mecA XpressFISH® is a qualitative nucleic acid fluorescence in situ hybridization assay intended for the detection of mecA mRNA on smears from blood cultures that are positive for Staphylococcus aureus by the Staphylococcus QuickFISH™ BC assay.

The mecA XpressFISH® assay is indicated for use in conjunction with other laboratory tests and clinical data available to the clinician as an aid in the detection of mecA mRNA from methicillin-resistant S. aureus (MRSA) from patient positive blood cultures. The mecA XpressFISH® assay is not intended to monitor treatment for MRSA infections or for use with mixed cultures including those containing both S. aureus and coagulase negative staphylococci.

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

Device Description

The mecA XpressFISH assay is a qualitative fluorescence in situ hybridization (FISH) assay which utilizes peptide nucleic acid (PNA) probes hybridizing to mecA messenger RNA (mRNA) on smears from blood cultures containing Staphylococcus aureus (SA). After the positive blood culture bottle is confirmed to have SA by Staphylococcus QuickFISH BC, an aliquot of the blood culture is incubated with media containing cefoxitin to induce mecA mRNA expression prior to mecA XpressFISH testing.

The assay consists of mecA mRNA inducing cefoxitin reagents, a fixation reagent, a hybridization reagent, a wash solution concentrate, coverslip mounting media and a microscope slide which has a sample well, and positive and negative control wells. Results are visualized and interpreted using fluorescence microscopy.

AI/ML Overview

Here's an analysis of the provided text, extracting the acceptance criteria and study details for the mecA XpressFISH device:

The document describes the mecA XpressFISH assay, a qualitative nucleic acid fluorescence in situ hybridization assay for the detection of mecA mRNA in Staphylococcus aureus (SA) positive blood cultures. The goal is to identify methicillin-resistant S. aureus (MRSA).

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied through the comparability with the predicate device (BinaxNOW PBP2a) and the detailed validation studies demonstrating high agreement, sensitivity, and specificity. Explicit, numerical acceptance criteria for each bench test are not always stated, but the results consistently show 100% agreement or very high percentages, indicating successful performance against internal or industry-standard benchmarks.

Acceptance Criteria (Implied/Derived)	Reported Device Performance
Clinical Performance:
High Agreement with Predicate Device (Cefoxitin Disk Diffusion) for MRSA Identification	99.1% (336/339) overall agreement with cefoxitin disk diffusion in clinical studies.
High Sensitivity for MRSA Detection	98.7% (151/153), 95% CI (95.4-99.6)
High Specificity for MRSA Detection	99.5% (185/186), 95% CI (97.0-99.9)
Positive Predictive Value (PPV)	99.3% (151/152), 95% CI (96.4-99.9)
Negative Predictive Value (NPV)	98.9% (185/187), 95% CI (96.2-99.7)
Bench Testing (Functional Requirements):
Limit of Detection (LoD) comparable to other slide-based tests	Lowest readable concentration for MRSA strains: 8.1x10^5 - 2.8x10^6 CFU/mL (comparable to Staphylococcus QuickFISH and Gram Stain).
Analytic Reactivity (Inclusivity) for MRSA strains	63/64 MRSA strains produced Green Positive results (1 strain known not to carry mecA did not).
Analytic Specificity (Exclusivity) against non-MRSA organisms	29/30 MSSA produced Negative results (1 mecA+ MSSA gave Green Positive). 34/40 other CoNS produced Negative results (6 produced Green Positive). 36/37 other Gram-positive were Negative (Lactococcus lactis Green Positive). 18/19 other clinically relevant species were Negative (Candida parapsilosis Green Positive).
No significant interference from co-infecting species	No False Positive results from methicillin-sensitive staphylococci; no interference observed in MRSA detection.
Robustness against various interfering substances	All MRSA tests produced Positive results (except weak positive with Linezolid, noted as limitation). All MSSA strains showed Negative results. Overall acceptable performance.
Compatibility with common blood culture media	100% agreement for all 9 tested media types (MRSA Positive, MSSA Negative).
Absence of cross-contamination during wash step	100% of MRSA samples Green Positive, 100% of MSSA samples Negative; no Green Positive in MSSA wells.
Effective Cefoxitin concentration range for mecA induction	100% of 20 MRSA strains Positive (60/60); 100% of 3 MSSA strains Negative (9/9) across concentrations 2.5, 6.1, and 15 µg/mL.
Effective Cefoxitin induction time range	100% of 20 MRSA strains Positive (60/60); 100% of 3 MSSA strains Negative (9/9) across 40, 45, 50 minutes.
Tolerance for Induction to Slide Preparation Interval	100% of 20 MRSA strains Positive (60/60); 100% of 3 MSSA strains Negative (9/9) across immediate, 30 min, 60 min delays.
Stability of Fixed Slides	100% of 158 MRSA strains Positive; 100% of 23 MSSA strains Negative across various storage conditions and times.
Effective Hybridization Time Range	100% of 20 MRSA strains Positive (60/60); 100% of 3 MSSA strains Negative (9/9) across 10, 15, 20 minutes.
Effective Slide Wash Time Range	100% of 20 MRSA strains Positive (60/60); 100% of 3 MSSA strains Negative (9/9) across 10, 15, 20 minutes.
Performance maintained across age of blood culture	100% of 20 MRSA strains Positive (80/80); 100% of 3 MSSA strains Negative (12/12) across bottle ring to ~60 hours. (One MRSA strain showed weak signal beyond 6-12 hr).
Reproducibility of results (intra-run, inter-run, inter-site, inter-operator)	100% Positive Green Agreement (120/120) and 100% Negative Agreement (60/60) across 3 sites, 2 operators, 5 days, and two different concentrations for 2 MRSA and 1 MSSA strain.

