
vanB:
- Fresh: 100% Negative Agreement (1251/1251)
- Frozen: 100% Positive Agreement (3/3), 100% Negative Agreement (172/172)
- Total: 100% Positive Agreement (3/3), 100% Negative Agreement (1423/1423)
- Simulated: 97.3% Positive Agreement (36/37), 100% Negative Agreement (179/179)
Streptococcus spp.:
- Fresh: 91.7% Positive Agreement (143/156), 99.5% Negative Agreement (1090/1095)
- Frozen: 98.4% Positive Agreement (63/64), 100% Negative Agreement (111/111)
- Total: 93.6% Positive Agreement (206/220), 99.6% Negative Agreement (1201/1206)
- Simulated: 100% Positive Agreement (92/92), 99.2% Negative Agreement (123/124)
S. pneumoniae:
- Fresh: 100% Positive Agreement (25/25), 99.6% Negative Agreement (1221/1226)
- Frozen: 100% Positive Agreement (13/13), 100% Negative Agreement (162/162)
- Total: 100% Positive Agreement (38/38), 99.6% Negative Agreement (1383/1388)
- Simulated: 100% Positive Agreement (8/8), 100% Negative Agreement (208/208)
S. agalactiae:
- Fresh: 97.5% Positive Agreement (39/40), 100% Negative Agreement (1211/1211)
- Frozen: 100% Positive Agreement (31/31), 100% Negative Agreement (144/144)
- Total: 98.6% Positive Agreement (70/71), 100% Negative Agreement (1355/1355)
- Simulated: 100% Positive Agreement (6/6), 100% Negative Agreement (210/210)
S. pyogenes:
- Fresh: 100% Positive Agreement (10/10), 100% Negative Agreement (1241/1241)
- Frozen: 92.9% Positive Agreement (13/14), 100% Negative Agreement (161/161)
- Total: 95.8% Positive Agreement (23/24), 100% Negative Agreement (1402/1402)
- Simulated: 98.2% Positive Agreement (53/54), 100% Negative Agreement (162/162)
S. anginosus group:
- Prospective Fresh: 100% Positive Agreement (9/9), 99.8% Negative Agreement (1239/1242)
- Prospective Frozen: 100% Positive Agreement (3/3), 100% Negative Agreement (172/172)
- Prospective Total: 100% Positive Agreement (12/12), 99.8% Negative Agreement (1411/1414)
- Simulated: 100% Positive Agreement (23/23), 99.5% Negative Agreement (192/193)

Detection of mecA with S. aureus and S. epidermidis, as Determined by Reference Method:

S. aureus (mecA):
- Positive [Cefoxitin (+)]: 97.5% (157/161)
- Negative [Cefoxitin (-) or no cefoxitin testing, as organism not isolated on culture]: 98.8% (172/174)
S. epidermidis (including 12 specimens identified as SE by BC-GP only) (mecA):
- Positive [Cefoxitin (+)]: 92.0% (219/238)
- Negative [Cefoxitin (-) or no cefoxitin testing, as organism not isolated on culture]: 81.5% (75/92)

Detection of vanA/vanB with E. faecalis and E. faecium, as Determined by Reference Method:

E. faecalis (vanA):
- Positive (Bi-directional Sequencing (+)): 85.7% (12/14)
- Negative (Bi-directional Sequencing (-)): 100% (95/95)
E. faecium (vanA):
- Positive (Bi-directional Sequencing (+)): 97.2% (69/71)
- Negative (Bi-directional Sequencing (-)): 93.0% (40/43)
E. faecalis (vanB):
- Positive (Bi-directional Sequencing (+)): 100% (7/7)
- Negative (Bi-directional Sequencing (-)): 100% (102/102)
E. faecium (vanB):
- Positive (Bi-directional Sequencing (+)): 97.0% (32/33)
- Negative (Bi-directional Sequencing (-)): 100% (81/81)

Mixed Culture Results:
There were 98 mixed specimens detected by either BC-GP, reference culture, or both.

BC-GP detected, reference methods did not (false positives): 6 discrepant mixed specimens.
Reference methods detected, BC-GP did not (false negatives): 25 discrepant mixed specimens. The majority (22/25) of these false negatives were organisms that are considered potential blood culture contaminants (Staphylococcus sp. (not SA), S. viridans, and S. salivarius).

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

The clinical study reports percent agreement with reference methods, broken down by positive agreement (sensitivity) and negative agreement (specificity). These are presented in the "Summary of Performance Studies" section.

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

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

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

Not Found

Regulation Number and Section

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

(a)
Identification. A multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures is a qualitative in vitro device intended to simultaneously detect and identify microorganism nucleic acids from blood cultures that test positive by Gram stain or other microbiological stains. The device detects specific nucleic acid sequences for microorganism identification as well as for antimicrobial resistance. This device aids in the diagnosis of bloodstream infections when used in conjunction with other clinical and laboratory findings. However, the device does not replace traditional methods for culture and susceptibility testing.(b)
Classification. Class II (special controls). The special control for this device is FDA's guideline document entitled “Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures.” For availability of the guideline document, see § 866.1(e).

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION (DE NOVO) FOR VERIGENE® GRAM POSITIVE BLOOD CULTURE NUCLEIC ACID TEST (BC-GP)

REGULATORY INFORMATION

FDA identifies this generic type of device as: Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures

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.

NEW REGULATION NUMBER: 21 CFR 866.3365

CLASSIFICATION: II

PRODUCT CODE: PAM

DEVICE NAME: VERIGENE® GRAM POSITIVE BLOOD CULTURE NUCLEIC ACID TEST (BC-GP)

BACKGROUND :

DEVICE NAME: VERIGENE® GRAM POSITIVE BLOOD CULTURE NUCLEIC ACID TEST (BC-GP)

510(K): K113450

DATE OF 510(K) NSE DECISION: JUNE 12, 2012

DATE OF DE NOVO PETITION: JUNE 14, 2012

PETITIONER CONTACT: NANOSPHERE, INC. - MR. MARK DEL VECHIO

PETITIONER'S RECOMMENDED CLASSIFICATION: II

PETITIONER'S RECOMMENDED CONTROLS:

· Class II Special Controls Guidance Document: Multiplex Nucleic Acid Assay for Identification of Microorganism and Resistance Markers from Positive Blood Cultures
General controls

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INDICATIONS FOR USE

The Verigene® Gram Positive Blood Culture Nucleic Acid Test (BC-GP) performed using the sample-to-result Verigene System is a qualitative, multiplexed in vitro diagnostic test for the simultaneous detection and identification of potentially pathogenic gram-positive bacteria which may cause bloodstream infection (BSI). BC-GP is performed directly on positive blood culture using BACTEC™ Plus Aerobic/F and BacT/ALERT FA FAN® Aerobic blood culture bottles, which contain gram positive bacteria. BC-GP is indicated for use in conjunction with other clinical and laboratory findings, such as culture, to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor bloodstream infections.

