ENTEROCOCCUS QUICKFISH BC

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K121991 B. Purpose for Submission: To obtain 510(k) SE determination for the Enterococcus QuickFISH BC Identification Kit for the identification of Enterococcus faecalis and other selected enterococci from positive blood cultures. C. Measurand: Enterococcus faecalis-specific ribosomal RNA sequences and ribosomal RNA sequences specific to other selected Enterococcus species D. Type of Test: Qualitative test using fluorescence *In Situ* nucleic acid hybridization (FISH) with peptide nucleic acid (PNA) probes E. Applicant: AdvanDx Inc. F. Proprietary and Established Names: Enterococcus QuickFISH BC G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | OAH | Class I | 866.3740 Streptococcus spp. serological reagents | Microbiology (83) | H. Intended Use: 1. Intended use(s): {1} Enterococcus QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and/or the detection of selected other enterococci on smears prepared from positive blood cultures containing gram positive cocci in pairs and chains observed on Gram stain. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing and/or differentiation of mixed growth. Enterococcus QuickFISH BC is indicated in an aid in the diagnosis of bacteremia caused by enterococci. 2. Indication(s) for use: Enterococcus QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and/or the detection of selected other enterococci on smears prepared from positive blood cultures containing gram positive cocci in pairs and chains observed on Gram stain. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing and/or differentiation of mixed growth. Enterococcus QuickFISH BC is indicated in an aid in the diagnosis of bacteremia caused by enterococci. 3. Special conditions for use statement(s): For prescription use. 4. Special instrument requirements: QuickFISH slides with controls (AdvanDx CS012) AdvanDx SlideStation 10 (AdvanDx AC028) Dual Band Microscope Filter (AdvanDx AC007) Additional equipment necessary for performance of the assay includes a fluorescent microscope equipped with a 60X or 100X-oil objective. I. Device Description: Enterococcus QuickFISH BC is a fluorescence in situ hybridization (FISH) assay which uses a PNA probe to hybridize to E. faecalis-specific ribosomal RNA sequences, and a PNA probe to hybridize to ribosomal RNA of selected other Enterococcus species. The test provides rapid (20 minutes) identification of E. faecalis and other selected enterococci on smears made from positive blood cultures. 2 {2} J. Substantial Equivalence Information: 1. Predicate device name(s): E. faecalis/OE PNA FISH 2. Predicate 510(k) number(s): K083074 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | Enterococcus QuickFISH BC | E. faecalis/OE PNA FISH (K083074) | | Intended Use | Enterococcus QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and/or the detection of selected other enterococci on smears prepared from positive blood cultures containing gram positive cocci in pairs and chains observed on Gram stain. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing and/or differentiation of mixed growth. Enterococcus QuickFISH BC is indicated in an aid in the diagnosis of bacteremia caused by enterococci. | E. faecalis/OE PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of Enterococcus faecalis and the detection of selected other enterococci (OE) on smears made from positive blood cultures containing gram-positive cocci in pairs and chains observed on Gram stain. Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth. | 3 {3} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | *Enterococcus* QuickFISH BC | *E. faecalis*/OE PNA FISH (K083074) | | Indication for Use | *Enterococcus* QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of *Enterococcus faecalis* and/or the detection of selected other enterococci on smears prepared from positive blood cultures containing gram positive cocci in pairs and chains observed on Gram stain. Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing and/or differentiation of mixed growth. *Enterococcus* QuickFISH BC is indicated in an aid in the diagnosis of bacteremia caused by enterococci. | *E. faecalis*/OE PNA FISH is a multicolor, qualitative nucleic acid hybridization assay intended for the identification of *Enterococcus faecalis* and the detection of selected other enterococci (OE) on smears made from positive blood cultures containing gram-positive cocci in pairs and chains observed on Gram stain. Subculturing of positive blood cultures is necessary for susceptibility testing and/or differentiation of mixed growth. | | Technology | Fluorescence *in situ* hybridization using PNA probes | same | | Sample type | Positive blood cultures from standard automated blood culture device | same | | Interpretation of | Qualitative | same | 4 {4} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | Enterococcus QuickFISH BC | E. faecalis/OE PNA FISH (K083074) | | Results | fluorescence microscopy | | | Differences | | | | --- | --- | --- | | Item | Enterococcus QuickFISH BC | E. faecalis/OE PNA FISH (K083074) | | Fixation Reagents | Fixation Two fixation solutions (QuickFix 1,2), at 55°C | One solution, at room temperature | | Probe Reagents | PNA probes in 2 solutions: 1) Enterococcus PNA Blue contains 4 quenching probes 2) Enterococcus PNA Yellow contains 1 PNA probe for E. faecalis and 1 PNA probe for selected other enterococci | 2 PNA probes in a single solution: one PNA probe for E. faecalis and 2 PNA probes for selected other enterococci | | Wash Reagent | No wash solution | Wash solution with Tris, NaCl and Triton X-100 | | Mounting Reagent | Not needed | 3 mL photobleaching inhibitor in glycerol | | Hybridization Time | 15-20 minutes | 30 minutes | | Time To Result | 20 minutes | 1.5 hours | | Assay Total Time | 20-25 minutes | 1.5 hours | | Assay controls | Positive and negative controls included on each slide | Positive and negative controls prepared separately | # K. Standard/Guidance Document Referenced (if applicable): Not applicable {5} L. Test Principle: The QuickFISH technology uses species-specific peptide nucleic acid (PNA) probes in a fluorescence *in situ* hybridization format. The probes are specific to rRNA targets which are present in high numbers in bacterial cells. The assay is performed directly on smears prepared from blood cultures that have been shown by Gram stain to contain gram-positive cocci in pairs and chains. A mixture of a fluorescein-labeled *E. faecalis*-specific PNA probe and a Tamra-labeled PNA probe targeting selected other enterococci is added to the smear. The probe mixture also contains quencher-labeled PNA probes that serve to bind unreacted fluorescent labeled probes to suppress unwanted signal. Hybridization is performed at 55°C for 15-20 minutes; smear is then ready for examination by fluorescent microscopy. While maintaining their cell morphology, *E. faecalis* or selected other *Enterococcus* species cells become fluorescent by the specific binding of the fluorophore-labeled PNA probes. The fluorescence microscopy is performed using a dual band microscope filter at 60 to 100X magnification. Using fluorescent microscopy, *E. faecalis* will appear as green cells and other selected enterococci will appear as red cells. M. Performance Characteristics (if/when applicable): 1. Analytical performance: a. Precision/Reproducibility: A reproducibility study was performed using 14 isolates including *E. faecalis* (5 isolates – green positive), *E. faecium* (2 isolates – red positive), one isolate each of *E. gallinarum*, *E. casseliflavus*, and *E. raffinosus* (red positive), and one isolate each of *Staphylococcus epidermidis*, *Streptococcus pneumoniae*, *Streptococcus agalactiae*, and *Streptococcus pyogenes* (red and green negative). Reproducibility was >95%. b. Linearity/assay reportable range: Not applicable c. Traceability, Stability, Expected values (controls, calibrators, or methods): Controls Each *Enterococcus* QuickFISH BC test slide contains both positive and negative controls ensuring that controls are run with each patient specimen. The slides are provided in individually sealed pouches with nitrogen and a dessicant. Slides are stored at 2-8°C and must be used immediately after breaking the pouch seal and prior to the expiration date. The controls of the *Enterococcus* QuickFISH BC assay slides contain the following mixtures of organisms specific for the *Enterococcus* test: {6} | Positive Controls | Expected Result | | --- | --- | | E. faecalis ATCC 29212 | Green Positive | | E. faecium ATCC 27270 | Red Positive | | | | | Negative Controls | Expected Result | | Micrococcus luteus ATCC 10240 | No fluorescence | | Cryptococcus neoformans ATCC 204092 | No