2. Sample Sizes Used for the Test Set and Data Provenance

Clinical Study Test Set Sample Size: 339 routine blood culture bottles that were positive for Staphylococcus aureus by Staphylococcus QuickFISH BC.
Data Provenance: The clinical study was conducted at seven clinical sites (6 in the US and 1 ex-US). This indicates a multi-center, prospective clinical study collecting real-world samples.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The ground truth for the clinical study was established using cefoxitin disk diffusion, which is a widely accepted laboratory method for determining methicillin resistance in clinical microbiology. The document does not specify the number or qualifications of experts who performed or interpreted the cefoxitin disk diffusion tests. It is assumed these were performed by trained laboratory personnel following standard clinical microbiology guidelines.

4. Adjudication Method for the Test Set

The document does not explicitly describe an adjudication method for the test set in the clinical study beyond stating the comparison was made against cefoxitin disk diffusion. Discrepant results are noted and briefly analyzed (e.g., "False Positive mecA (weak Green Positive); Negative upon repeat testing. Cefoxitin disk=28 mm."), suggesting individual review of such cases, but a formal adjudication process (e.g., 2+1, 3+1 consensus) is not detailed.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a multi-reader multi-case (MRMC) comparative effectiveness study comparing human readers with AI assistance versus human readers without AI assistance was not performed or described in this document.
The device is a diagnostic assay interpreted visually by a single operator, not an AI-assisted diagnostic.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

The mecA XpressFISH assay is a standalone diagnostic assay in the context that its results are interpreted by an operator viewing fluorescence microscopy images. It is not an "algorithm only" device in the AI sense.
The "standalone" performance here refers to the direct output of the assay itself, interpreted by a human, without further AI algorithms altering or assisting that interpretation. The clinical study precisely evaluates this standalone performance.

7. Type of Ground Truth Used

Clinical Study Ground Truth: Cefoxitin Disk Diffusion for identification of methicillin-resistant S. aureus (MRSA). This is a well-established phenotypic method used in clinical microbiology as a standard of care for resistance testing.
Bench Testing Ground Truth: Laboratory-characterized strains (e.g., ATCC strains, well-characterized clinical isolates) with known mecA gene status or methicillin resistance phenotypes were used for inclusivity, exclusivity, LOD, and reproducibility studies.

8. Sample Size for the Training Set

The document does not specify a distinct "training set" in the context of machine learning.
This device is a traditional in vitro diagnostic (IVD) assay, not an AI/ML-based device that would typically have a separate training set. Development and internal bench testing used various characterized strains, but these do not constitute a "training set" in the AI sense.

9. How the Ground Truth for the Training Set Was Established

As there isn't a "training set" in the AI sense, this question isn't directly applicable.
For the development and bench marking activities, ground truth was established through:
- Using reference strains (e.g., ATCC strains with known characteristics).
- Characterization of clinical isolates using standard microbiological methods to determine their mecA status or methicillin resistance profile.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

ADVANDX, INC. RON EISENWINTER DIRECTOR RA/CA 400 TRADECENTER SUITE 6990 WOBURN, MA 01801

October 10, 2014

Re: K140619

Trade/Device Name: mecA XpressFISH Regulation Number: 21 CFR 866.1640 Regulation Name: Antimicrobial susceptibility test powder Regulatory Class: II Product Code: MYI Dated: September 8, 2014 Received: September 9, 2014

Dear Mr. Eisenwinter:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21. Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours.

Uwe Scherf -S for

Sally Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K140619

Device Name mecA XpressFISH

Indications for Use (Describe)

The mecA XpressFISH® assay is indicated for use in conjunction with other laboratory tests and clinical data available to the clinician as an aid in the detection of mecA mRNA from methicillin-resistant S. aureus (MRSA) from patient positive blood cultures. The mecA XpressFlSH® assay is not intended to monitor treatment for MRSA infections or for use with mixed cultures including those containing both S. aureus and coagulase negative staphylococci.

