STAPHYLOCOCCUS QUICKFISH BC

K113371 · Advandx, Inc. · NXX · Apr 3, 2012 · Microbiology

Device Facts

• Record ID: K113371
• Device Name: STAPHYLOCOCCUS QUICKFISH BC
• Applicant: Advandx, Inc.
• Product Code: NXX · Microbiology
• Decision Date: Apr 3, 2012
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3700
• Device Class: Class 1

Indications for Use

The Staphylococcus QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Staphylococcus aureus and/or coagulasenegative staphylococci commonly isolated from human blood cultures, on smears prepared from positive blood cultures containing gram-positive cocci in clusters observed on Gram stain. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing, and/or differentiation of mixed growth. Staphylococcus QuickFISH BC is indicated as an aid in the diagnosis of S. aureus bacteremia and/or coagulase-negative staphylococci commonly isolated from human blood cultures.

Device Story

Staphylococcus QuickFISH BC is a qualitative nucleic acid hybridization assay; utilizes fluorescent in situ hybridization (FISH) technology. Input: smears prepared from positive blood cultures containing gram-positive cocci in clusters. Process: multicolor hybridization to identify specific staphylococcal species. Output: visual identification of S. aureus and/or coagulase-negative staphylococci via fluorescence. Used in clinical laboratories by trained personnel. Results aid clinicians in diagnosing bacteremia; facilitates faster identification compared to traditional culture methods; enables earlier targeted therapy. Sub-culturing remains required for susceptibility testing and mixed growth differentiation.

Clinical Evidence

No clinical data provided in the document; bench testing only.

Technological Characteristics

Multicolor, qualitative nucleic acid hybridization assay (FISH). Utilizes fluorescent probes for identification of S. aureus and coagulase-negative staphylococci. Form factor: kit-based reagents for smear preparation and hybridization. Standalone diagnostic test.

Indications for Use

Indicated for identification of S. aureus and/or coagulase-negative staphylococci in human blood cultures for patients with positive blood cultures showing gram-positive cocci in clusters.

Regulatory Classification

Identification

Staphylococcus aureus serological reagents are devices that consist of antigens and antisera used in serological tests to identify enterotoxin (toxin affecting the intestine) producing staphylococci from cultured isolates. The identification aids in the diagnosis of disease caused by this bacterium belonging to the genus Staphylococcus and provides epidemiological information on these diseases. Certain strains of Staphylococcus aureus produce an enterotoxin while growing in meat, dairy, or bakery products. After ingestion, this enterotoxin is absorbed in the gut and causes destruction of the intestinal lining (gastroenteritis).

Submission Summary (Full Text)

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

A. 510(k) Number

k113371

B. Purpose for Submission:

To determine substantial equivalence of the device for the identification of *Staphylococcus aureus* and other *Staphylococci* on smears from positive blood cultures containing Gram positive cocci in clusters.

C. Measurand:

*Staphylococcus aureus*, and other *Staphylococci* species (CoNS) 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:

*Staphylococcus QuickFISH™* BC

G. Regulatory Information:

1. Regulation section:

21 CFR 866.3700

2. Classification:

Class I

3. Product code:

NXX

FISH (Fluorescent *in situ* hybridization) kit, Protein Nuclei Acid, RNA, *Staphylococcus aureus*

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4. Panel:

83 Microbiology

H. Intended Use:

1. Intended use(s):

The Staphylococcus QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Staphylococcus aureus and/or coagulase-negative staphylococci commonly isolated from human blood cultures, on smears prepared from positive blood cultures containing Gram-positive cocci in clusters observed on Gram stain.

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

Staphylococcus QuickFISH BC is indicated as an aid in the diagnosis of S. aureus bacteremia and/or coagulase-negative staphylococci commonly isolated from human blood cultures.

2. Indication(s) for use:

The Staphylococcus QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Staphylococcus aureus and/or coagulase-negative staphylococci commonly isolated from human blood cultures, on smears prepared from positive blood cultures containing Gram-positive cocci in clusters observed on Gram stain.

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

Staphylococcus QuickFISH BC is indicated as an aid in the diagnosis of S. aureus bacteremia and/or coagulase-negative staphylococci commonly isolated from human blood cultures.

