AVANDX E. FAECALIS/OE PNA FISH CULTURE IDENTIFICATION KIT

K083074 · Advandx, Inc. · OAH · Jul 24, 2009 · Microbiology

Device Facts

• Record ID: K083074
• Device Name: AVANDX E. FAECALIS/OE PNA FISH CULTURE IDENTIFICATION KIT
• Applicant: Advandx, Inc.
• Product Code: OAH · Microbiology
• Decision Date: Jul 24, 2009
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3740
• Device Class: Class 1

Intended Use

E. faecalis/OE PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and the detection of selected other enterococci (OE) on smears made from positive blood cultures containing Grampositive cocci in pairs and chains observed on Gram stain. Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth.

Device Story

Qualitative nucleic acid hybridization assay; identifies E. faecalis and other enterococci (OE) in positive blood cultures. Input: smear prepared from blood culture bottle containing Gram-positive cocci. Process: mixture of fluorescein-labeled E. faecalis-specific PNA probe and rhodamine-labeled OE-specific PNA probe added to smear; 30-minute hybridization at 55°C; stringent wash; mounting. Output: fluorescence microscopy visualization. Interpretation: bright green fluorescent cocci indicate E. faecalis; bright red fluorescent cocci indicate other enterococci. Used in clinical microbiology laboratories; performed by trained laboratory personnel. Enables rapid identification of enterococci directly from blood culture, facilitating timely clinical decision-making regarding antibiotic therapy.

Clinical Evidence

No clinical data provided in the document; substantial equivalence is based on bench testing and performance validation of the PNA FISH assay methodology.

Technological Characteristics

Multicolor nucleic acid hybridization assay using Peptide Nucleic Acid (PNA) probes. Principle: fluorescence in situ hybridization (FISH) targeting rRNA. Form factor: kit-based reagents for slide preparation and staining. Requires fluorescence microscopy for detection. Standalone diagnostic test.

Indications for Use

Indicated for identification of Enterococcus faecalis and detection of other enterococci (OE) in patients with positive blood cultures showing Gram-positive cocci in pairs and chains. For professional use only.

Regulatory Classification

Identification

Streptococcus spp. serological reagents are devices that consist of antigens and antisera (excluding streptococcal exoenzyme reagents made from enzymes secreted by streptococci) used in serological tests to identify Streptococcus spp. from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria belonging to the genus Streptococcus and provides epidemiological information on these diseases. Pathogenic streptococci are associated with infections, such as sore throat, impetigo (an infection characterized by small pustules on the skin), urinary tract infections, rheumatic fever, and kidney disease.

Predicate Devices

• E. faecalis/OE PNA FISH (K063127)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY TEMPLATE

A. 510(k) Number:
K083074

B. Purpose for Submission:
Modify the assay procedure of *E. faecalis*/OE PNA FISH. The specific modifications are: A reduction of the hybridization step from 90 minutes to 30 minutes; removal of the 5-10 minutes ethanol step during smear preparation.

C. Measurand:
*E. faecalis* and other enterococci (OE) specific ribosomal RNA sequences

D. Type of Test:
Fluorescence *In Situ* Hybridization (FISH) using protein nucleic acid (PNA) probes

E. Applicant:
AdvanDx, Inc

F. Proprietary and Established Names:
*E. faecalis*/OE PNA FISH™

G. Regulatory Information:
1. Regulation section:
866.3740
2. Classification:
Class I
3. Product code:
OAH
4. Panel:

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83 Microbiology

## H. Intended Use:

1. Intended use(s):

E. faecalis/OE PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and the detection of selected other enterococci (OE) on smears made from positive blood cultures containing Gram-positive cocci in pairs and chains observed on Gram stain. Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth.

2. Indication(s) for use:

E. faecalis/OE PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and the detection of selected other enterococci (OE) on smears made from positive blood cultures containing Gram-positive cocci in pairs and chains observed on Gram stain. Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth.

3. Special conditions for use statement(s):

Prescription use only

4. Special instrument requirements:

Dual Band Filter (Cat. No. AC003)

Microscope Slides (Cat. No. AC001)

## I. Device Description:

A mixture of a fluorescein-labeled, E. faecalis-specific PNA probe and a rhodamine-labeled PNA probe specific for OE is added to a smear prepared from a blood culture. Hybridization is performed at 55°C for 30 minutes. The hybridization is followed by a post-hybridization wash at 55°C for 30 min with a stringent Wash Solution. Finally, the smear is mounted with Mounting Medium and examined by fluorescence microscopy.