BC-GP detects and identifies the following bacterial genera and species:

Staphylococcus spp.	Streptococcus spp.	Enterococcus
Staphylococcus aureus	Streptococcus	faecalis
Staphylococcus	pneumoniae	Enterococcus
epidermidis	Streptococcus pyogenes	faecium
Staphylococcus	Streptococcus agalactiae
lugdunensis	Streptococcus anginosus	Listeria spp.
	group

In addition, BC-GP 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, BC-GP does not specifically attribute van-mediated vancomycin resistance to either E. faecium, or mecA-mediated methicillin resistance to either S. aureus or S. epidermidis.

BC-GP is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial bloodstream infections; however, is not to be used to monitor these infections. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing. identification of organisms not detected by BC-GP, 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 BC-GP LABELING FOR A MORE COMPLETE LIST OF WARNINGS, PRECAUTIONS AND CONTRAINDICATIONS.

DEVICE DESCRIPTION

The Verigene® Gram Positive Blood Culture Nucleic Acid Test (BC-GP) is a molecular assav which relies on detection of specific nucleic acid targets in a microarray format. For each of the

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bacterial nucleic acid sequences detected by the BC-GP test, Capture and Mediator oligonucleotides are utilized for gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides bind to a specific portion of the nucleic acid target and are themselves bound onto a substrate in the microarray. The Mediator oligonucleotides bind to a different portion of the same nucleic acid target and allow binding of a gold nanoparticle probe to a portion complementary to a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency.

The BC-GP test is performed on the Verigene® System, a 'sample-to-result', fully automated, bench-top molecular diagnostics workstation. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP. The BC-GP test utilizes single-use disposable test consumables and a self-contained Verigene Test Cartridge for each sample tested. For the BC-GP test, the Verigene System allows automated nucleic acid extraction from Gram-positive bacteria-containing blood culture specimens and target detection of bacteria-specific DNA.

The Reader is the Verigene System's user interface, which serves as the central control unit for all aspects of test processing and results generation. The Reader's graphical user interface guides the user through test processing and test results using a barcode scanner. The user inserts the Test Cartridge into the Verigene Processor SP, which executes the test procedure. automating the steps of (1) Sample Preparation - Cell lysis and magnetic bead-based bacterial DNA isolation from blood culture samples and (2) Verigene Hybridization Test - Detection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology.

After test processing is complete, to obtain the test results the user removes the Test Cartridge from the Processor SP, removes the reagent pack from the substrate holder, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make decisions regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte.

SUMMARY OF NONCLINICAL/BENCH STUDIES

a. Limit of Detection Studies:
The limit of detection (LoD) of the BC-GP assay was assessed and confirmed by using bacterial strains with established titers. By definition, the LoD is the lowest target concentration that can be detected approximately 95% of the time. For each strain, the dilution series began with the sample obtained at 'bottle positivity'. The dilution series were prepared by using a diluent matrix that was comprised of blood culture broth containing charcoal, human blood, and a common commensal skin bacterium at a minimum concentration of ~10 CFU/mL. Each dilution was tested in replicates of four. The putative LoD was the lowest concentration level where all the replicates for the analyte were

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'Detected'. Once the putative LOD was established, 20 replicate samples were tested to confirm the LoD.

Thirteen (13) strains of S. aureus (SA) and S. epidermidis (SE), both methicillin sensitive and resistant, ranged from 1.9 x 10 to 5.7 x 10 CFU/mL for S. aureus and 2.0 x 106 to 7.5 x 10° CFU/mL for S. epidermidis.

Twelve (12) strains of E. faecalis and E. faecium, both vancomycin sensitive and resistant, ranged from 1.1 x 10' to 5.7 x 10' CFU/mL for E. faecalis and 2.4 x 10' to 3.7 x 10' CFU/mL for E. faecium.

Sixteen (16) strains of Streptococcus spp., Staphylococcus spp. (other than S. aureus and S. epidermidis), and Listeria spp. ranged from (CFU/mL):

Streptococcus spp.	1.8 x 106 to 1.2 x 108
S. agalactiae	1.2 x 107 to 2.2 x 107
S. pyogenes	9.5 x 106 to 6.3 x 107
S. anginosus group	1.4 x 107 to 1.2 x 108
S. lugdunensis	3.4 x 106 to 4.0 x 106
S. pneumoniae	1.8 x 106 to 9.9 x 106
Staphylococcus spp.	2.9 x 106 to 4.0 x 106
Listeria spp.	7.5 x 106 to 1.2 x 107

b. Inclusivity Studies:

In addition to the strains tested in the LoD studies, testing was performed on additional strains of each targeted organism/resistance marker. Bottles were spiked with each organism in blood culture bottles containing resins and whole blood. The inoculated blood culture bottles were placed in the automated blood culture system and incubated until positive by audible alarm (bottle ring). Positive specimens were plated to determine purity and quantitation (CFU/ml). Concentrations of tested positive blood culture specimens ranged from ~10° to ~10° CFU/mL at bottle ring. Testing was performed in duplicate with the BC-GP test. The following describes the organisms tested in the inclusivity studies:

SA and SE (both with and without the resistance marker mecA) - 98 methicillin-resistant S. aureus (MRSA) strains (including 65 representative NARSA strains), 18 methicillinsensitive Staphylococcus aureus (SA), eight (8) borderline oxacillin-resistant Staphylococcus aureus (BORSA) strains, six (6) methicillin-resistant Staphylococcus epidermidis (MRSE), and seven (7) methicillin-sensitive Staphylococcus epidermidis (SE).