3. Special conditions for use statement(s):

Prescription use only

4. Special instrument requirements:

AdvanDx Microscope Dual Band Filter (Cat. No. AC007)

AdvanDx QuickFISH Slides (Cat. No. CS012)

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I. Device Description:

Staphylococcus QuickFISH BC is a fluorescence in situ hybridization (FISH) assay which uses PNA probes hybridizing to S. aureus-specific ribosomal RNA sequences and PNA probes hybridizing to ribosomal RNA of other Staphylococcus species (CoNS). The test provides rapid (20 minutes assay time) identification of S. aureus and CoNS on smears made from positive blood cultures.

J. Substantial Equivalence Information:

1. Predicate device name(s):

S. aureus/CNS PNA FISH

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

k092166

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Technology | Fluorescence In Situ Hybridization (FISH) using protein nucleic acid (PNA) probe | Same |
| Function | Identification of S. aureus and other Staphylococci | Same |
| Sample | Positive blood culture | Same |
| Interpretation of results | Qualitative Fluorescence microscope | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Reagents | Two fixation solutions (QuickFix 1,2), at 55°C | One fixation solution, at room temperature |
| | Two PNA probe solutions (PNA Blue, and PNA Yellow) | One single solution |
| | Wash solution, and mounting fluid not required | Require wash solution and mounting media |
| Control Organisms | Pre-fixed on the same slide as sample | Positive/negative control organisms prepared separately |
| Sample preparation | Secondary vessel required to hold the sample for pipetting 10μL onto a slide | One drop of sample from positive blood culture bottle |
| Hybridization | 15 minutes | 30 minutes |

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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Assay time | 20 minutes | 1.5 hrs |

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

Not applicable

# L. Test Principle:

A mixture of fluorescein-labeled S. aureus specific PNA probe and two Tamra-labeled PNA probes targeting other staphylococci (CoNS) is added to a smear prepared from a positive blood culture.

The fluorophore-labeled PNA probes and quencher-labeled PNA probes are added to a smear prepared from a culture. Hybridization is performed at $55^{\circ}\mathrm{C}$ for 15-20 minutes then the smear is ready for examination by fluorescence microscopy. While maintaining the morphology of the cells, S. aureus and other Staphylococcus species (CoNS) cells become fluorescent by specific binding of the fluorophore-labeled PNA probes. With the Staphylococcus QuickFISH BC, Staphylococcus aureus cells yield green fluorescence whereas CoNS cells yield bright red fluorescence.

The fluorescence microscopy is performed using a dual band microscope filter (AdvanDx, Cat. No. AC007).

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

# 1. Analytical performance:

a. Precision/Reproducibility:

The assay was performed on 14 slides consisting of five S. aureus (pos-green), five CoNS (pos- red), and four non-Staphylococci species (negative), in triplicate on three separate days at three separate sites. Each batch was run 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 QuickFISH slides are provided in individually sealed pouches with nitrogen and a desiccant. Slides must be used immediately after breaking pouch seal and within expiration. Slides are store s at $2 - 8^{\circ}\mathrm{C}$ .

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The QuickFISH microscope slides with controls (i.e. CS012) consist of the following for the Staphylococcus QuickFISH BC:

**Positive Control:**

Staphylococcus aureus ATCC 29213 Green

Staphylococcus lugdunensis ATCC 700328 Red

Staphylococcus epidermidis ATCC 12228 Red

**Negative Control:**

Micrococcus luteus ATCC 10240 No fluorescence

Cryptococcus neoformans ATCC 204092 No fluorescence

Klebsiella oxytoca ATCC 43086 No fluorescence

Streptococcus pyogenes ATCC 12384 No fluorescence

All results were as expected.

**Compatibility Study**

A compatibility study was performed with BacT/Alert (SA, SN), BACTEC (Lytic 10 anaerobic, aerobic plus, anaerobic plus, PEDS Plus, Standard 10 aerobic, Standard anaerobic) and VERSA TREK (REDOX 1 aerobic) on a panel of six S. aureus, seven CoNS, and four negatives. The study demonstrated that compatibility among these bottle types.

The Staphylococcus QuickFISH BC is not compatible with bottles supplemented with charcoal and VERSA TREK Redox-2 anaerobic bottles.

