GNR TRAFFIC LIGHT PNA FISH

K101558 · Advandx, Inc. · JSS · Dec 16, 2010 · Microbiology

Device Facts

• Record ID: K101558
• Device Name: GNR TRAFFIC LIGHT PNA FISH
• Applicant: Advandx, Inc.
• Product Code: JSS · Microbiology
• Decision Date: Dec 16, 2010
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.2660
• Device Class: Class 1

Intended Use

GNR Traffic Light PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Escherichia coli, and/or Klebsiella pneumoniae, and/or Pseudomonas aeruginosa on smears made from positive blood cultures containing Gram-negative rods observed on Gram stain. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing, and/or differentiation of mixed growth. GNR Traffic Light PNA FISH is indicated as an aid in the diagnosis of Escherichia coli, and/or Klebsiella pneumoniae, and/or Pseudomonas aeruginosa bacteremia.

Device Story

GNR Traffic Light PNA FISH is a qualitative nucleic acid hybridization assay. Input: smears from positive blood cultures containing Gram-negative rods. Principle: Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) uses fluorescently labeled PNA probes to target specific ribosomal RNA sequences within the microorganisms. Output: multicolor fluorescent signals visualized via fluorescence microscopy. Used in clinical microbiology laboratories by trained technicians/microbiologists. Provides rapid identification of specific pathogens (E. coli, K. pneumoniae, P. aeruginosa) directly from positive blood cultures, enabling faster targeted antimicrobial therapy compared to traditional culture-based methods. Results assist clinicians in optimizing patient management for bacteremia.

Clinical Evidence

Prospective study (358 samples) and 32 spiked samples (N=395 total) across four U.S. sites. Compared to conventional culture methods. Results: E. coli sensitivity 100% (135/135), K. pneumoniae 98.7% (77/78), P. aeruginosa 96.9% (62/64). Specificity 97.5% (115/118). 95% CIs provided for all metrics. Bench testing confirmed analytical specificity across 163 microorganisms and a detection limit of 10^5 CFU/mL.

Technological Characteristics

Multicolor PNA FISH assay. Uses fluorescently labeled PNA probes for nucleic acid hybridization. Requires fluorescence microscope for visualization. Standalone diagnostic kit. No software or electronic components.

Indications for Use

Indicated for identification of E. coli, K. pneumoniae, and/or P. aeruginosa in patients with positive blood cultures containing Gram-negative rods. Used as an aid in diagnosis of bacteremia. Requires subculturing for susceptibility testing.

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 (K092393)

Submission Summary (Full Text)

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

A. 510(k) Number:
K101558

B. Purpose for Submission:
To determine substantial equivalence for the identification of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) on smears from positive blood cultures containing Gram negative rods.

C. Measurand:
E. coli, K. 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:
GNR Traffic Light PNA FISH Culture Identification Kit

G. Regulatory Information:
1. Regulation section:
866.2660
2. Classification:
Class I
3. Product code:
JSS- Kit, Identification, Enterobacteriaceae

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4. Panel:

83- Microbiology

H. Intended Use:

1. Intended use(s):

GNR Traffic Light PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Escherichia coli, and/or Klebsiella pneumoniae, and/or Pseudomonas aeruginosa on smears made from positive blood cultures containing Gram-negative rods observed on Gram stain.

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

GNR Traffic Light PNA FISH is indicated as an aid in the diagnosis of Escherichia coli, and/or Klebsiella pneumoniae, and/or Pseudomonas aeruginosa bacteremia.

2. Indication(s) for use:

GNR Traffic Light PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Escherichia coli, and/or Klebsiella pneumoniae, and/or P. aeruginosa on smears made from positive blood cultures containing Gram-negative rods observed on Gram stain.

Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing.

GNR Traffic Light PNA FISH is indicated as an aid in the diagnosis of Escherichia coli, and/or Klebsiella pneumoniae, and/or P. aeruginosa bacteremia.

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:

GNR Traffic Light PNA FISH is a fluorescence in situ hybridization (FISH) method using PNA probes hybridizing to E. coli, K. pneumoniae and P. aeruginosa specific ribosomal RNA sequences. The test provides rapid (within 90 minutes)

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identification of $E$ coli, $K$ pneumoniae and $P$ aeruginosa on smears made from positive blood cultures.

# J. Substantial Equivalence Information:

1. Predicate device name(s):

EK/P. aeruginosa PNA FISH

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

K092393

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 |
| Interpretation of results | Qualitative Fluorescence microscope | Same |
| Time to result | 1.5 hours | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Function | Identification of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa separately | Identification of Escherichia coli/Klebsiella pneumoniae combined, and Pseudomonas aeruginosa separately |
| Control organisms | Positive Control E. coli, K. pneumoniae P. aeruginosa Negative Control K. oxytoca | Positive Control E. coli, P. aeruginosa Negative Control K. oxytoca |

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

Not applicable

# L. Test Principle:

A mixture of fluorescein-labeled $E.$ coli specific PNA probe and

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tetramethylrhodamine and double fluorescein-labeled $K.$ pneumoniae specific PNA probe and Texas Red labeled, $P.$ aeruginosa specific PNA probe is added to a smear prepared from a positive blood culture. Hybridization is performed at $55^{\circ}\mathrm{C}$ for 30 minutes. The hybridization is followed by a rinse step to remove the cover slip and a post-hybridization wash at $55^{\circ}\mathrm{C}$ for $30\mathrm{min}$ with a stringent Wash Solution to remove unbound PNA probe. The smear is mounted with Mounting Medium and examined by fluorescence microscopy.