E. faecium and E. faecalis (both with and without resistance markers vanA and vanB) - 79 strains including 14 vancomycin-resistant Enterococcus faecalis (11 of which were classified as E. faecalis, vanA and 3 were classified as vanB). 17 vancomvcin-sensitive Enterococcus faecalis, 33 vancomycin-resistant Enterococcus faecium (26 of which were

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classified as vanA and 7 were classified as vanB), and 15 vancomycin-sensitive Enterococcus faecium.

Streptococcus spp., Staphylococcus spp. and Listeria spp. - 233 bacterial strains, 184 of which belonged to the Streptococcus genus, including 34 Streptococcus agalactiae, 10 Streptococcus anginosus, 60 Streptococcus pneumoniae, and 16 Streptococcus pyogenes, 12 Listeria sp., and 45 Staphylococcus strains (of which four were Staphylococcus lugdunensis). One or more strains each of 13 different Staphylococcus species (other than SA, SE, and S. lugdenensis), 18 different Streptococcus species (other than S. pneumoniae, S. agalactiae, and S. pyogenes, and 6 different Listeria species were tested in the studies. For the Streptococcus anginosus group, 10 total strains covering the three species S. anginosus, S. intermedius, and S. constellatus were tested. All strains tested gave positive results with the BC-GP assay, with the exception of Listeria grayi and BORSA strains. Two strains of L. grayi were tested and both were not detected. Subsequent in silico sequence analysis verified that this species will likely not be detected by the BC-GP assay. Therefore, the following limitation was placed in the package insert: "For Listeria species detection, L. grayi should not be detected by the test, as suggested by in silico analysis and confirmed with analytical testing." Tested BORSA strains were characterized as mecA negative and expectantly identified by the BC-GP test as mecA negative.

In order to support the genus level claims, a separate in silico analysis was performed by comparing the homology of the BC-GP assay probes against available GenBank entries for Streptococcus spp. Staphylococcus sp. and Listeria sp. The analysis, combined with wettesting results, suggested that the BC-GP assay should detect 41 different species of Staphylococcus, 54 species of Streptococcus, and six species of Listeria.

c. Analytical Specificity (Exclusivity)

Analytical specificity was assessed using organisms phylogenetically related to panel organisms detected by the BC-GP test as well as those commonly present as contaminants in blood culture specimens. The exclusivity samples were divided into two distinct panels of organisms. The first panel consisted of 127 "non-BC-GP panel" organisms which would not have been expected to be detected by the BC-GP test.

. Fifty-two (79) gram-positive bacteria. including 15 Micrococcus strains, two each of seven Enterococcus strains and one of an eighth, a Kytococcus, a Kocuria, a Peptostreptococcus, two each of Planococcus, Rothia, Leuconostoc, Granulicatella, Lactococcus, and Pediococcus strains
Forty (40) gram-negative bacteria ●
One (1) acid-fast bacillus (attenuated Mycobacterium tuberculosis) ●
One (1) mollicute (Mycoplasma pneumonia) ●
Six (6) yeast strains

The second panel consisted of over 450 "BC-GP panel" organisms, which in total comprised the analytical inclusivity study samples. The size and characteristics of the second exclusivity sample set is as follows:

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. Staphylococcus strains (178), including MRSA (98), MSSA (18), BORSA (8), MRSE (6), MSSE (7), and 45 "non-SA/SE" Staphylococcus spp
Enterococcus strains (79). including E. faecium VS (15). E. faecium VRE (33). E. . faecalis VS (17), and E. faecalis VRE (14)
Streptococcus strains (184), including S. agalactiae (34), S. pneumoniae (60), S. ● anginosus (10), and S. pyogenes (16)
. Listeria strains (12)

All testing was performed in BACTEC plus bottles. Positive blood cultures were checked for purity and quantitated with final concentrations determined to be between 10° to 10° CFU/ml. Testing was performed in duplicate for each strain and depending on the organism, bottle preparation was performed in one of the following ways:

Organisms were spiked into blood culture bottles containing resins and whole blood, ● and then incubated on the blood culture system until bottle ring, followed by testing with the BC-GP assay.
. High concentrations of organisms were spiked into blood culture bottles and immediately tested with the BC-GP assay.
. Previously-isolated genomic DNA was spiked into blood culture bottles for both Yersinia enterocolitica and Cryptococcus neoformans due to the risks associated with handling live cultures of these organisms.

The BC-GP test demonstrated acceptable specificity when challenged with these two panels, with no cross-reactivity observed for any of the organisms and/or strains tested with the exception of two strains of Enterococcus avium, which were identified as E. faecium, and Lactococcus lactis, which was identified as Streptococcus species. This information was presented in the package insert.

d. Interference

The potential inhibitory effects of substances that may be encountered in blood and associated with the blood culturing process were tested with the BC-GP test at the concentration levels listed in the table below.

Testing was performed using positive blood cultures containing representative strains of SA and SE (both with and without the resistance marker mecA), E. faecium (with and without vanA and vanB), Streptococcus spp., S. agalactiae, S. anginosus group, S. pneumoniae, S. pyogenes, Staphylococcus spp., S. lugdunensis and Listeria spp. A negative blood culture sample was also tested with each interferent to assess the impact of the interferent on the processing of the BC-GP. For each organism and for the negative samples, the potential interferents (in table below) were added directly into individual positive blood cultures (or negative) and tested in triplicate. All the replicates for all the strains tested (and

6

the negative sample controls) gave the expected results indicating that these potential interferents do not interfere with the BC-GP test.