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

Type of Use (Select one or both, as applicable)

2 Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

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FOR FDA USE ONLY

Concurrence of Center for Devices and Radiological Health (CDRH) (Signature)

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510(k) Summary

510(k) Number: K140619

Applicant

AdvanDx, Inc. 400 TradeCenter, Suite 6990 Woburn, MA 01801

Contact Information:

Ronald K. Eisenwinter Director, Regulatory and Clinical Affairs AdvanDx, Inc. 400 TradeCenter, Suite 6990 Woburn, MA 01801 781-376-0009 extension 1027 Phone: Fax: 781-376-0111 E-mail: rke@advandx.com

Establishment Registration Number

3004080598

Manufacturer Establishment Number:

3003994627

Device Trade Name:

mecA XpressFISH (formerly mecA PNA FISH)

Device Common Name:

System, Test, Genotypic Detection, Resistant Markers, Staphylococcus Colonies

Device Classification:

AdvanDx mecA XpressFISH is a Class II device and is classified by FDA under 21 CFR §866.1640. Antimicrobial Susceptibility Test Powder. The Product Code is MYI.

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Intended Use and Indications For Use

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

Special conditions for use statement(s)

For Prescription Use only.

Special instrument requirements

Fluorescence microscope with appropriate filter and light source.

Device Description

Substantial Equivalence:

For the identification of mecA-mediated methicillin (Oxacillin) resistance in S. aureus, the mecA XpressFISH assay is substantially equivalent to the BinaxNOW PBP2a test (Alere, Maine, K090301), which detects the PBP2a protein responsible for methicillin resistance.

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Similarities
Item	mecA XpressFISHK140619	BinaxNOW PBP2aK090301
IntendedUse	mecA XpressFISH ® is aqualitative nucleic acidfluorescence in situhybridization assay intended forthe detection of mecA mRNAon smears from blood culturesthat are positive forStaphylococcus aureus by theStaphylococcus QuickFISH ™BC assay.The mecA XpressFISH ® assayis indicated for use inconjunction with otherlaboratory tests and clinicaldata available to the clinicianas an aid in the detection ofmecA mRNA from methicillin-resistant S. aureus (MRSA)from patient positive bloodcultures. The mecAXpressFISH ® assay is notintended to monitor treatmentfor MRSA infections or for usewith mixed cultures includingthose containing both S. aureusand coagulase negativestaphylococci.Sub-culturing of positive bloodcultures is necessary to recoverorganisms for susceptibilitytesting, epidemiological typing,and/or differentiation of mixedgrowth.	The BinaxNOW® PBP2a Testis a qualitative, in vitroimmunochromatographic assayfor the rapid detection ofpenicillin-binding protein 2a(PBP2a) present in methicillin-resistant Staphylococcusaureus (MRSA). The test isperformed directly on bloodculture samples positive for S.aureus .The BinaxNOW PBP2a Test isnot intended to diagnoseMRSA nor to guide or monitortreatment for MRSA infections.Subculturing positive bloodcultures is necessary to recoverorganisms for susceptibilitytesting or epidemiologicaltyping.
Sample Type	S. aureus positive bloodcultures	Same
TestPrinciple	Detection of mecA geneexpression	Same
Method ofResult	Visual	Same

Comparison Tables of Similarities and Differences

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Similarities
Item	mecA XpressFISHK140619	BinaxNOW PBP2aK090301
Interpretation
Mode ofOperation	Manual	Same

Differences
Item	mecA XpressFISHK140619	BinaxNOW PBP2aK090301
TargetAnalyte	mecA mRNA	PBP2a protein
Test Method	Molecular detection of geneexpression	Phenotypic detection of geneexpression
Technology	In situ hybridization offluorescently labeled peptidenucleic acid (PNA) probe	Immunochromatography withmonoclonal antibodies that bindto the penicillin binding proteinPBP2a
SamplePreparation	Cefoxitin induced expression ofmecA expression	Recovery and washing of cellsby centrifugation, followed bylysis
Controls	Integrated Positive andNegative Controls to monitorhybridization, washing andresult interpretation	Separate preparation of teststrip(s) for control organisms

Bench Testing

AdvanDx has conducted in-house bench testing of the mecA XpressFISH assay. The types of testing included limit of detection, analytic inclusivity, cross-reactivity, microbial interference, interfering substances (including different blood culture media), crosscontamination, tolerance, and reproducibility. Additionally, tests were conducted to validate the timing of each step of the testing procedure as well as the storage conditions of prepared slides.

The results of this testing verify that for the detection of mecA-mediated methicillin resistance in S. aureus in blood cultures, mecA XpressFISH is substantially equivalent to the BinaxNOW PBP2a predicate device.

