E.COLI/P. AERUGINOSA PNA FISH

K092236 · Advandx, Inc. · JSS · Dec 16, 2009 · Microbiology

Device Facts

• Record ID: K092236
• Device Name: E.COLI/P. AERUGINOSA PNA FISH
• Applicant: Advandx, Inc.
• Product Code: JSS · Microbiology
• Decision Date: Dec 16, 2009
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.2660
• Device Class: Class 1

Intended Use

Escherichia coli/Pseudomonas aeruginosa PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for identification of Escherichia coli and Pseudomonas aeruginosa on smears from positive blood cultures containing Gram-negative rods. The E. coli/P. aeruginosa PNA FISH assay is indicated for use as an aid in the diagnosis of E. coli and/or P. aeruginosa bacteremia. Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth.

Device Story

Qualitative nucleic acid hybridization assay; uses fluorescence in situ hybridization (FISH) with protein nucleic acid (PNA) probes to identify E. coli and P. aeruginosa in positive blood culture smears. Input: smear from positive blood culture containing Gram-negative rods. Process: heat fixation; hybridization with fluorescein-labeled E. coli-specific PNA and Texas Red-labeled P. aeruginosa-specific PNA; stringent wash; mounting. Output: visual fluorescence signal (green for E. coli, red for P. aeruginosa) viewed via fluorescence microscope. Used in clinical laboratories by trained personnel. Provides rapid identification (1.5 hours) to assist clinicians in targeted antimicrobial therapy decisions for bacteremia patients.

Clinical Evidence

No clinical data provided in the document. Substantial equivalence is based on technological characteristics and performance validation via bench testing.

Technological Characteristics

Qualitative nucleic acid hybridization assay using PNA probes. Principle: Fluorescence in situ hybridization (FISH). Form factor: Reagent kit for slide-based microscopy. Detection: Fluorescence microscopy. No software or electronic components; manual diagnostic procedure.

Indications for Use

Indicated for identification of E. coli and P. aeruginosa in patients with positive blood cultures containing Gram-negative rods. Used as an aid in diagnosing E. coli and/or P. aeruginosa bacteremia.

Regulatory Classification

Identification

A microorganism differentiation and identification device is a device intended for medical purposes that consists of one or more components, such as differential culture media, biochemical reagents, and paper discs or paper strips impregnated with test reagents, that are usually contained in individual compartments and used to differentiate and identify selected microorganisms. The device aids in the diagnosis of disease.

Predicate Devices

• S. aureus/CNS PNA FISH (K061336)

Submission Summary (Full Text)

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

A. 510(k) Number:
K092236

B. Purpose for Submission:
To obtain a substantial equivalence determination for a modification of the assay procedure for the *E. coli* and/or *P. aeruginosa* PNA FISH. The specific modifications are: elimination of the 5-10 minutes ethanol step in smear preparation and a reduction of the hybridization time from 90 minutes to 30 minutes.

C. Measurand:
*E. coli* and *P. aeruginosa* 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. coli/P. aeruginosa* PNA FISH™

G. Regulatory Information:
1. Regulation section: 866.2660
2. Classification: Class I
3. Product code: JSS
4. Panel: 83-Microbiology

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H. Intended Use:

1. Intended use:

Escherichia coli/Pseudomonas aeruginosa PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for identification of Escherichia coli and Pseudomonas aeruginosa on smears from positive blood cultures containing Gram-negative rods. The E. coli/P. aeruginosa PNA FISH assay is indicated for use as an aid in the diagnosis of E. coli and/or P. aeruginosa bacteremia.

Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth.

2. Indications for use:

Escherichia coli/Pseudomonas aeruginosa PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for identification of Escherichia coli and Pseudomonas aeruginosa on smears from positive blood cultures containing Gram-negative rods. The E. coli/P. aeruginosa PNA FISH assay is indicated for use as an aid in the diagnosis of E. coli and/or P. aeruginosa bacteremia.

Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth.

3. Special conditions for use statement:

Prescription use only

4. Special instrument requirements:

Dual Band Filter (Cat. No. AC003)
Microscope Slides (Cat. No. AC001)

I. Device Description:

The Escherichia coli/Pseudomonas aeruginosa PNA FISH is a multicolor, qualitative, nucleic acid hybridization assay intended for identification of E. coli and P. aeruginosa on smears made from positive blood culture. This new proposed model of the assay, purported to provide rapid (within 1.5 hours) identification, consists of the following reagents:

- GN Fixation Solution
3 mL phosphate-buffered saline

- E. coli/P. aeruginosa PNA
1.5 mL fluorescein-labeled, E. coli/P. aeruginosa specific PNA probes in hybridization solution. Contains 30% foramide

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- 60X Wash Solution
50 mL Tris-buffered saline with detergent
- Mounting Medium
3 mL photobleaching inhibitor in glycerol

Materials required but not provided:
- Water, deionized or distilled
- Fluorescence microscope equipped with a 60x or 100x oil objective lens
- Immersion oil. Must comply with the microscope objective and be non-fluorescent