| Interferent | Reference
Level | Concentrat
ion Tested |
|-------------------------------------------|----------------------------------------------------------------------|--------------------------|
| Hemoglobin | 1-2 g/L | 14 g/L |
| Triglyceride
(Intralipid) | Normal Staphylococcus aureus (SA) | | Positive | Negative | Culture and Conventional Biochemical and Vitek2 | Staphylococcus species | | Positive | Negative | Culture and Conventional Biochemical and Vitek2 | |
| | Fresh 1251 | 99.1%
322/325
(97.3-99.8) | 100%
926/926
(99.6-100) | | | Fresh 1251 | 97.9%
895/914
(96.8-98.7) | 99.4%
335/337
(97.9-99.9) | | |
| | Frozen 175 | 100%
10/10
(69.2-100) | 100%
165/165
(97.8-100) | | | Frozen 175 | 100%
30/30
(88.4-100) | 99.3%
144/145
(96.2-99.9) | | |
| | Total 1426 | 99.1%
332/335
(97.4-99.8) | 100%
1091/1091
(99.7-100) | | | Total 1426 | 98.0%
925/944
(96.9-98.8) | 99.4%
479/482
(98.2-99.9) | | |
| | Simulated 216 | | 100%
216/216
(98.3-100) | | Simulated 216 | | 100%
25/25
(86.3-100) | 100%
191/191
(98.1-100) | | |
| Staphylococcus epidermidis (SE) | | | | Culture and Conventional Biochemical and Vitek2 | Staphylococcus lugdunensis | | | | Culture and Conventional Biochemical and Vitek2 | |
| | Fresh 1251 | 93.0%
294/316
(89.6-95.6) | 98.7%
923/935
(97.8-99.3) | | | Fresh 1251 | 87.5%
7/8
(47.4-99.7) | 100%
1243/1243
(99.7-100) | | |
| | Frozen 175 | 100%
2/2
(15.8-100) | 100%
173/173
(97.9-100) | | | Frozen 175 | 100%
12/12
(73.5-100) | 100%
163/163
(97.8-100) | | |
| | Total 1426 | 93.1%
296/318
(89.7-95.6) | 98.9%
1096/1108
(98.1-99.4) | | | Total 1426 | 95.0%
19/20
(75.1-99.9) | 100%
1406/1406
(99.7-99.9) | | |
| | Simulated 216 | | 100%
2/2
(15.8-100) | 100%
214/214
(98.3-100) | | Simulated 216 | | 100%
20/20
(83.2-100) | 99.5%
195/196
(97.2-99.9) | |
| mecA | | | | Culture and Cefoxitin Disk Diffusion | Listeria species | | | | Culture and Cefoxitin Disk Diffusion | |
| Fresh 1251 | 94.1%
366/389
(91.2-96.2) | 97.8%
843/862
(96.6-98.7) | Fresh 1251 | | | 100%
3/3
(29.2-100) | 100%
1248/1248
(99.7-100) | | | |
| Frozen 175 | 100%
9/9
(66.4-100) | 100%
166/166
(97.8-100) | Frozen 175 | | | | 100%
175/175
(97.9-100) | | | |
| Total 1426 | 94.2%
375/398
(91.5-96.3) | 98.2%
1009/1028
(97.1-98.9) | Total 1426 | | | 100%
3/3
(29.2-100) | 100%
1423/1423
(99.7-100) | | | |
| | Simulated 216 | | 100%
216/216
(98.3-100) | | Simulated 216 | | 100%
34/34
(89.7-100) | 100%
182/182
(98.0-100) | | |

Summary of Clinical Test Performance versus Reference Method(s) – Staphylococcus and Listeria

14

| Specimen
Type | n= | % Agreement (95% CI) | | Reference
Method | Specimen
Type | n= | % Agreement (95% CI) | | Reference
Method |
|-----------------------------|------|-------------------------------|-----------------------------------|-------------------------------------------------|------------------|------|-------------------------------|-----------------------------------|--------------------------------------|
| | | Positive | Negative | | | | Positive | Negative | |
| Enterococcus faecalis (EFL) | | | | Culture and Conventional Biochemical and Vitek2 | vanA | | | | Culture and Bidirectional Sequencing |
| Fresh | 1251 | 96.0%
72/75
(88.8-99.2) | 99.9%
1175/1176
(99.5-99.9) | | Fresh | 1251 | 91.9%
34/37
(78.1-98.3) | 100%
1214/1214
(99.7-100) | |
| Frozen | 175 | 100%
21/21
(83.9-100) | 100%
154/154
(97.6-100) | | Frozen | 175 | 96.9%
31/32
(83.8-99.9) | 97.9%
140/143
(94.0-99.6) | |
| Total | 1426 | 96.9%
93/96
(91.1-99.4) | 99.9%
1329/1330
(99.6-99.9) | | Total | 1426 | 94.2%
65/69
(85.8-98.4) | 99.8%
1354/1357
(99.4-99.9) | |
| Simulated | 216 | 92.3%
12/13
(64.0-99.8) | 100%
203/203
(98.2-100) | | Simulated | 216 | 100%
15/15
(78.2-100) | 100%
201/201
(98.2-100) | |
| Enterococcus faecium (EFC) | | | | Culture and Conventional Biochemical and Vitek2 | vanB | | | | Culture and Bidirectional Sequencing |
| Fresh | 1251 | 94.4%
34/36
(81.3-99.3) | 100%
1215/1215
(99.7-100) | | Fresh | 1251 | 0/0 | 100%
1251/1251
(99.7-100) | |
| Frozen | 175 | 100%
32/32
(89.1-100) | 100%
143/143
(97.5-100) | | Frozen | 175 | 100%
3/3
(29.2-100) | 100%
172/172
(97.9-100) | |
| Total | 1426 | 97.1%
66/68
(89.8-99.6) | 100%
1358/1358
(99.7-100) | | Total | 1426 | 100%
3/3
(29.2-100) | 100%
1423/1423
(99.7-100) | |
| Simulated | 216 | 100%
46/46
(92.3-100) | 99.4%
169/170
(96.8-99.9) | | Simulated | 216 | 97.3%
36/37
(85.8-99.9) | 100%
179/179
(98.0-100) | |