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Limit of Detection ("LoD")

Three different MRSA strains were used for the LoD study, MRSA ATCC 33591, ATCC 43300, and NRS383. Organisms were grown under simulated blood culture conditions.

The Limit of Detection was measured by serially diluting the MRSA strains in quadruplicate. The lowest readable concentration for each strain was re-tested in replicates of 20 and found to be 8.1x10 - 2.8x10 CFU/mL. This LoD is comparable to that of Staphylococcus QuickFISH and other slide based microbiological tests such as Gram Stain.

Analytic Reactivity (Inclusivity) and Analytic Specificity (Exclusivity)

The panel of organisms used to evaluate the analytical inclusivity (reactivity) of the mecA XpressFISH assay was comprised of 64 MRSA strains representing a broad spectrum of genotypes, clonal complexes, isolation dates, and locations. Of the 64 MRSA strains tested, only one, which is known not to carry the mecA gene, did not produce Green Positive results (mecC type).

For exclusivity (specificity), the panel included 30 strains of MSSA, 40 strains of other Staphylococcus species (i.e., coagulase-negative staphylococci [CoNS]), 36 strains of other gram-positive organisms, and 20 other clinically relevant species (i.e. gramnegatives and yeasts). Organisms were grown under simulated blood culture conditions. 29 of 30 MSSA produced Negative mecA XpressFISH results, while 1 MSSA, known to be mecA+, gave a Green Positive result. Six of the 40 other CoNS produced Green Positive results while the remaining 34 were Negative. Of the 37 other Gram positive organisms. 36 tested Negative. Lactococcus lactis was Green Positive. Subsequent additional analytical testing also produced weak Green Positive fluorescence with Micrococcus luteus. One of the 19 other orqanisms tested (Candida parapsilosis) produced a Green Positive result, while the other 18 were Negative. Because mecA XpressFISH is only indicated for use with blood cultures that contain Gram Positive Cocci in Clusters and which test Positive for S. aureus alone by Staphylococcus QuickFISH BC (i.e., pure cultures of S. aureus), the risk of False Positive results due to cross-reaction with other species is considered low.

Co-Infection

mecA XpressFISH is intended to be used with blood cultures of S. aureus as determined by Staphylococcus QuickFISH BC and is not intended to be used with mixed cultures identified either by Gram stain or Staphylococcus QuickFISH BC. Because of the inherent analytic sensitivity of slide based microbial tests and the variable rate of growth of microbes, there exists a possibility that a blood culture which appears to be positive solely for S. aureus by Gram stain and Staphylococcus QuickFISH BC is in fact a mixed culture (i.e., S. aureus and another species). In addition, should MRSA and MSSA occur together in the same blood culture, the culture would not be distinguishable as a mixed culture by either Gram stain or Staphylococcus QuickFISH BC. A microbial interference study was therefore conducted in order to

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evaluate whether the presence of a non-MRSA organism would interfere with the detection of MRSA by mecA XpressFISH.

Testing was performed with simulated blood cultures to determine the impact of mixed populations of MRSA and MSSA or MRSA and coaqulase negative staphylococci on the mecA XpressFISH assay. Two strains of MRSA were tested in the presence of different "co-infecting" species of methicillin-sensitive staphylococci: S. aureus, S. epidermidis and S. simulans. The strains of MRSA were both tested near the previously determined LoD of the assay, which is ~10° CFU/mL. The other organisms were all tested at about 10° CFU/mL. No False Positive results were obtained from the methicillin-sensitive staphylococci and no interference was observed in detection of MRSA in the presence of the co-infecting species.

Interfering Substances

Testing of potential interfering substances was performed at the concentrations shown in the following Table. Two MRSA strains. ATC 43300 (MRSA 1) and NRS 674 (MRSA 2), and two MSSA strains, ATCC 29213 (MSSA 1) and ATCC 11632 (MSSA 2), were tested. All tests with MRSA produced Positive results, although only weak fluorescence was observed in the presence of Linezolid. Testing of samples from patients treated with Linezolid could therefore produce False Negative results. This is reflected as a limitation in the mecA XpressFISH Package Insert.

All MSSA strains showed Negative results.