J. Substantial Equivalence Information:

1. Predicate device name:
E. coli/P. aeruginosa PNA FISH™

2. Predicate 510(k) number:
K081309

3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device (K092236) | Predicate (K081309) |
| Function | Identification of E. coli and P. aeruginosa | Same |
| Technology | Fluorescence In Situ Hybridization (FISH) using protein nucleic acid (PNA) probe | Same |
| Sample Type | Positive Blood Cultures | Same |
| Controls | E. coli, P. aeruginosa and Klebsiella spp. | Same |
| Interpretation of Results | Qualitative Fluorescence microscope | Same |
| PNA Probes | Eco 16S06-Flu
Pse23S32-TXR | Same |

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| Differences | | |
| --- | --- | --- |
| Item | Device (K092236) | Predicate (K081309) |
| Fixed smear treatment | None | Ethanol for 10 minutes and air dried |
| Hybridization Time | 30 minutes | 90 minutes |
| Time to Result | 1.5 hours | 2.5 hours |

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

Not applicable

## L. Test Principle:

A mixture of fluorescein-labeled, *E. coli* specific PNA probe and a Texas Red labeled, *P. aeruginosa* specific PNA probe is added to a smear prepared from a positive blood culture. Hybridization is performed at 55°C for 30 minutes. The hybridization is followed by a rinse step to remove the cover slip followed by a wash at 55°C for 30 minutes with a stringent wash solution to remove unbound PNA probe. Subsequently, the smear is mounted with Mounting Medium and examined by fluorescence microscopy.

## M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Reproducibility:

A Reproducibility study for *E. coli*/ *P. aeruginosa* PNA FISH assay was performed by using 16 reference isolates of Gram negative rods, once per day with positive and negative controls, over a period of three days at three different sites. Each batch was run independently by at least two blinded operators at each site.

Results showed > 97.9 % reproducibility between and within sites. This is acceptable for this type device.

b. Linearity/assay reportable range:

Not applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

**Quality Control**

Positive and negative control slides were performed at each testing site. All results were as expected

d. Detection limit:

The detection limit was determined to be approximately 10⁵ CFU/mL by serial dilutions of *E. coli* or *P. aeruginosa* positive cultures. Serial dilutions of exponentially growing cultures were prepared. Then 0.01 mL was plated on growth

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media and 0.01 mL was used for preparation of smears. The smears were run through PNA FISH and scored positive or negative. The following day, colonies were counted on the plates and the average number of colonies per dilution was calculated. The data sets showed a minimum of 10⁵ CFU/mL to produce a positive result for the E. coli/P. aeruginosa PNA FISH™ assay.

e. Analytical specificity:

E. coli/P. aeruginosa PNA FISH has also been tested on laboratory and reference strains comprising of 14 E. coli, 17 P. aeruginosa, 60 additional Gram negative organisms, 12 Gram positive organisms and 6 yeasts, representing phylogenetically closely related strains. All (14/14) E. coli strains were green-positive and all (17/17) P. aeruginosa strains were red-positive. Shigella spp. (serogroup A, B, C, or D), Escherichia albertii and Escherichia fergusonii cross-reacted to create a green signal, Brevundimonas diminuta, Herbaspirillum huttiense, Pseudomonas nitroreducens, and Pseudomonas fulva cross-reacted to create a red signal. All other strains were negative.

Interference

A study consisting of 10 Gram negative rods were tested on four types of blood culture bottles, BD BACTEC, BacTAlert SA and BacTAlert FA, and VersaTREK for Heat Fixation at 70°. Fifteen organisms were tested at 55° and 80° for at for the interference from charcoal and different temperatures for Heat Fixation. No interferences were observed.

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 and the conventional culture methods.

b. Matrix comparison:

Not applicable

3. Clinical studies:

a. Clinical Sensitivity:

The performance of E. coli/P. aeruginosa PNA FISH (new procedure) versus

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E. coli/P. aeruginosa PNA FISH (K081309), along with conventional routine methods, was assessed in five clinical laboratory studies. A total of 385 blood culture bottles with Gram negative rods were included in the studies. Performance results were as follows:

Performance data E. coli/P. aeruginosa PNA FISH (proposed device) versus Routine Conventional Methods on GNR-positive Blood Culture Bottles

| Study | Sensitivity
E. coli | Sensitivity
P. aeruginosa | Specificity | Blood Culture System |
| --- | --- | --- | --- | --- |
| A | 51/51 | 12/12 | 54/54 | BACTEC |
| B | 51/51 | 9/9 | 40/40 | BacT/Alert |
| C | 17/17 | 7/7 | 51/51 | BACTEC |
| D | 32/32 | 7/8 | 36/36 | BACTEC |
| E | 7/7 | 4/4 | 6/6 | VersaTREK |
| Total | 100% (158/158) | 97.5% (39/40) | 100% (187/187) | |
| | 95% CI (98.0-100) | 95% CI (87-99.9) | 95% CI (98.4-100) | |

b. Clinical specificity

Refer to table in section 3a.

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:

Positive E. coli cells - green fluorescence

Positive P. aeruginosa cells - red fluorescence

The expected positive rates from positive blood culture bottles for E.coli and P. aeruginosa are 37% and 13%, respectively.

N. Proposed Labeling:

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

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O. Conclusion:

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

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