Summary of Clinical Test Performance versus Reference Method(s) – Enterococcus

Summary of Clinical Test Performance versus Reference Method(s) – Streptococcus

Specimen Type	n=	% Agreement (95% CI)		Reference Method	Specimen Type	n=	% Agreement (95% CI)		Reference Method
Streptococcus spp.	1251	Positive	Negative	Culture and Conventional Biochemical and Vitek2	S. pneumoniae	1251	Positive	Negative	Culture and Conventional Biochemical and Vitek2
Fresh	1251	91.7%
143/156
(86.2-95.5)	99.5%
1090/1095
(98.9-99.9)		Fresh	1251	100%
25/25
(86.3-100)	99.6%
1221/1226
(99.1-99.9)
Frozen	175	98.4%
63/64
(91.6-99.9)	100%
111/111
(96.7-100)		Frozen	175	100%
13/13
(75.3-100)	100%
162/162
(97.8-100)
Total	1426	93.6%
206/220
(89.6-96.5)	99.6%
1201/1206
(99.0-99.9)		Total	1426	100%
38/38
(90.8-100)	99.6%
1383/1388
(99.2-99.9)
Simulated	216	100%
92/92
(96.1-100)	99.2%
123/124
(95.6-99.9)	Culture and Conventional Biochemical and Vitek2	Simulated	216	100%
8/8
(63.1-100)	100%
208/208
(98.2-100)	Culture and Conventional Biochemical and Vitek2
S. agalactiae	1251	Positive	Negative		S. pyogenes	1251	Positive	Negative
Fresh	1251	97.5%
39/40
(86.8-99.9)	100%
1211/1211
(99.7-100)		Fresh	1251	100%
10/10
(69.2-100)	100%
1241/1241
(99.7-100)
Frozen	175	100%
31/31
(88.8-100)	100%
144/144
(97.5-100)		Frozen	175	92.9%
13/14
(66.1-99.8)	100%
161/161
(97.7-100)
Total	1426	98.6%
70/71
(92.4-99.9)	100%
1355/1355
(99.7-100)	Culture and Conventional Biochemical and Vitek2	Total	1426	95.8%
23/24
(78.9-99.9)	100%
1402/1402
(99.7-100)	Culture and Conventional Biochemical and Vitek2
Simulated	216	100%
6/6
(54.1-100)	100%
210/210
(98.3-100)		Simulated	216	98.2%
53/54
(90.1-99.9)	100%
162/162
(97.8-100)

15

S. anginosus group
Prospective	Fresh	1251	100%
9/9
(66.4-100)	99.8%
1239/1242
(99.3-99.9)
	Frozen	175	100%
3/3
(29.2-100)	100%
172/172
(97.9-100)
	Total	1426	100%
12/12
(73.5-100)	99.8%
1411/1414
(99.4-99.9)
Simulated	216	100%
23/23
(85.2-100)	99.5%
192/193
(97.2-99.9)

The following tables provide the clinical performance of BC-GP for mecA detection with S. aureus and S. epidermidis (as determined by the reference method) and vanA/vanB detection with E. faecalis and E. faecium (as determined by the reference method). In the study protocol, Staphylococcus species (other than S. aureus and S. epidermidis) were not tested for methicillin resistance by cefoxitin disk analysis. However, 12 Staphylococcus species (as determined by the reference method) were positive for S. epidermidis and mecA by the BC-GP test and have been counted as false positives for mecA. These 12 specimens are accounted for in the total negative agreement calculation for the mecA target.

| Organism | Resistance
Marker | n= | % Agreement (95% CI) with Reference Method | |
|---------------------------------------------------------------------------------|---------------------------------|-----|--------------------------------------------|------------------------------------------------------------------------------------------------|
| | | | Positive
[Cefoxitin (+)] | Negative
[Cefoxitin (-) or no cefoxitin
testing, as organism not isolated
on culture] |
| S. aureus | mecA | 335 | 97.5%
157/161
(93.8-99.3) | 98.8%
172/174
(95.9-99.9) |
| S. epidermidis
(including 12 specimens
identified as SE by BC-GP
only) | 94.2%
375/398
(91.5-96.3) | 330 | 92.0%
219/238
(87.8-95.1) | 81.5%
75/92
(72.1-88.9) |

Detection of mecA with S. aureus and S. evidermidis, as Determined by Reference Method

Detection of vanA/vanB with E. faecalis and E. faecium, as Determined by Reference Method

| Organism | Resistance
Marker | n= | % Agreement (95% CI) with Reference Method | |
|-------------|-------------------------------|-----|------------------------------------------------|------------------------------------------------|
| | | | Positive
(Bi-directional Sequencing
(+)) | Negative
(Bi-directional Sequencing
(-)) |
| E. faecalis | vanA | 109 | 85.7%
12/14
(57.2-98.2) | 100%
95/95
(96.2-100) |
| E. faecium | 95.2%
80/84
(88.3-98.7) | 114 | 97.2%
69/71
(90.2-99.7) | 93.0%
40/43
(80.9-98.5) |
| E. faecalis | vanB | 109 | 100%
7/7
(59.0-100) | 100%
102/102
(96.5-100) |
| E. faecium | 97.5%
39/40
(86.8-99.9) | 114 | 97.0%
32/33
(84.2-99.9) | 100%
81/81
(95.6-100) |

16

The following table contains additional genus/group-level specific BC-GP clinical and analytical performance data stratified by individual species within each genus (i.e.; Staphylococcus sp. (other than SA, SE, S. lugdunensis), Streptococcus sp. (other than S. agalactiae, S. pyogenes, S. pneumoniae) Listeria sp. and Streptococcus anginosus group.