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Substance	Concentra-tion	MRSA 1	MRSA 2	MSSA 1	MSSA 2
Amoxicillin	12 µg/mL	Pos.	Pos.	Neg.	Neg.
Clavulanate	3 µg/mL	Pos.	Pos.	Neg.	Neg.
Sulbactam	45 µg/mL	Pos.	Pos.	Neg.	Neg.
Ampicillin	120 µg/mL	Pos.	Pos.	Neg.	Neg.
Clindamycin	10 µg/mL	Pos.	Pos.	Neg.	Neg.
Daptomycin	130 µg/mL	Pos.	Pos.	Neg.	Neg.
Ibuprofen	50 µg/mL	Pos.	Pos.	Neg.	Neg.
Linezolid	38 µg/mL	Weak Pos.	Weak Pos.	Neg.	Neg.
Oxacillin	230 µg/mL	Pos.	Pos.	Neg.	Neg.
Vancomycin	50 µg/mL	Pos.	Pos.	Neg.	Neg.
Amoxicillin andClavulanate	12 µg/mL3 µg/mL	Pos.	Pos.	Neg.	Neg.
Sulbactam andAmpicillin	45 µg/mL120 µg/mL	Pos.	Pos.	Neg.	Neg.
Bilirubin	150 µg/mL	Pos.	Pos.	Neg.	Neg.
Hemoglobin	~30 mg/mL	Pos.	Pos.	Neg.	Neg.
Triglycerides	3500 µg/mL	Pos.	Pos.	Neg.	Neg.
SPS	0.04%	Pos.	Pos.	Neg.	Neg.
Saponin	~0.2%	Pos.	Pos.	Neg.	Neg.

Interference Testing Results

Compatible Blood Culture Media

An in-house study was conducted to assess the compatibility of mecA XpressFISH with certain of the most commonly used blood culture media of the three predominant blood culture systems. Testing was performed using multiple clinical strains of S. aureus representing a wide range of geographic and genotypic strain diversity.

In all, there were nine different blood culture media bottles tested. mecA XpressFISH testing for each MRSA produced a Positive result for all media types and each MSSA produced a Negative result for all media types, with an overall agreement of 100%. It is concluded that mecA XpressFISH is compatible with the nine commonly utilized blood culture media that were tested.

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Manufacturer	Blood Culture System	Type of Media Bottle	Catalog Number
Becton Dickinson	BD BACTEC™	Plus Aerobic/F	442192
		Plus Anaerobic/F*	442193
		Standard/10 Aerobic/F*	442260
		Standard Anaerobic/F*	442191
		Lytic/10 Anaerobic/F	442265
		Peds Plus*	442194
bioMérieux	BacT/ALERT®	SA-Standard Aerobic	259789
		SN-StandardAnaerobic*	259790

Thermo Scientific	VersaTREK	REDOX 1 aerobic*	7102-44

Blood Culture Media Types Tested

*Clinical performance with mecA XpressFISH not established.

Cross-contamination (Wash Step)

According to the instructions for use, mecA XpressFISH slides can be washed in batches of up to five slides following the hybridization step. This is the only step in the mecA XpressFISH procedure where slides prepared from different samples (patients) are present and exposed together to a common reagent and where they might become cross-contaminated. AdvanDx conducted an in-house study with the mecA XpressFISH assay to assess the potential for cross-contamination during the wash step leading to False Positive results.

A well characterized mecA positive MRSA strain (NRS 383) and a strain of MSSA (ATCC BAA 1718) were used for testing. For each run, a set of four MRSA slides and one MSSA slide was prepared from simulated blood cultures at >10° CFU/mL. Three sets of five slides were processed through the hybridization step according to the mecA XpressFISH procedure and each slide set was then placed into a slide holder for the wash step. The placement of the MSSA slide relative to the four MRSA slides within the holder was varied between sets (i.e., the MSSA slide within a set was placed at a different middle or outside position relative to the four MRSA slides within the holder). The slide sets were each then washed according to the mecA XpressFISH procedure. and the slides were mounted and examined by fluorescence microscopy. All MRSA samples were Green Positive (12/12, 100%) and all MSSA samples were Negative (3/3, 100%) with no Green Positive organisms found in the MSSA sample wells (or Internal Negative Control wells). The results verify that cross-contamination of slides is unlikely to occur when up to five slides are washed together.

Cefoxitin Concentration

It was established during the mecA XpressFISH product development that a cefoxitin concentration of between 2.5 and 15 ug/mL in TSB is optimal for induction of the mecA gene within 40-50 min. at 35°C. AdvanDx conducted an in-house study to verify the functional range of cefoxitin concentration in TSB for the mecA XpressFISH assay. A

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panel of 20 diverse MRSA and three MSSA strains were grown under simulated blood culture conditions. The mecA XpressFISH assay was then conducted with each sample where the cefoxitin concentration in TSB was set at three different levels: 2.5ug/mL, 6.1µg/mL and 15µg/mL. All 20 of the MRSA tested under the three cefoxitin concentrations for the induction step produced Positive mecA XpressFISH test results (60/60; 100%) and all three MSSA produced Negative test results (9/9, 100%). The results verify that a cefoxitin concentration range of 2.5-15 ug/mL is suitable for induction of mecA mRNA expression.

