ENTEROCOCCUS FAECALIS PNA FISH
Device Facts
| Record ID | K063127 |
|---|---|
| Device Name | ENTEROCOCCUS FAECALIS PNA FISH |
| Applicant | Advandx, Inc. |
| Product Code | OAH · Microbiology |
| Decision Date | Dec 22, 2006 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3740 |
| Device Class | Class 1 |
Intended Use
E. faecalis PNA FISH™ is a multicolor qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and selected other enterococci from blood cultures. E. faecalis PNA FISH™ is a qualitative nucleic acid hybridization assay intended for identification of Enterococcus faecalis and selected other enterococci (OE) from blood cultures. E. faecalis PNA FISH™ is indicated for testing positive blood cultures with Gram-positive cocci in pairs and chains.
Device Story
E. faecalis PNA FISH is a qualitative nucleic acid hybridization assay used in clinical laboratories. It utilizes Peptide Nucleic Acid (PNA) probes to target specific ribosomal RNA sequences within Enterococcus faecalis and other enterococci present in positive blood culture samples. The assay involves hybridization of fluorescently labeled PNA probes to the target organisms, followed by a wash step to remove unbound probes. Results are visualized via fluorescence microscopy. The device provides rapid identification of E. faecalis, enabling clinicians to optimize antimicrobial therapy for patients with suspected bacteremia, potentially improving clinical outcomes by facilitating targeted treatment.
Clinical Evidence
No clinical data provided in the document.
Technological Characteristics
Qualitative nucleic acid hybridization assay using fluorescently labeled Peptide Nucleic Acid (PNA) probes. Designed for use with positive blood culture samples. Visualization via fluorescence microscopy.
Indications for Use
Indicated for qualitative identification of Enterococcus faecalis and selected other enterococci (OE) from positive blood cultures in patients suspected of having enterococcal bacteremia.
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.
Related Devices
- K083074 — AVANDX E. FAECALIS/OE PNA FISH CULTURE IDENTIFICATION KIT · Advandx, Inc. · Jul 24, 2009
- K121991 — ENTEROCOCCUS QUICKFISH BC · Advandx, Inc. · Jan 7, 2013
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
A. 510(k) Number:
k063127
B. Purpose for Submission:
New product *E. faecalis* PNA FISH™
C. Measurand:
*Enterococcus faecalis* specific 16 S ribosomal RNA and selected other enterococci-specific 16S ribosomal RNA
D. Type of Test:
Fluorescent In Situ Hybridization (FISH) using protein nucleic acid (PNA) probes
E. Applicant:
AdvanDx, Inc.
F. Proprietary and Established Names:
*E. faecalis* PNA FISH™, *Enterococcus faecalis* Culture Identification Kit
G. Regulatory Information:
1. Regulation section:
21 CFR Part 866.3740 – Streptococcus spp. serological reagents
2. Classification:
Class I
3. Product code:
OAH - FISH (fluorescent in situ hybridization) kit, protein nucleic acid, *Enterococcus faecalis*
4. Panel:
83 Microbiology
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H. Intended Use:
1. Intended use(s):
E. faecalis PNA FISH™ is a multicolor qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and selected other enterococci from blood cultures.
2. Indication(s) for use:
E. faecalis PNA FISH™ is a qualitative nucleic acid hybridization assay intended for identification of Enterococcus faecalis and selected other enterococci (OE) from blood cultures. E. faecalis PNA FISH™ is indicated for testing positive blood cultures with Gram-positive cocci in pairs and chains.
3. Special conditions for use statement(s):
Prescription use only.
Limitations: The following organisms are not detected by the PNA probe hybridizing to selected other enterococci: Enterococcus asinii, E. dispar, E. haemoperoxidus, E. raffinosus, E. saccharolyticus, E. solitarius and E. sulfurous.
Enterococcus moraviensis is identified as Enterococcus faecalis due to sequence identity.
4. Special instrument requirements:
AdvanDx Teflon-coated Microscope Slides. A fluorescent microscope equipped with an AdvanDx Dual Band Filter.