**Fixed Smear Stability (BacT/ALERT SA)**

An in-house study was conducted to determine the stability of fixed slides. There were six S. aureus (green pos), six CoNS (red pos), and four negatives (no fluorescence). Positive and negative controls were also included in the slides.

The data demonstrated that fixed QuickFISH smears were stable on the slide warmer at 55°C for up to 5 minutes, at room temperature for 1 hour, and at 2-8 °C for up to 24 hrs before testing.

**d. Detection limit:**

The detection limit was determined to be approximately 10⁵ CFU/mL by serial dilutions of S. aureus and S. epidermidis cultures from BacT/ALERT blood culture bottles. The average number of colonies per mL (CFU/mL) was calculated from three plates.

The data sets support the LoD of 10⁵ CFU/mL for positive results (green- S. aureus, red- S. epidermidis) with the Staphylococcus QuickFISH BC assay.

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# Co-infection Study

Co- infection studies were performed using BacT/ALERT SA blood culture bottle with sterile human blood. The target organism was held at LoD while the competing organisms (i.e. Enterococcus faecalis, E. coli, Candida albicans) in increased concentrations, which varied and were up to $10^{8}$ , $10^{9}$ , $10^{10}$ , $10^{11}$ CFU/mL. Results demonstrated that the target organisms at LoD were detected in the presence of other competing organisms; both S. aureus and S. epidermidis were detected at and above LoD in co-infection studies.

# Analytical sensitivity

The Staphylococcus QuickFISH BC was tested on 29 S. aureus and 40 Staphylococcus spp that were not S. aureus, including three S. cohnii subspp. cohnii strains. All 29 S. aureus tested were Green-positive and 38 out of the 40 Staphylococcus spp, not S. aureus were Red-positive. The two negative results were S. felis and S. simulans.

The following CoNS were tested in the analytic studies:

| Organism | Strain |
| --- | --- |
| Staphylococcus arlettae | ATCC-43957 |
| Staphylococcus auricularis | ATCC-33753 |
| Staphylococcus capitis | ATCC-27840 |
| Staphylococcus caprae | ATCC-51548 |
| Staphylococcus chromogenes | ATCC-43764 |
| Staphylococcus cohnii | ATCC-29974 |
| Staphylococcus cohnii subsp. cohnii | ATCC 29972 |
| Staphylococcus cohnii subsp. cohnii | ATCC 29973 |
| Staphylococcus cohnii subsp. urealyticus | ATCC 49328 |
| Staphylococcus cohnii subsp. urealyticus | ATCC 49329 |
| Staphylococcus cohnii subsp. urealyticus | ATCC 49330 |
| Staphylococcus cohnii subsp. urealyticus | ATCC 49331 |
| Staphylococcus delphini | ATCC-49171 |
| Staphylococcus epidermidis | ATCC-14990 |
| Staphylococcus epidermidis | ATCC-49461 |
| Staphylococcus epidermidis | ATCC-51625 |
| Staphylococcus equorum | ATCC-43958 |
| Staphylococcus felis | ATCC-49168 |
| Staphylococcus fleurettii | BAA-274 |
| Staphylococcus haemolyticus | ATCC-29970 |
| Staphylococcus hominis | ATCC-27844 |
| Staphylococcus intermedius | ATCC-49052 |
| Staphylococcus kloosii | ATCC-43959 |
| Staphylococcus lentus | ATCC-29070 |

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| Organism | Strain |
| --- | --- |
| Staphylococcus lugdunensis | ATCC-49576 |
| Staphylococcus lutrae | ATCC-700373 |
| Staphylococcus muscae | ATCC-49910 |
| Staphylococcus pasteuri | ATCC-51128 |
| Staphylococcus piscifermentans | ATCC-51136 |
| Staphylococcus pseudintermedius | ATCC 49444 |
| Staphylococcus pulvereri | ATCC-51699 |
| Staphylococcus saccharolyticus | ATCC-14953 |
| Staphylococcus saprophyticus | ATCC-15305 |
| Staphylococcus schleiferi | ATCC-43808 |
| Staphylococcus schleiferi | ATCC-49545 |
| Staphylococcus sciuri | ATCC-29061 |
| Staphylococcus simulans | ATCC-27851 |
| Staphylococcus succinus | ATCC-700337 |
| Staphylococcus warneri | ATCC-49454 |
| Staphylococcus xylosus | ATCC-29971 |

e. Analytical specificity:

The study included ten Gram positive cocci in clusters including Rothia (Stomatococcus) mucilaginosa, twelve yeasts with six strains of C. krusei and 51 strains of bacteria. They all tested negative with the Staphylococcus QuickFISH BC assay. However, two Macrococcus spp, namely M. caseolyticus (formerly Staphylococcus cohnii subspp. cohnii), and M. equipericus (formerly Staphylococcus equipericus) were all formerly Staphylococcus spp.

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 Staphylococcus QuickFISH BC assay was compared to the routine blood culture identification methods.

b. Matrix comparison:

Not applicable

3. Clinical studies:

The performance of Staphylococcus QuickFISH BC versus routine laboratory methods has been assessed in five clinical laboratory studies. A total of 516 routine Gram Positive Cocci in clusters (GPCC) positive blood culture bottles (from 431 patients) and 31 spiked samples were included in the studies. The

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studies showed 99.3% (150/151) positive percent agreement for S. aureus and 98.3% (351/357) for CoNS. The negative percent agreement was 95.6% (45/47) from positive blood culture bottles containing GPCC.

| | S. aureus | CoNS* | Other |
| --- | --- | --- | --- |
| Green
(S. aureus) | 150 | 0 | 1^{4} |
| Red (CoNS) | 1^{1} | 351 | 1^{5} |
| Negative (Non-Staphylococcus spp.) | 0 | 6^{2,3} | 43 |
| Total | Positive Percent Agreement
99.3% (150/151)^{6}
95% CI
(96.4-100) | Positive Percent Agreement
98.3% (351/357)^{6}
95% CI
(96.4-99.4) | Negative Percent Agreement
95.6%
(43/45)
95% CI
(84.9-99.5) |

1 False positive red result, culture ID was S. aureus. Result of retest was green fluorescence.
2 Result of retesting of 2 false negatives was red fluorescence for each.
3 Includes 4 samples identified as S. simulans, a known limitation of the assay.
4 Repeat testing of one false positive (green) was negative. Culture identification was Micrococcus spp.
5 Results of one test (S. aureus by culture ID) were both green and red. Technically a false positive red result; however, the test was correctly positive (green) for S. aureus. Specimen was not available for retesting.
6 Includes five mixed cultures (S. aureus and CoNS) correctly identified as green and red.

*The following CoNS were identified in the clinical studies:

| Organism | Number |
| --- | --- |
| Coagulase negative staphylococci (not further speciated) | 212 |
| S. auricularis | 2 |
| S. capitis | 9 |
| S. caprae | 4 |
| S. epidermidis | 96 |
| S. haemolyticus | 5 |
| S. hominis | 18 |
| S. hyicus^{1} | 1 |
| S. intermedius^{1} | 1 |
| S. lugdunensis | 1 |
| S. saccharolyticus | 1 |
| S. schleiferi | 1 |
| S. simulans | 4 |
| S. warneri | 1 |
| S. xylosus | 1 |
| 1 S. intermedius and S. hyicus are coagulase positive | |

In the clinical studies, the time between routine Gram stain and Staphylococcus QuickFISH BC testing varied for each of the laboratories. Bottles were stored at room temperature after Gram stain and before QuickFISH testing: tested within 2 hours 13% (67/516) of the time, 31% (159/516) within 4 hours and 48% (248/516) within 8 hours. Fifty percent (256/516) of the samples were tested between 8 and 48 hours from Gram stain and 2% (12/516) were greater than 48 hours when tested with QuickFISH. No discrepancies were reported within the

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first 6-hour time frame and only one in less than 8 hours (at 6 ½ hours). The four other discrepancies (not counting S. simulans, a known limitation) occurred at greater than 8 hours.

a. Clinical Sensitivity:
See table above

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:
S. aureus: multiple bright green fluorescent cocci in multiple fields
CoNS: multiple bright red fluorescent cocci in multiple fields
Non-staphylococci are non-fluorescent

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.