Cefoxitin Induction Time Range

Exposure of MRSA to cefoxitin (or other methicillin-type drugs) induces the production of mecA mRNA. As part of the product development for mecA XpressFISH, it was determined that 40 to 50 minutes of drug exposure is required to activate mecA gene transcription for MRSA strains that do not express (or only weakly express) mecA mRNA in the absence of drug exposure. In order to verify this time range, AdvanDx conducted an in-house study with the mecA XpressFISH assay and a panel of wellcharacterized MRSA strains to assess test performance within the prescribed time range. A panel of 20 MRSA and three MSSA were tested at each of three time points: 40, 45 and 50 minutes. All 20 of the MRSA gave a Positive result for mecA XpressFISH at each of the three time points tested (60/60, 100%). All three of the MSSA produced a Negative result for each time point (9/9, 100%). The results verify that a 40-50 min. induction time is suitable for induction of mecA mRNA.

Induction to Slide Preparation Interval

During the design of mecA XpressFISH it was determined that users wished to have flexibility in the timing between completion of the cefoxitin induction step and preparation of smears from the induced culture. AdvanDx conducted an in-house study to assess whether a delay of up to one hour between induction and slide preparation would affect the performance of mecA XpressFISH. A panel of 20 MRSA and 3 MSSA were grown under simulated blood culture conditions. Following the induction step. slides were prepared immediately and then at 30 and 60 minutes. Induced cultures were stored at room temperature prior to slide preparation. All 20 of the MRSA gave a Positive test result for mecA XpressFISH at the three time points tested (60/60, 100%) and the three MSSA produced Negative results (9/9, 100%). The results verify that slides may be prepared for up to one hour after the completion of the induction step.

Fixed Slide Stability Testing

During the design of the mecA XpressFISH assay it was determined that users wished to have flexibility in the timing between completion step and the start of the hybridization step of the assay procedure. According to the instructions for use, fixed slides may be stored under the following conditions prior to the hybridization step:

. Store mecA XpressFISH control slides in their original, individually sealed pouches at 2-8°C. Slides must be used immediately after breaking pouch seal. Do not use slides after the expiration date.

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. Fixed mecA XpressFISH smears should be hybridized within 5 minutes following fixation and may be left on the slide warmer at 55 ± 1°C for up to 5 minutes before addition of the hybridization reagent (mecA PNA).
- Prepared smears may be stored at room temperature for up to 1 hour prior to testing or o may be stored at 2-8 °C for up to 24 hours before testing.
Note: If smears were stored after fixation at 2-8°C or room temperature they must be placed on . the slide warmer for approximately 5 minutes at 55 ± 1℃ before adding the hybridization reagents.

AdvanDx conducted an in-house study of the mecA XpressFISH assay to assess test performance within the prescribed fixed slide storage times and conditions. Multiple clinically relevant strains of S. aureus were tested under the described conditions. All MRSA tested under the various storage conditions and times produced Positive mecA XpressFISH test results (158/158; 100%) and all three MSSA produced Negative test results (23/23, 100%). The results verify that following the fixation step, slides may be stored and processed according to the instructions for use, without affecting test performance.

Hybridization Time Range

During the design of the mecA XpressFISH assay, it was determined that users would need to have flexibility in the duration of the hybridization step in order to accommodate batch processing of small numbers of slides and for other reasons. According to the instructions for use, the hybridization for mecA XpressFISH can be performed within 10 to 20 minutes. AdvanDx conducted an in-house study to assess test performance within this prescribed time range. A panel of 20 MRSA and three MSSA were tested at each of three time points (10, 15, and 20 minutes of hybridization). The twenty MRSA gave a Positive test result for mecA XpressFISH at the three time points tested (60/60. 100%) and the three MSSA produced Negative test results (9/9, 100%). The results verify that the hybridization time of between 10 and 20 minutes is suitable.

Slide Wash Time Range

According to the instructions for use, mecA XpressFISH slides may be washed for between 10 to 20 minutes at 57°C following the hybridization step. An AdvanDx inhouse study was conducted with the mecA XpressFISH assay to assess test performance within this prescribed time range. A panel of 20 MRSA and three MSSA were tested at each of three time points: 10, 15, and 20 minutes. The twenty MRSA gave a Positive test result for mecA XpressFISH at the three time points tested (60/60, 100%) and the three MSSA produced Negative test results (9/9, 100%). The results verify that a slide wash time of between 10 and 20 minutes is suitable.