## J. Substantial Equivalence Information:

1. Predicate device name(s):

E. faecalis/OE PNA FISH

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2. Predicate 510(k) number(s):

K063127

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Technology | Fluorescence In Situ Hybridization (FISH) using protein nucleic acid (PNA) probe | Same |
| Sample | Positive blood culture | Same |
| PNA Probes | Fluorescein-labeled E. faecalis specific PNA probe and rhodamine-labeled specific for other enterococci | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Fixed smear treatment | None | Ethanol for 10 minutes and air dried |
| Hybridization at 55°C | 30 minutes | 90 minutes |

K. Standard/Guidance Document Referenced (if applicable):

Non applicable

L. Test Principle:

A mixture of a fluorescein-labeled, E. faecalis-specific PNA probe and a rhodamine-labeled PNA probe specific for OE is added to a smear prepared from a blood culture. Hybridization is performed at 55°C for 30 minutes. The hybridization is followed by a post-hybridization wash at 55°C for 30 min with a stringent Wash Solution. Finally, the smear is mounted with Mounting Medium and examined by fluorescence microscopy.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

A reproducibility study for E. faecalis/OE PNA FISH assay was performed by using ten reference Gram positive cocci, once per day with positive and

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negative controls, over a period of three days at three different sites, by at least two different operators at each site. Results showed 100% precision and reproducibility between and within sites.

b. Linearity/assay reportable range:
Not applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Positive and negative control slides were performed at each testing site on each day of testing. All results were as expected.

d. Detection limit:
The detection limit was determined to be approximately 10⁵ CFU/mL by serial dilutions of E. faecalis and E. faecium positive cultures. Average number of colonies per mL (CFU/mL) was calculated from three plates. The data sets showed a minimum of 10⁵ CFU/mL to produce a positive result for the E. faecalis/OE PNA FISH™ assay.

e. Analytical specificity:
The modified assay procedure was tested and compared to the original assay procedure. E. faecalis/OE PNA FISH has been evaluated on (11) E. faecalis, and (22) Enterococcus spp, not faecalis; (17) Gram negative organisms, (21) Gram positive organisms and (7) yeasts representing phylogenetically closely related organisms and a variety of clinically significant organisms. All eleven E. faecalis showed green fluorescence in both procedures; E. cecorum, E. columbae, and E. dispar were negative (no fluorescence) and E. saccharolyticus showed yellow fluorescence in both procedures.

f. Assay cut-off:
Not applicable

2. Comparison studies:

a. Method comparison of device to conventional methods, as the reference method:
The modified assay procedure was compared to the original assay procedure.

b. Matrix comparison:
Not applicable

3. Clinical studies:
Clinical Study was conducted at three sites, directly on blood culture bottles

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containing Gram positive cocci in pairs and chains (GPCPC). A total of 152 GPCPC-positive blood bottles, from two commercial continuously monitoring blood culture systems (BacT/Alert and BACTEC) were included in the study. A total of (50) BACTEC Standard 10A Aerobic and (50) BACTEC Standard Anaerobic, (23) BacT/Alert SA and (29) BacT/Alert SN blood culture bottles were tested. Performance results of the modified assay procedure (i.e. 30 minutes hybridization, prepared smears not treated with ethanol) compare to the original assay procedure was summarized below.

| Study | Positive Agreement (Green) E. faecalis | Positive Agreement (Red) Other Enterococci | Negative Agreement | Blood Culture System |
| --- | --- | --- | --- | --- |
| A | 100% | 100% | 100% | BACTEC |
| | (19/19) | (11/11) | (18/18) | |
| B | 100% | 100% | 96.6% | BacT/Alert |
| | (9/9) | (12/12) | (31/31) | |
| C | 100% | 92.3% | 93.1% | BACTEC |
| | (13/13) | (8/8) | (29/29) | |
| Total | 100% | 100% | 100% | |
| | (41/41) | (33/33) | (78/78) | |
| | 95% CI (93.0 - 100) | 95% CI (91.3 - 100) | 95% CI (96.2 - 100) | |

a. Clinical Sensitivity:
Not applicable

b. Clinical specificity:
Not applicable

c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable

4. Clinical cut-off:
Not applicable

5. Expected values/Reference range:
E. faecalis cells: bright green fluorescent cocci in multiple fields
Enterococcus spp, not E. faecalis cells: bright red fluorescent cocci in multiple

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fields

The expected positive rate from positive blood culture bottles is 40-50% for *E. faecalis* and 15-25% for other Enterococci.

## N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

## O. Conclusion:

The information submitted in this premarket notification is complete and supports a substantial equivalence decision.