Staphylococcus Genus			Streptococcus Genus			Streptococcus anginosus Group
Organism	% (+) Agreement (95% CI)	Analytical	Organism	% (+) Agreement (95% CI)	Analytical	Organism	% (+) Agreement (95% CI)
	Clinical			Clinical			Clinical	Analytical
Combined
Staphylococcus spp.	98.0%
950/969
(97.0-98.8)	100%
37/37
(90.5-100)	Combined
Streptococcus spp.	95.5%
298/312
(92.6-97.5)	100%
183/183
(98.0-100)	Streptococcus.
anginosus group	100%
35/35
(90.0-100)	100%
10/10
(69.2-100)
Staphylococcus
arlettae	100%
1/1
(2.5-100)	NT*	Streptococcus
bovis	100%
1/1
(2.5-100)	100%
4/4
(39.8-100)	Streptococcus
anginosus	100%
7/7
(59.0-100)	100%
5/5
(47.8-100)
Staphylococcus
auricularis	100%
16/16
(79.4-100)	100%
2/2
(15.8-100)	Streptococcus
dysgalactiae	100%
5/5
(47.8-100)	100%
2/2
(15.8-100)	Streptococcus
constellatus	100%
9/9
(66.4-100)	100%
3/3
(29.2-100)
Staphylococcus
capitis	96.4%
54/56
(87.7-99.6)	100%
2/2
(15.8-100)	Streptococcus
dysgalactiae
equisimilis	100%
2/2
(15.8-100)	100%
3/3
(29.2-100)	Streptococcus
intermedius	100%
8/8
(63.1-100)	100%
2/2
(15.8-100)
Staphylococcus
caprae	100%
5/5
(47.8-100)	100%
2/2
(15.8-100)	Streptococcus
equi	NT	100%
2/2
(15.8-100)	Streptococcus.
anginosus group
(not further Identified)	100%
11/11
(71.5-100)	NT
Staphylococcus cohnii	NT	100%
2/2
(15.8-100)	Streptococcus
equinus	NT	100%
2/2
(15.8-100)
Staphylococcus
chromogenes	100%
2/2
(15.8-100)	NT	Streptococcus
gallolyricus	NT	100%
3/3
(29.2-100)	Listeria Genus
Staphylococcus
haemolyticus	91.4%
32/35
(76.9-98.2)	100%
2/2
(15.8-100)	Streptococcus
gallolyticus
pasteurianus	NT	100%
1/1
(2.5-100)	Combined
Listeria spp.	100%
37/37
(90.5-100)	100%
12/12
(73.5-100)
Staphylococcus
hominis	98.5%
133/135
(94.8-99.8)	100%
3/3
(29.2-100)	Streptococcus
gordonii	100%
1/1
(2.5-100)	100%
2/2
(15.8-100)	Listeria grayi	NT	0%
0/2
(15.8-100)
Staphylococcus
intermedius	100%
3/3
(29.2-100)	100%
1/1
(2.5-100)	Streptococcus
infantarius
infantarius	NT	100%
1/1
(2.5-100)	Listeria innocua	100%
8/8
(63.1-100)	100%
1/1
(2.5-100)
Staphylococcus
muscae	NT	100%
1/1
(2.5-100)	Streptococcus
infantarius subsp.
coli	NT	100%
1/1
(2.5-100)	Listeria ivanovii	100%
2/2
(15.8-100)	100%
1/1
(2.5-100)
Staphylococcus
pasteuri	NT	100%
2/2
(15.8-100)	Streptococcus
infantis	NT	100%
2/2
(15.8-100)	Listeria
monocytogenes	100%
24/24
(85.8-100)	100%
6/6
(54.1-100)
Staphylococcus
saccharolyticus	NT	100%
4/4
(39.8-100)	S. mitis, S. oralis,
or S. mitis/oralis	95.2%
20/22
(70.8-98.9)	100%
19/19
(82.4-100)	Listeria seeligeri	100%
1/1
(2.5-100)	100%
1/1
(2.5-100)
Staphylococcus
saprophyticus	100%
4/4
(39.8-100)	100%
2/2
(15.8-100)	Streptococcus
mutans	50%
1/2
(1.3-98.7)	100%
2/2
(15.8-100)	Listeria welshimeri	100%
1/1
(2.5-100)	100%
1/1
(2.5-100)
Staphylococcus
schleiferi	100%
1/1
(2.5-100)	100%
2/2
(15.8-100)	Streptococcus
parasanguinis	100%
4/4
(39.8-100)	100%
4/4
(39.8-100)	Listeria spp.
unidentified	100%
1/1
(2.5-100)	NT
Staphylococcus sciuri	NT	100%
2/2
(15.8-100)	Streptococcus
peroris	NT	100%
1/1
(2.5-100)
Staphylococcus
simulans	100%
5/5
(47.8-100)	100%
2/2
(15.8-100)	Streptococcus
salivarius	50%
4/8
(15.7-84.3)	100%
5/5
(47.8-100)

Summary of Genus/Group-level Test Performance versus Reference Method(s) – Stratified by Species

17

| Staphylococcus
warneri | 90.0%
18/20
(68.3-98.8) | 100%
2/2
(15.8-100) | Streptococcus
sanguinis | 75%
3/4
(19.4-99.4) | 100%
3/3
(29.2-100) |
|----------------------------------|-------------------------------|---------------------------|-------------------------------|-------------------------------|---------------------------|
| Staphylococcus
xylosus | 100%
2/2
(15.8-100) | 100%
2/2
(15.8-100) | Streptococcus
thoraltensis | NT | 100%
1/1
(2.5-100) |
| Staphylococcus
species (CONS) | 100%
29/29
(88.1-100) | NT | Streptococcus
viridans | 83.3%
20/24
(62.6-95.3) | NT |
| | | | Streptococcus
uberis | 100%
1/1
(2.5-100) | NT |

*NT = "Not Tested

Mixed Culture Results: In total, there were 98 mixed specimens that were detected either by BC-GP, the reference culture methods or both. The two tables below list the distinct mixed specimen combinations detected by BC-GP in the clinical study and the additional distinct mixed specimen combinations detected by the reference/comparator methods, but not detected by BC-GP. In the clinical study, there were a total of six discrepant mixed specimens for which the BC-GP test detected organisms that were not detected by the reference culture (false positives). Additionally, there were 25 discrepant mixed specimens for which the reference culture detected organisms that were not detected by the BC-GP Test (false negatives). The majority (22/25) of the organisms not detected by the BC-GP Test in the false negative mixed specimens were organisms that can sometimes be considered as potential blood culture contaminants (Staphylococcus sp. (not SA), S. viridans, and S. salivarius.) For any mixed blood culture, there may be different rates of growth for each organism present, and therefore the organism present in lower concentrations (below "bottle ring") may not be detected. The following limitation was placed in the package insert regarding detection of organism by the BC-GP Test in mixed blood cultures:

. In mixed cultures containing Gram positive bacteria and other organisms, BC-GP may not identify all the detectable organisms in the specimen, depending upon the concentration of each target present.