Age of Blood Culture at mecA XpressFISH Testing

ldeally in the clinical setting, mecA XpressFISH should be performed as soon as possible after blood culture bottle positivity and the completion of Staphylococcus QuickFISH BC testing. However, the elapsed time from blood culture positivity to mecA XpressFISH testing could vary from lab to lab, depending on such factors as laboratory staffing, reporting policies, and time of day at which bottles signal Positive. Hence, the age of a Positive blood culture bottle at the time of mecA XpressFISH testing could be

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as little as < 1 hour to greater than 48 hours. AdvanDx conducted an in-house bridging study to assess the performance of the test on blood cultures ranging from the time of bottle positivity to ~60 hours of age. A panel of 20 MRSA and three MSSA was used for testing and mecA XpressFISH testing was performed at each of four time points: "Bottle Ring" (≤ 2 hrs.) . "Bottle Ring" + 6-12 hours, "Bottle Ring" + 24-28 hours and "Bottle Ring" + 54-64 hours. All of the 20 MRSA gave a Green Positive result for mecA XpressFISH at all four time points tested (80/80, 100%). One strain, ATCC 700699, produced a weak (but detectable) Green Positive signal beyond the 6-12 hr. testing time. The three MSSA all produced Negative test results (12/12, 100%). The results verify that bottles may be tested up to 60 hours following positivity (Bottle Ring) without affecting the performance of mecA XpressFISH.

Time to Viewing Results

According to the Package Insert, mounted slides prepared using the mecA XpressFISH assay may be viewed for interpretation up to two hours after completion of the assay. An in-house study was conducted to assess the ability to interpret results within this prescribed time range. A panel of 20 MRSA and three MSSA was grown under simulated blood culture conditions and samples were processed with the mecA XpressFISH assay as per the instructions for use. Slides were interpreted immediately following the procedure, one hour after the procedure, two hours after the procedure and 18-24 hours after the procedure. The 20 MRSA gave a Positive test result for mecA XpressFISH at the four viewing time points (80/80, 100%) with some reduction in signal observed for some strains at the 18-24 hour time point. The three MSSA produced Negative test results for all time points (12/12, 100%). The results verify that viewing slides up to two hours following completion of the assay does not affect the performance of the mecA XpressFISH assay.

Tolerance Testing

The critical steps of the mecA XpressFISH assay were tested at different temperature ranges with a specified organism panel. The lower and upper inner limits of the required incubation temperatures, along with lower and upper outer limits of the incubation temperatures, were tested for the induction, hybridization, and wash steps. 20 MRSA, 3 MSSA, and 1 M. luteus were tested for these conditions.

All MRSA tested were Green Positive with mecA XpressFISH, reqardless of the tolerance conditions. All MSSA tested were Negative with mecA XpressFISH, regardless of the tolerance conditions. Throughout the development of the mecA XpressFISH assay, it has been noticed that Micrococcus luteus ATCC 49732 has exhibited sporadic green auto-fluorescence. Micrococcus luteus synthesizes weakly fluorescent chromophores as part of its normal physiology, as may be observed in the yellow pigmentation of colonies grown in TBS agar. Weak Green Positive results were observed with M. luteus, even with the low and high temperature conditions that are within tolerance. False Positive results may therefore be obtained with M. luteus. This is reflected as a Limitation in the mecA XpressFISH Package Insert.

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Reproducibilitv

A reproducibility study was performed with mecA XpressFISH. Testing was performed in triplicate by two operators (per site) over five days.* The test panel consisted of two strains of MRSA and one of MSSA grown to two different concentrations. Operators were blinded to the identification of all samples. The results are presented below by site across 5 days and by five days across three sites.

Strain	Strain ID	Set 1 InoculumCFU/Bottle	Set 1 CFU/mL atBottle Ring	Set 2 InoculumCFU/Bottle	Set 2 CFU/mL atBottle Ring + 8Hours
MRSA	NRS 674	128	$4.5 x 10^7$	75	$9.6 x 10^8$
MRSA	ATCC 43300	59	$3.4 x 10^7$	64	$1.1 x 10^8$
MSSA	ATCC 29213	80	$7.7 x 10^7$	122	$2.8 x 10^7$

Sample Matrix for mecA XpressFISH Reproducibility

mecA XpressFISH Reproducibility Results by Site for 5 Test Days

	Site 1	Site 2	Site 3	Total
Positive GreenAgreement	40/40	40/40	40/40	100%120/120
NegativeAgreement	20/20	20/20	20/20	100%60/60
Total Agreement	100%60/60	100%60/60	100%60/60	100%180/180

mecA XpressFISH Reproducibility Results by Day for 3 study Sites

	Day 1	Day 2	Day 3	Day 4	Day 5	Total
Positive GreenAgreement	24/24	24/24	24/24	24/24	24/24	100%120/120
NegativeAgreement	12/12	12/12	12/12	12/12	12/12	100%60/60
Total Agreement	100%36/36	100%36/36	100%36/36	100%36/36	100%36/36	100%180/180

Note: This study was repeated because of several False Positive results obtained by one operator on one day at one site, the root cause for which could not be established. Only results from the repeat study are shown.

Clinical Studies

The performance of the mecA XpressFISH assay compared to cefoxitin disk diffusion for identification of methicillin-resistant SA (MRSA) was determined in seven clinical sites (6 US and 1 ex-US). A total of 339 routine blood culture bottles that were determined to be positive for only Staphylococcus aureus by Staphylococcus QuickFISH BC were included in the study. The results showed 99.1% (336/339) agreement between mecA XpressFISH and cefoxitin disk diffusion for identification of MRSA. The sensitivity and specificity were determined to be 98.7% and 99.5%.