$E.$ coli cells yield green fluorescence by specific binding of the fluorescein-labeled PNA probes whereas $P.$ aeruginosa yield red fluorescence by specific binding of the Texas Red-labeled PNA probes; $K.$ pneumoniae cells yield yellow fluorescence by specific binding of the tetramethylrhodamine and double fluorescein-labeled PNA probes.

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

### 1. Analytical performance:

a. Precision/Reproducibility:

The assay was performed on 14 slides (19 isolates) in triplicate on three separate days at three separate sites. Each batch was performed independently by at least two different operators at each site. The reproducibility was $>95\%$.

b. Linearity/assay reportable range:

Not applicable

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

The GNR Traffic Control slide set (i.e. CS001) is:

Positive Control:

E. coli ATCC 35218 Green

P. aeruginosa ATCC 10145 Red

K. pneumoniae ATCC 13882 Yellow

Negative Control:

K. oxytoca ATCC 43086 No fluorescence

All results were as expected.

d. Detection limit:

The detection limit was determined to be approximately $10^{5}$ 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 $10^{5}$ CFU/mL to produce a positive result for the GNR Traffic Light PNA FISH™ assay.

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e. Analytical specificity:

The GNR Traffic Light PNA FISH has been evaluated on a total of 163 microorganisms including 144 Gram-negative strains, 13 Gram-positive organisms, and 6 yeasts. The results demonstrated:

All (19/19) *E. coli* were green-positive

All (20/20) *K. pneumoniae* (including the three subspecies: *pneumoniae*, *ozaenae* and *rhinosclermatis*) were yellow-positive,

All (20/20) *P. aeruginosa* were red-positive

All four *Shigella* spp serogroups (i.e. A, B, C and D), one *Escherichia albertii*, and one *Escherichia fergusonii* cross-reacted, producing a green signal. One *Brevundimonas diminuta*, one *Herbaspirillum huttiense* and one *Acinetobacter radioresistens* cross-reacted, producing a red signal. One *Escherichia vulneris* cross-reacted, producing a yellow signal. The other 75 Gram-negative rods, 13 Gram-positive bacteria and 6 yeast species all yielded negative results.

f. Assay cut-off:

Not applicable

2. Comparison studies:

a. Method comparison of device to conventional methods, as the reference method:

The performance of the GNR Traffic Light PNA FISH™ assay was compared to the conventional culture methods.

b. Matrix comparison:

Not applicable

3. Clinical studies:

A total of 358 prospective samples, and 32 spiked samples were evaluated at four U.S. sites resulting in 395 results. There were five mixed positive cultures.

a. Clinical Sensitivity:

Performance Data for GNR Traffic Light PNA FISH vs. Routine Identification Methods (Combined) on GNR-positive Blood Culture Bottles

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| Study | Sensitivity
E. coli | Sensitivity
K. pneumoniae | Sensitivity
P. aeruginosa | Specificity | Blood Culture System |
| --- | --- | --- | --- | --- | --- |
| A | 35/35 | 17/17 | 7/9^{3} | 35/37^{4} | BACTEC |
| B | 31/31 | 30/30 | 39/39^{6} | 36/36 | BacT/ALERT |
| C | 48/48 | 28/28 | 8/8 | 29/30^{5} | BACTEC |
| D | 21/21 | 2/3^{2} | 8/8^{1} | 15/15 | VersaTREK |
| | 100% (135/135) | 98.7% (77/78) | 96.9% (62/64) | 97.5% (115/118) | |
| Total | 95% CI | 95% CI | 95% CI | 95% CI | N=395 |
| | (97.8-100) | (93.1-100) | (89.2-99.6) | (92.8-99.5) | |

1 Includes 4 samples spiked with *P. aeruginosa* clinical isolates.
2 One *K. pneumoniae* in a mixed culture of *E. coli* and *K. pneumoniae* was negative by PNA FISH and identified following subculturing.
3 Two *P. aeruginosa* in mixed cultures of *P. aeruginosa* and *K. pneumoniae* were negative by PNA FISH and identified following subculturing.
4 One false green positive *E. cloacae* (negative upon re-test) and one false red positive *A. baumannii*
5 One false red positive *Acinetobacter radioresistens* in a mixed culture with *E. faecalis*.
6 Includes 28 samples spiked with *P. aeruginosa* isolates

## b. Clinical specificity:

See table above

## 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. coli*: multiple bright green fluorescent rods in multiple fields
*K. pneumoniae*: multiple bright yellow fluorescent rods in multiple fields
*P. aeruginosa*: multiple bright red fluorescent rods in multiple fields

The expected *E. coli*, *K. pneumoniae*, and *P. aeruginosa* positive rates for Gram negative rod positive blood cultures as determined by the clinical studies are approximately 37%, 22%, and 9%, respectively. Rates may vary depending on institution and patient population.

## 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.