18

| Analyte 1 | Analyte 2 | Ob
ser
ved
Fre
que
ncy | No. of
Specime
ns with
Discrepa
nt Co-
analytes | Discrepant Analyte(s)1 |
|--------------------------------------------------------|--------------------------------------------------------|---------------------------------------|----------------------------------------------------------------|------------------------|
| E. faecalis and vanA | E. faecium | 1 | 0 | |
| E. faecalis | Staphylococcus species | 3 | 0 | |
| E. faecalis | Streptococcus species | 1 | 1 | Streptococcus species |
| E. faecium and vanA | Staphylococcus species | 1 | 0 | |
| Listeria | S. anginosus group | 1 | 1 | S. anginosus group |
| S. lugdunensis | Listeria species | 1 | 0 | |
| Staphylococcus species | E. faecalis | 2 | 1 | Staphylococcus species |
| Staphylococcus species and S.
epidermidis and mecA+ | E. faecalis | 1 | 0 | |
| Staphylococcus species and S.
aureus | Staphylococcus species and S. epidermidis and
mecA+ | 1 | 0 | |
| Streptococcus species and
S. agalactiae | Staphylococcus species and
S. aureus | 1 | 0 | |
| Streptococcus species and
S. pneumoniae | Streptococcus species and
S. anginosus group | 1 | 1 | S. anginosus group |
| Streptococcus species and S.
pyogenes | E. faecalis | 1 | 1 | E. faecalis |
| Streptococcus species | Staphylococcus species | 2 | 1 | Staphylococcus species |
| | TOTAL | 17 | 6 | |

Clinical Mixed Specimen Combinations Detected by BC-GP, but not by Reference Methods

1 Defined as an analyte that was detected by BC-GP, but not detected by the reference methods.

Additional Clinical Mixed Specimens Detected by Reference Methods, but not Detected by BC-GP

| Analyte 1 | Analyte 2 | Analyt
e 3 | Observed
Frequency | No. Specimens
w/ Discrepant
Co-analytes | Discrepant
Analyte(s)¹ |
|----------------------------------|-------------------------------------|-------------------|-----------------------|-----------------------------------------------|---------------------------|
| E. faecalis (vanS) | S. haemolyticus | | 2 | 2 | S. haemolyticus |
| E. faecalis (vanS) | S. aureus (methR) | | 1 | 1 | S. aureus
(methR) |
| E. faecalis (vanS) | GNR | | 2 | 0 | - |
| E. faecalis (vanS) | S. epidermidis (methR) | | 1 | 1 | S. epidermidis
(methR) |
| E. faecalis (vanS) | yeast | S.
warneri | 1 | 1 | S. warneri |
| E. faecalis (vanS) | Bacillus species (not
anthracis) | | 1 | 0 | - |
| E. faecalis (vanR) | S. epidermidis (methR) | | 2 | 2 | S. epidermidis
(methR) |
| E. faecalis (vanR) | S. agalactiae | | 1 | 1 | (vanR) |
| E. faecium (vanS) | S. aureus (methR) | | 1 | 1 | S. aureus
(methR) |
| E. faecium | GNR | | 1 | 0 | - |
| E. faecium (vanR) | S. warneri | | 1 | 1 | S. warneri |
| E. faecium (vanR) | S. capitis | | 1 | 1 | S. capitis |
| E. faecium (vanR) | S. epidermidis (methR) | | 1 | 1 | S. epidermidis
(methR) |
| E. faecium (vanR) | GNR | | 1 | 0 | - |
| E. faecium (vanR) | Corynebacterium | | 1 | 0 | - |
| M. lylae | M. luteus | | 1 | 0 | - |
| Streptococcus viridans
group | GNR | | 1 | 1 | Strep viridans
group |
| K. rosea | K. kristinae | | 1 | 0 | - |
| E. casseliflavus | GNR | | 1 | 0 | - |
| Leuconostoc
mesenteroides | S. salivarius | | 1 | 1 | S. salivarius |
| S. hominis | S. warneri | | 1 | 0 | - |
| S. capitis | S. epidermidis (methS) | | 2 | 1 | S. epidermidis
(methS) |
| Analyte 1 | Analyte 2 | Analyt e 3 | Observed
Frequency | No. Specimens
w/ Discrepant
Co-analytes | Discrepant
Analyte(s)¹ |
| S. haemolyticus | K. kristinae | | 1 | 0 | - |
| S. hominis | S. salivarius | | 1 | 1 | Streptococcus
spp. |
| S. simulans | S. hominis | | 1 | 0 | - |
| S. epidermidis (methR) | S. hominis | S.
auricularis | 1 | 1 | S. epidermidis
(methR) |
| S. simulans | S. warneri | | 1 | 0 | - |
| S. epidermidis (methS) | S. hominis | | 3 | 2 | S. epidermidis
(methS) |
| S. hominis | Staphylococcus species
(CoNS) | | 2 | 0 | - |
| S. hominis | S. schleiferi | | 1 | 0 | - |
| S. haemolyticus | S. capitis | | 1 | 0 | - |
| S. haemolyticus | S. hominis | | 1 | 0 | - |
| S. auricularis | GNR | | 1 | 0 | - |
| S. hominis | S. epidermidis (methR) | | 6 | 2 | S. epidermidis
(methR) |
| S. hominis | S. capitis | | 2 | 0 | - |
| S. viridans group | S. hominis | | 1 | 1 | Streptococcus
species |
| S. epidermidis (methS) | S. warneri | | 1 | 0 | - |
| S. epidermidis | Staphylococcus species
(CoNS) | | 1 | 0 | - |
| Corynebacterium | S. epidermidis (methR) | | 1 | 0 | - |
| S. capitis | S. epidermidis (methR) | | 4 | 0 | - |
| S. epidermidis (methR) | S. hominis | | 5 | 0 | - |
| S. hominis | S. capitis | | 1 | 0 | - |
| S. lugdunensis | S. warneri | | 1 | 1 | S. lugdunensis |
| Staphylococcus species
(CoNS) | S. epidermidis (methS) | | 1 | 0 | - |
| S. aureus (methR) | S. hominis | | 2 | 0 | - |
| S. hominis | S. aureus (methS) | | 1 | 0 | - |
| Staphylococcus spp
(CoNS) | S.epidermidis (methR) | | 1 | 0 | - |
| S. capitis | Globicatella sp. | | 1 | 0 | - |
| S. salivarius | R. mucilaginosa | | 1 | 0 | - |
| Viridans Streptococcus | S. mitis | | 1 | 0 | - |
| S. agalactiae | Proteus species | | 1 | 0 | - |
| S. epidermidis (methR) | S. agalactiae | | 1 | 1 | S. epidermidis
(methR) |
| S. constellatus | GNR | | 1 | 0 | - |
| S. epidermidis (methR) | S. pyogenes | | 1 | 1 | S. epidermidis
(methR) |
| S. salivarius | Viridans strep | Neisseria spp. | 1 | 0 | - |
| S. salivarius | K. kristinae | | 1 | 0 | - |
| Neisseria spp. | S. mitis | S.
sanguinis | 1 | 0 | - |
| S. mitis | Granulicatella | | 1 | 0 | - |
| L. pseudomesenteroides | S. mitis | | 1 | 0 | - |
| S. parasanguinis | R. muciliginosa | | 1 | 0 | - |
| | | TOTAL | 81 | 25 | |

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1 Defined as an analyte that was detected by the reference methods, but not detected by BC-GP.