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respectively. The performance of mecA XpressFISH versus cefoxitin disk diffusion is presented in the following Table:

mecA XpressFISH Performance vs. Cefoxitin Disk Diffusion
	Cefoxitin Disk Diffusion
	Methicillin Resistant(≤21mm)	Methicillin Susceptible(≥ 22mm)
mecA XpressFISHPositive	151	11	PPV99.3%(151/152)95% CI(96.4-99.9)
mecA XpressFISHNegative	22	185	NPV98.9%(185/187)95% CI96.2-99.7
N=339	Sensitivity98.7% (151/153)95%CI(95.4-99.6)	Specificity99.5% (185/186)95% CI(97.0-99.9)

False Positive mecA (weak Green Positive); Negative upon repeat testing. Cefoxitin disk=28 mm. 2Two False Negative mecA: For one, repeat testing was Negative, and for the other it was Positive (weak Green Positive). Cefoxitin disk diffusion results were19mm and 11mm, respectively.

An analysis of the results of the clinical study by site and type of culture medium is presented in the table below.

Bottle Type	Site	MethicillinResistant(mecA+)	MethicillinSusceptible(mecA-)	Total(%; 95% CI)
BACTEC Plus Aerobic/F	A	2/2	12/12	14/14(100)
	B	18/19	17/17	35/36(97.2)
	C	7/7	18/19	25/26(96.2)
	D	7/7	14/14	21/21(100)
	E	19/19	19/19	38/38(100)
	Total(%; 95% CI)	53/54(98.1; 90.2-99.7)	80/81(98.8; 93.3-99.8)	133/135(98.5; 94.8-99.6)
BACTEC Lytic/10Anaerobic/F	A	7/7	10/10	17/17(100)
	B	10/10	17/17	27/27(100)
	C	10/10	14/14	24/24(100)
	D	19/19	4/4	23/23(100)
	E	13/14	13/13	26/27(96.3)
Total(%; 95% CI)	59/60(98.3; 91.1-99.7)	58/58(100; 93.8-100)	117/118(99.2; 95.4-99.9)

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Bottle Type	Site	MethicillinResistant(mecA+)	MethicillinSusceptible(mecA-)	Total(%; 95% CI)
BACTEC Peds Plus	B	1/1	1/1	2/2(100)
	C	2/2	5/5	7/7(100)
	D	0	1/1	1/1(100)
	Total(%; 95% CI)	3/3(100; 43.9-100)	7/7(100; 64.6-100)	10/10(100; 72.3-100)
BACTEC PlusAnaerobic/F	B [Total](%; 95% CI)	7/7(100; 64.6-100)	5/5(100; 56.6-100)	12/12(100; 75.8-100)
BACTEC Standard/10Aerobic/F	D [Total](%; 95% CI)	0	1/1(100; 20.7-100)	1/1(100; 20.7-100)
	Total(%; 95% CI)	122/124(98.4; 94.3-99.6)	151/152(99.3; 96.4-99.9)	273/276(98.9; 96.9-99.6)
BACTEC All Media	F	24/24	18/18	42/42(100)
	G	1/1	8/8	9/9(100)
		Total(%; 95% CI)	25/25(100; 86.7-100)	26/26(100; 87.1-100)
BacT/ALERTSA Standard Aerobic	F	4/4	2/2	6/6(100)
	G	0	6/6	6/6(100)
		Total(%; 95% CI)	4/4(100; 51.0-100)	8/8(100; 67.6-100)
BacT/ALERTSN Standard Anaerobic	Total(%; 95% CI)	29/29(100; 88.3-100)	34/34(100; 89.9-100)	63/63(100; 94.2-100)
BacT/ALERT All Media	Total(%; 95% CI)	29/29(100; 88.3-100)	34/34(100; 89.9-100)	63/63(100; 94.2-100)

Conclusion

It is concluded from the data presented above, that the identification of mecA-mediated methicillin resistance in Staphylococcus aureus by the mecA XpressFISH assay from S. aureus positive blood culture bottles as identified by Staphylococcus QuickFISH BC is substantially equivalent to identification a previously cleared predicate method and does not raise new types of safety or effectiveness questions when used as labelled.

Regulation Number and Section

§ 866.1640 Antimicrobial susceptibility test powder.

(a)
Identification. An antimicrobial susceptibility test powder is a device that consists of an antimicrobial drug powder packaged in vials in specified amounts and intended for use in clinical laboratories for determining in vitro susceptibility of bacterial pathogens to these therapeutic agents. Test results are used to determine the antimicrobial agent of choice in the treatment of bacterial diseases.(b)
Classification. Class II (performance standards).