EK/P. AERUGINOSA PNA FISH

K092393 · Advandx, Inc. · JSS · Sep 1, 2009 · Microbiology

Device Facts

• Record ID: K092393
• Device Name: EK/P. AERUGINOSA PNA FISH
• Applicant: Advandx, Inc.
• Product Code: JSS · Microbiology
• Decision Date: Sep 1, 2009
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.2660
• Device Class: Class 1

Intended Use

EK/P. aeruginosa PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for identification of Escherichia coli and/or Klebsiella pneumoniae and Pseudomonas aeruginosa on smears made from positive blood cultures containing Gram-negative rods. The test does not distinguish between E. coli and K. pneumoniae. Further testing is needed to differentiate E. coli and K. pneumoniae. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and/or differentiation of mixed growth. The EK/P. aeruginosa PNA FISH assay is indicated for use as an aid in the diagnosis of E. coli and/or K. pneumoniae and P. aeruginosa bacteremia.

Device Story

Fluorescence in situ hybridization (FISH) assay using PNA probes; identifies E. coli/K. pneumoniae (green fluorescence) and P. aeruginosa (red fluorescence) in positive blood culture smears. Procedure: smear fixation on slide; hybridization with PNA probe mixture at 55°C for 30 minutes; stringent wash at 55°C; mounting; examination via fluorescence microscopy using dual band filter. Used in clinical microbiology laboratories by trained technicians. Provides rapid identification of pathogens to assist clinical decision-making regarding antibiotic therapy. Modification from predicate includes elimination of 10-minute ethanol fixation step and reduction of hybridization time from 90 to 30 minutes.

Clinical Evidence

No clinical data provided in the summary document; substantial equivalence is based on analytical performance and technological characteristics.

Technological Characteristics

Qualitative nucleic acid hybridization assay using PNA probes. Multicolor fluorescence detection. Form factor: kit containing reagents for slide preparation and hybridization. Principle: PNA probes bind to specific rRNA sequences in target bacteria. Visualization via fluorescence microscopy. Standalone diagnostic test.

Indications for Use

Indicated for qualitative identification of E. coli, K. pneumoniae, and P. aeruginosa in patients with positive blood cultures containing Gram-negative rods. Requires subculturing for susceptibility testing and differentiation of E. coli/K. pneumoniae.

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

• EK/P. aeruginosa PNA FISH (K081433)

Submission Summary (Full Text)

{0}

1

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K092393

B. Purpose for Submission:
SE determination for a modification of the assay procedure for $EK/P$. aeruginosa PNA FISH. The specific modifications are: elimination of the 5-10 minutes ethanol step in smear preparation; a reduction of the hybridization time from 90 minutes to 30 minutes.

C. Measurand:
E. coli + Klebsiella pneumoniae 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:
EK/P aeruginosa PNA FISH™

G. Regulatory Information:
1. Regulation section:
866.2660
2. Classification:
Class I
3. Product code:
JSS, JSZ

{1}

4. Panel:
83 Microbiology

H. Intended Use:

1. Intended use(s):

EK/P aeruginosa PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for identification of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa on smears from positive blood cultures containing Gram negative rods. The test does not distinguish between E. coli, and K. pneumoniae. Further testing is needed to differentiate E. coli, and K. pneumoniae. The EK/P aeruginosa PNA FISH assay is indicated for use in conjunction with positive blood subcultures as an aid in the identification of E.coli/Klebsiella pneumoniae and/or P. aeruginosa

2. Indication(s) for use:

EK/P aeruginosa PNA FISH is a multicolor qualitative nucleic acid hybridization assay intended for identification of Escherichia coli/ Klebsiella pneumoniae and Pseudomonas aeruginosa on smears from positive blood cultures containing Gram-negative rods. The test does not distinguish between E coli and K pneumoniae. Further testing is needed to differentiate E coli and K pneumoniae. The EK/P aeruginosa PNA FISH assay is indicated for use in conjunction with positive blood subcultures as an aid in the identification of E.coli/Klebsiella pneumoniae and/or P aeruginosa.

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)

{2}

3

I. Device Description:

The EK/P. aeruginosa PNA FISH is a fluorescence *in situ* hybridization (FISH) method using PNA probes hybridizing to *E. coli*, fluorescein labeled *K. pneumoniae* specific PNA probe and a Texas Red labeled *P. aeruginosa* specific PNA probe. The test is performed directly on smears fixed onto a microscope slide prepared from a positive blood culture. Hybridization is performed at 55°C for 30 minutes. The hybridization is followed by a water rinse at 55°C to remove the cover slips, followed by a wash at 55°C for 30 min with a stringent wash solution. The smear is finally mounted with Mounting Medium for examination with fluorescence microscopy (Dual Band Filter) *E. coli*, and *K. pneumoniae* are bright green fluorescent rods whereas *P. aeruginosa* are bright red fluorescent rods. The test does not distinguish between *E. coli* and *K. pneumoniae*.