I. Device Description
The E. faecalis PNA FISH™ Culture Identification Kit contains a 3 mL bottle of fixation solution, a 1.5 mL bottle of fluorescein-labeled and rhodamine-labeled PNA probe in hybridization solution, a 50 mL bottle of concentrated wash solution, which must be diluted prior to use, and a 3 mL bottle of mounting medium. The one-well, Teflon-coated microscope slides are sold separately. User- prepared quality control organism slides are acceptable. After processing, the slides must be examined within two hours by using a fluorescent microscope equipped with a dual band filter available from the manufacturer.
J. Substantial Equivalence Information:
1. Predicate device name(s):
a. Conventional Methods-similar Indication for Use
b. Gen Probe AccuProbe Enterococcus – similar Indication For Use
c. S. aureus PNA FISH – similar technology
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2. Predicate K number(s):
a. K895103
b. K060099
Note: Pre-amendment devices were also referenced.
3. Comparison with predicate(s):
| Item | Test Device | Pre-Amendment | Primary Predicate | Secondary Predicate |
| --- | --- | --- | --- | --- |
| Product Name | E. faecalis PNA FISH | Conventional Routine Methods | Accuprobe Enterococcus | S. aureus PNA FISH |
| Intended Use | Identification of Enterococcus faecalis and selected other enterococci | Identification of enterococci from colonies isolated on solid media | Detection and identification of Enterococcus species | Identification of Staphylococcus aureus |
| Technology Method | Fluorescence in situ hybridization | Phenotypic, Biochemical | DNA probe hybridization | Fluorescence in situ hybridization |
| Time to result | 2.5 hours from time of smear preparation | 1 – 2 days, including subculture | 18 - 24 hours including subculture | 2.5 hours from time of smear preparation |
| Sample | Smear of blood cultures | Colonies isolated on solid media | Isolated cultures | Smear of blood cultures |
| Control organisms | Positive Controls: E. faecalis, and E. faecium Negative Controls: Streptococcus or Staphylococcus | Test dependent | E. faecalis and S. bovis | S. aureus and S. epidermidis |
| Mechanism of identification | E. faecalis specific 16S rRNA and also other enterococci - specific 16S rRNA | Test dependent | DNA capture and probe which utilizes nucleic acid hybridization | S. aureus specific 16 S rRNA |
| Interpretation of results | Qualitative fluorescent microscopy | Qualitative-Test dependent | Quantitative luminometer | Qualitative fluorescent microscopy |
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K. Standard/Guidance Document Referenced (if applicable):
Not applicable
L. Test Principle:
Add one drop of *E. faecalis*-specific PNA probe (which contains both fluorescein-labeled *E. faecalis* PNA probe and rhodamine-labeled other *Enterococcus* spp. probe) to a methanol, heat, or flame fixed smear, prepared from liquid blood culture media with Gram-positive cocci in pairs or chains. Hybridization is performed during a 90 +/- 5 minutes incubation at 55 +/- 1° C, in an incubator or on a slide warmer. The slide is examined by fluorescent microscopy within two hours of staining. *Enterococcus faecalis* is identified as multiple bright green fluorescent cocci in multiple fields on a dark reddish background. Selected other *Enterococcus* species are identified as multiple bright red fluorescent cocci on a dark red background. Non-*Enterococcus* cells will not fluoresce.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
The performance data were generated by multiple, geographically distinct, clinical laboratories. Each laboratory compared the device results to corresponding results obtained by standard culture identification procedures. These laboratories reported the sensitivity and specificity results in peer reviewed literature articles, and during presentations performed at professional meetings (posters).
a. Precision/Reproducibility:
Inter-laboratory and Intra-laboratory (one internal site and two external sites) testing demonstrated >95% reproducibility and precision. The ten isolate study was used (10 organisms tested 3 times on 3 days at 3 sites.).
b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
The recommended Positive Control QC isolates -- *Enterococcus faecalis* and *Enterococcus faecium* -- may be combined onto one slide, or prepared as separate slides. Negative Control QC isolates can be either *Staphylococcus* species or *Streptococcus* species. User-prepared slides from stock or liquid blood cultures are recommended for testing in parallel for each batch of tests performed.
Liquid culture organism control slide testing was performed at three sites (one internal and two external sites) a sufficient number of times to demonstrate that the device can produce acceptable quality control results >95% of the time.