20

LABELING

Labeling has been provided which is adequate and satisfies 21 CFR Parts 801 and 809 as well as the special control requirements.

RISKS TO HEALTH

FDA has identified the following risks to health associated with use of the Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures: false positive results, false negative results, and errors in interpretation. Failure of the device to detect and identify a targeted microorganism when such microorganism is present in the specimen (false negative result) may lead to a delay in finding the true cause of the bloodstream infection/bacteremia and to inappropriate antibiotic use. An incorrect positive test result (false positive result) also may lead to unnecessary or ineffective antibiotic therapy and delay in determining the true cause of the patient's illness. Failure of the device to detect a targeted gene associated with resistance when such gene is present in the detected microorganism (e.g., false negative results for mecA, vanA, vanB) may lead to treatment with ineffective antibiotics and lapses in infection control measures. An incorrect positive result for the presence of a targeted gene associated with resistance when such gene is present in the detected microorganism (false positive result) may also lead to inappropriate antibiotic therapy (frequently overly broad) to cover resistant microorganisms that are not present. The more potent antibiotics may have more side effects (e.g., renal toxicity, etc), and may lead to unnecessary and often costly implementation of infection control measures. Failure to correctly interpret test results in the context of other clinical and laboratory findings may lead to inappropriate or delayed treatment. For example, positive assay results do not rule out viral or other bacterial co-infections.

All risks to health have been identified for the subject device.

Risks to health have been addressed for this device in the special controls guideline document entitled "Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures."

Identified Risks	Required Mitigations
False negative result	The FDA document entitled "Class II Special Controls
Guideline: Multiplex Nucleic Acid Assay for Identification
of Microorganisms and Resistance Markers from Positive
Blood Cultures," which addresses this risk through: Device
Description Containing the Information Specified in the
Special Control Guideline, Performance Characteristics, and
Labeling
False positive result	The FDA document entitled "Class II Special Controls
Guideline: Multiplex Nucleic Acid Assay for Identification
of Microorganisms and Resistance Markers from Positive
Blood Cultures," which addresses this risk through: Device
Description Containing the Information Specified in the
Special Control Guideline, Performance Characteristics, and

Identified Risks and Required Mitigations

21

Identified Risks	Required Mitigations
Errors in Interpretation	Labeling
The FDA document entitled "Class II Special Controls
Guideline: Multiplex Nucleic Acid Assay for Identification
of Microorganisms and Resistance Markers from Positive
Blood Cultures," which addresses this risk through: Device
Description Containing the Information Specified in the
Special Control Guideline, Performance Characteristics, and
Labeling

BENEFITS RISKS ANALYSIS

We considered the following factors in our analysis of benefit: the accuracy of the device, availability of alternative treatments or diagnostics, the magnitude of the benefits, and the probability that patients would experience more than one benefit.

We considered the following factors in our analysis of risk; the probability and the consequences of false-positive or false-negative test results; the quality of the study design and robustness of the data; the risk mitigation by use of product labeling; and an analysis of available postmarket data for other similar multiplex assays.

The direct benefit of the device, established through pre-clinical studies, rests on accurate detection of gram-positive bacteria, including the test's specified drug resistance markers, in blood cultures. Analytical performance characteristics (limit of detection, inclusivity, interference, competitive inhibition, carry-over, precision/reproducibility and fresh/frozen matrix studies) were adequately validated for each of the BC-GP assay's analytes. The clinical study with over 1600 evaluable samples showed high concordance between results from the BC-GP assay and results from conventional methods (culture and identification) for the analytes.

Rapid availability of results from the BC-GP assay, compared to conventional methods for the BC-GP assay's analytes, presents a plausible additional benefit arising from earlier treatment prompted by the detection analytes. Ordinarily, the most readily available information to the physician (within ~12-24 hours) is the Gram stain performed after blood culture bottle positivity. The antibiotic may be changed based on the Gram stain results (to cover for either Gram-positive or Gram-negative organisms or both). Definitive organism targeted therapy is delayed an additional 1-2 days until the process for final identification of the organism and antimicrobial susceptibility is completed by traditional culture methods. The BC-GP test allows for simultaneous identification of these twelve types of bacteria and their resistance genes within about 2 1/2 hours after bacterial growth is first detected in the sample and after a Gram stain is performed and shows Gram-positive organisms.

The risks for this device are associated with inaccurate test results, which may lead to error in diagnosis and treatment. The probability of such events is small (from the analytical and clinical data presented), as has been observed for already similar marketed devices. The small risk for the BC-GP assay is mitigated through labeling specifying that the intended use of the BC-GP assay is "for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial bloodstream infections; Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility

22

testing, identification of organisms not detected by BC-GP, differentiation of mixed growth, association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing." Additionally, a limitation has been provided in the product labeling stating that a trained health care professional should interpret assay results together with the patient's medical history, clinical signs and symptoms, and the results of other diagnostic tests.

The benefits of the BC-GP assay outweigh the risks. Analytical and clinical performance characteristics are comparable to those from already marketed devices. Use of this device would allow faster identification of causative pathogens and their resistance markers in cases of bacteremia, which may lead to earlier and more definitive targeted therapy for patients with bloodstream infections/bacteremia likely resulting in improved clinical outcomes. Studies have shown that delayed administration of antibiotics is associated with a 7.6% decrease in survival rate for each hour that therapy is delayed. I The identified risks posed by the device have a low probability and are adequately mitigated.

Kumar A., Roberts D., Wood KE. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006;34(6): 1589-1596.

SPECIAL CONTROLS:

In combination with the general controls of the FD&C Act, the Verigene® Gram Positive Blood Culture Nucleic Acid Test is subject to the following special controls:

The special controls for the BC-GP Assay are contained in the guideline document entitled "Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures."

CONCLUSION

The De Novo petition for the Verigene® Gram Positive Blood Culture BC-GP Nucleic Acid Test is granted and the device is classified under the following:

Product Code: PAM, Gram-positive bacteria and their resistance markers Device Type: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures Class: II Regulation: 21 CFR 866.3365