J. Substantial Equivalence Information:

1. Predicate device name(s):

*EK/P. aeruginosa PNA FISH*

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

K081433

3. Comparison with predicate:

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

{3}

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

Non applicable

L. Test Principle:

A mixture of fluorescein-labeled, E. coli, a fluorescein-labeled K pneumoniae specific PNA probe and a Texas Red label, 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 water rinse at 55°C to remove the cover slips followed by a wash at 55°C for 30 minutes 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 EK/P aeruginosa PNA FISH assay was performed by using ten reference Gram negative rods, once per day with positive and negative controls, over a period of three days at three different sites, by one operator 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 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, K. pneumoniae, and P. aeruginosa cultures. The 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 EK/P aeruginosa PNA FISH™ assay.

4

{4}

e. Analytical specificity:

The analytical specificity of the EK/P aeruginosa PNA FISH™ was determined by BLAST search and sequence alignments and experimentally by testing of well characterized laboratory and reference strains comprising of (19) E.coli, (12) K. pneumoniae, (20) P. aeruginosa, (51) other additional Gram negative organisms, (13) Gram positive organisms, and (6) yeasts representing phylogenetically closely related organisms and a variety of clinically significant organisms. All (19/19) E.coli strains and (12/12) K. pneumoniae showed green-positive fluorescence and all (20/20) P. aeruginosa strains showed red-positive. Shigella spp (serogroup A, B, or D), Escherichia albertii and Escherichia fergusonii cross reacted to create a green signal. Brevundimonas diminuta, Herbaspirillum huttiense, Pseudomonas nitroreducens, and Pseudomonas fuva cross-reacted to create a red signal. All other strains were negative.

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:

The Clinical Study was conducted at three sites: the performance of EK/P aeruginosa PNA FISH (New) versus EK/P aeruginosa PNA FISH, was compared to conventional routine methods. A total of 368 blood culture bottles with Gram negative rods (GNR) were included in the studies.

Performance results of the modified, shortened assay procedure (i.e. 30 minutes hybridization, prepared smears not treated with ethanol) compared to the original assay procedure, and compared to the conventional methods are summarized below.

{5}

Performance Data for EK/P. aeruginosa PNA FISH (New) vs. EK/P. aeruginosa PNA FISH (K081433).

| Study | Positive Agreement E.coli/K. pneumoniae | Positive Agreement P. aeruginosa | Negative Agreement | Blood Culture System |
| --- | --- | --- | --- | --- |
| A | 65/65 | 12/12* | 42/42 | BACTEC |
| B | 69/69 | 9/9 | 22/22 | BacT/Alert |
| C | 34/34 | 7/7 | 34/34 | BACTEC |
| D | 47/47 | 7/7 | 22/22 | BACTEC |
| Total | 100% (215/215)
95%CI (98.6-100) | 100% (35/35)
95% CI (91.8-100) | 100% (120/120)
95% CI (97.5-100) | N=370 |

*2 mix P. aeruginosa and K. pneumoniae samples

Performance Data for EK/P. aeruginosa PNA FISH (New) vs Conventional Methods.

| Study | Sensitivity E.coli/K. pneumoniae | Sensitivity P. aeruginosa | Specificity | Blood Culture System |
| --- | --- | --- | --- | --- |
| A | 65/65 | 12/12* | 42/42 | BACTEC |
| B | 69/69 | 9/9 | 22/22 | BacT/Alert |
| C | 34/34 | 7/7 | 34/34 | BACTEC |
| D | 47/47 | 7/8 | 21/21 | BACTEC |
| Total | 100% (215/215)
95%CI (98.6-100) | 97.2% (35/36)
95% CI (85.5-99.9) | 100% (120/120)
95% CI (97.5-100) | N=370 |

*2 mix P. aeruginosa and K. pneumoniae samples

a. Clinical Sensitivity:
Not applicable

b. Clinical specificity:
Not applicable

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

4. Clinical cut-off:
Not applicable

{6}

5. Expected values/Reference range:

The expected *E. coli / K. pneumoniae* results are multiple bright green fluorescent rods in multiple fields. *P. aeruginosa* cells are multiple bright red fluorescent rods in multiple fields. In the studies conducted, *E. coli*, *K. pneumoniae* and *P. aeruginosa* had positive result rates for Gram negative rods with positive blood culture bottles of approximately 37%, 18% and 13%, respectively. This may differ depending on the patient population and the institution.

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.

7