The stability of the *E. faecalis* PNA FISH reagents were evaluated using real-time data. The sponsor periodically removed samples from five lots of *E. faecalis* PNA FISH reagent from storage for testing. Both the fluorescein and the rhodamine
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fluorescence over time were evaluated. The analytical fluorescence performance and a functional performance of each lot were tested 5 times over 30 months after the manufacturing date. The fluorescent dyes on the probes are light sensitive and are considered the least stable components of the kit. There was no change in performance for at least 24 months when stored at $2 - 8^{\circ}\mathrm{C}$ and protected from light, as indicated in the product labeling. This study is on-going and data are being collected to potentially extend the shelf life.
# d. Detection limit:
The claimed detection limit for $E$ faecalis in blood cultures was determined to be approximately $1 \times 10^{5}$ colony forming units (CFU) per mL by serial dilutions of an $E$ faecalis positive culture. This is consistent with the analytical sensitivity of slide-based staining techniques and is not limited by the test itself, but rather by the general requirement for $1 \times 10^{5}$ CFU/mL for interpretation by standard light microscopy.
# e. Analytical specificity:
Specificity of $E$ faecalis PNA FISH probe was evaluated using cultures of 16 selected Enterococcus spp. reference strains, excluding species listed under the Limitations section. The results are presented in the table below.
| Species | PNA FISH result |
| --- | --- |
| Enterococcus faecalis | E. faecalis |
| Enterococcus faecium | Other Enterococci |
| E. avium, E casseliflavus, E. cecorum, E. columbae, E. durans, E. flavescens, E. gallinarium, E. hirae, E. malodoratus, E. mundtii, E. porcinus (reclassified as E. villorum), E. pseudoavium, E. ratti. E. villorum | Other Enterococci |
The analytical specificity by organism group is $E$ faecalis at $100\%$ (1/1) and for other Enterococci is $100\%$ (15/15).
E. faecalis PNA FISH has also been evaluated on 24 reference strains representing Gram-positive cocci, other bacteria and yeast species. The analytical specificity for E. faecalis strains positive by PNA FISH was E. faecalis (3/3) $100\%$ , selected other enterococci (6/6) $100\%$ and the other species (15/15) were negative.
A BLAST search and Sequence Alignments search of the GeneBank nr-database (www.nim.nih.gov/blast) showed that the target sequence matched exactly for 61 E. faecalis 16S rRNA sequences stored in the database. The E. faecalis PNA probe and the other Enterococcus species PNA probe target ribosomal sequences, which are well-suited for the design of species-specific probes.
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f. Assay cut-off:
Not applicable
2. Comparison studies:
a. Method comparison of device to conventional methods, as the reference method:
The performance of E. faecalis PNA FISH was evaluated using three different automated blood culture media systems at multiple sites. The data demonstrates that the E. faecalis PNA FISH™ is compatible with three major blood culture systems, and results from testing are comparable to results obtained by conventional methods. The results are displayed in the table below.
Automated blood culture system media evaluation comparing device results to results obtained by subculture and subsequent identification by standard methods, by study site
| Blood Culture System Evaluated | Sensitivity E. faecalis | Sensitivity Other Enterococci | Specificity | Study |
| --- | --- | --- | --- | --- |
| ESP | 100% (26/26) | 93.3% (14/15) | 92.3% (12/13) | A |
| BACTEC, ESP, BacT/Alert | 92.9% (26/28) | 92.9% (13/14) | 100% (79/79) | B |
| BACTEC | 100% (16/16) | N/A (0/0) | 100% 4/4 | C |
| BacT/Alert | 100% 14/14 | N/A^{1} | N/A^{1} | D |
| BACTEC | 100% (43/43) | 100% (29/29) | 100% (32/32) | E |
| Total | 98.4% (125/127) | 96.6% (56/58) | 99.2% (127/128) | |
BacT/Alert = BacT/Alert Blood culture system (bioMérieux, Durham, NC)
BACTEC = BACTEC Blood culture system (Becton Dickenson, Sparks, MD)
ESP = ESP Blood culture system (Trek Diagnostics, Cleveland, OH)
N/A¹ Data on 41 non-E.faecalis were reported as 100% specificity, but were not differentiated between other enterococci and non-enterococci.
b. Matrix comparison:
Not applicable
3. Clinical studies:
a. Clinical Sensitivity:
Not applicable
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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:
The expected E. faecalis positive result rate from positive blood culture bottles showing Gram-positive cocci in pairs and chains is 40% - 50%, where as selected other enterococci account for 15% - 25%, depending on institutional and patient population.
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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