fluorescence | | Klebsiella oxytoca ATCC43086 | No fluorescence | | Streptococcus pyogenes ATCC 12384 | No fluorescence | ## Fixed Smear Stability The stability of the slides prepared for the Enterococcus QuickFISH BC assay were analyzed under a variety of conditions to support the labeling claim that fixed smears can be kept at room temperature for 1 hour prior to initiation of testing by the Enterococcus QuickFISH assay or stored at $2 - 8^{\circ}$ C for up to one day before initiation of testing. The evaluation included 6 strains of E. faecalis, 2 strains of E. faecium, 4 strains of other enterococcal species, 1 strain of Staphylococcus and 3 species of streptococci (not enterococci). Slides were prepared for each strain and read at time zero and 5 minutes at $55^{\circ}$ C; 5, 15 and 30 minutes at room temperature; and at 4, 18 and 24 hours at $2 - 8^{\circ}$ C. Controls were run with each test and gave the expected results. The data demonstrated that fixed Enterococcus QuickFISH smears were stable at $55^{\circ}$ C for 5 minutes, at room temperature for 1 hour, and for 24 hours at $2 - 8^{\circ}$ C. ## d. Detection limit: The detection limit for the Enterococcus QuickFISH was determined to be approximately $1 - 2 \times 10^{5}$ CFU/mL. Half-log serial dilutions of aliquots from positive blood cultures containing E. faecalis and E. faecium were prepared using blood/culture media. Aliquots of each dilution were plated in triplicate to determine the average concentration of organisms in each aliquot (CFU/mL); each aliquot was tested using the Enterococcus QuickFISH BC assay. ## Co-Infection Studies Co-infection studies were performed for the Enterococcus QuickFISH BC assay using growth from BacT/ALERT SA blood culture bottles with sterile human blood added. The concentration of the target organism (E. faecalis) was tested at or near the LoD while the competing organisms were introduced at increasing 10-fold concentrations (E. faecium $2.0 \times 10^{3}$ to $2.0 \times 10^{10}$; Streptococcus mitis, $6.4 \times 10^{2}$ to $6.4 \times 10^{9}$; Escherichia coli, $8.0 \times 10^{3}$ to $8.0 \times 10^{10}$ and Candida albicans, $1.2 \times 10^{1}$ to $1.2 \times 10^{7}$). Results demonstrated that the target organism was detected in the presence of even high concentrations of competing organisms. {7} 8 # Analytical Sensitivity The sensitivity of the Enterococcus QuickFISH was tested with a variety of Enterococcus species. All 16 Enterococcus faecalis isolates tested gave the expected green positive result. Three species of Enterococcus gave false green positive results: Enterococcus caccae, Enterococcus haemoperoxidus and Enterococcus moraviensis. Fourteen species of Enterococcus gave red positive results and 7 species gave negative results with both the green and red probe. See tables below. The following table lists the Enterococcus species that gave green positive results: | Species | Strain ID | Enterococcus QuickFISH BC | | --- | --- | --- | | Enterococcus faecalis | ATCC 51299 | Green | | Enterococcus faecalis | NCTC 775 | Green | | Enterococcus faecalis | ATCC 19433 | Green | | Enterococcus faecalis | NCIMB 13280 | Green | | Enterococcus faecalis | Clinical isolate | Green | | Enterococcus faecalis | Clinical isolate | Green | | Enterococcus faecalis | ATCC 29212 | Green | | Enterococcus faecalis | ATCC 49533 | Green | | Enterococcus faecalis | ATCC 14506 | Green | | Enterococcus faecalis | ATCC 51188 | Green | | Enterococcus faecalis | ATCC 7080 | Green | | Enterococcus faecalis | ATCC 49532 | Green | | Enterococcus faecalis | ATCC 49452 | Green | | Enterococcus faecalis | ATCC 33186 | Green | | Enterococcus faecalis | ATCC 13280 | Green | | Enterococcus faecalis | NCTC 13379 | Green | The following table lists the Enterococcus species that gave false green positive results: | Species | Strain ID | Enterococcus QuickFISH BC | | --- | --- | --- | | Enterococcus caccae | ATCC BAA-1240 | Green | | Enterococcus haemoperoxidus | ATCC BAA-382 | Green | | Enterococcus moraviensis | ATCC BAA-383 | Green | {8} The following table lists the Enterococcus species that gave red positive results: | Species | Strain ID | Enterococcus QuickFISH BC | | --- | --- | --- | | Enterococcus faecium | ATCC 27270 | Red | | Enterococcus faecium | ATCC 35667 | Red | | Enterococcus faecium | ATCC 51559 | Red | | Enterococcus faecium | ATCC 19434 | Red | | Enterococcus faecium | ATCC 49224 | Red | | Enterococcus faecium | ATCC BAA-472 | Red | | Enterococcus faecium | ATCC 51858 | Red | | Enterococcus faecium | ATCC 6569 | Red | | Enterococcus flavescens | ATCC 49996 | Red | | Enterococcus avium | ATCC 49463 | Red | | Enterococcus casseliflavus | ATCC25788 | Red | | Enterococcus durans | ATCC 6056 | Red | | Enterococcus gallinarum | ATCC 49573 | Red | | Enterococcus gilvus | ATCC BAA-350 | Red | | Enterococcus hirae | ATCC 8043 | Red | | Enterococcus hirae | ATCC 49135 | Red | | Enterococcus malodoratus | ATCC 43197 | Red | | Enterococcus mundtii | ATCC 43187 | Red | | Enterococcus phoenicullicola | ATCC BAA-412 | Red | | Enterococcus raffinosus | ATCC 49464 | Red | | Enterococcus ratti | ATCC 700914 | Red | | Enterococcus villorum | ATCC 700913 | Red | The following table lists the Enterococcus species that gave negative results with both the green and red probes: | Species | Strain ID | Enterococcus QuickFISH BC | | --- | --- | --- | | Enterococcus asini | ATCC700915 | Negative | | Enterococcus cecorum | ATCC BAA-597 | Negative | | Enterococcus columbae | ATCC 51263 | Negative | | Enterococcus dispar | ATCC51266 | Negative | | Enterococcus pallens | ATCC BAA-351 | Negative | | Enterococcus saccharolyticus | ATCC 43076 | Negative | | Enterococcus sulfureus | ATCC 49903 | Negative | {9} e. Analytical specificity: The specificity of the Enterococcus QuickFISH BC assay was determined using a panel of 76 gram positive (non-enterococci) and gram negative organisms and 6 Candida species. All isolates tested negative with the assay with the exception of Granicatella adiacens which gave a fluorescent orange signal. In addition weak green signals were seen with Enterobacter cloacae, Proteus mirabilis, Granicatella elegans and 2 strains of Streptococcus anginosus. f. Assay cut-off: Not applicable g. Media Interference Studies A media compatibility study was performed using the following blood culture bottle types: BacT/Alert (SA and SN), BACTEC (Lytic 10, Aerobic Plus, Anaerobic Plus, PEDS Plus, Standard 10 Aerobic, Standard Anaerobic) and VersaTREK REDOX-1 Aerobic. Organisms used included 6 strains of E. faecalis, 2 strains of E. faecium as well as 4 strains of additional Enterococcus species expected to give positive results, one strain of Staphylococcus epidermidis and 3 Streptococcus species. Results showed the Enterococcus QuickFISH to be compatible with all of the above mentioned blood culture bottle types. The Enterococcus QuickFISH is not compatible with blood culture media containing charcoal or with VersaTREK REDOX 2 blood culture bottles. h. Validation studies: Not applicable 2. Comparison studies: a. Method comparison with predicate device: Not Applicable 3. Clinical studies: The performance of the Enterococcus QuickFISH BC assay was compared to results obtained from routine identification methods for the identification of enterococci from blood cultures. The clinical studies were performed at 5 clinical laboratory sites in the U.S. A total of 244 blood culture bottles growing gram positive cocci in pairs and chains (as assessed by Gram stain) from 244 patients were included in the study. One blood culture contained a mixture of 2 species of Enterococcus (one red positive and one green positive) to provide a total of 245 test results. Blood cultures were performed using two commercially available, continuously monitoring blood 10 {10} culture systems, BacT/ALERT (BioMérieux) and BACTEC (Becton Dickinson and Co). The identification comparator methods included BD Phoenix Automated Microbiology System (1 site), Siemens Microscan (1 site) and BioMérieux VITEK (3 sites). The positive agreement for $E$ faecalis was $100\%$ (70/70). For other enterococci the positive agreement was $97.5\%$ (39/40). Negative percent agreement was $100\%$ (135/135). # Performance Data for Enterococcus QuickFISH BC vs. Reference Identification Methods on Blood Cultures Positive with Gram-Positive Cocci in Pairs and Chains (all sites) | Enterococcus QuickFISH BC | Reference Method | | | | --- | --- | --- | --- | | | E. faecalis | Other Enterococci | Other (non enterococci)3 | | E. faecalis | 70 | 0 | 0 | | Other Enterococci | 0 | 39 | 0 | | Negative | 0 | 11 | 135 | | Total | Positive Percent Agreement | Positive Percent Agreement | Negative Percent Agreement | | | 100% (70/70)2 | 97.5% (39/40)2 | 100% (135/135) | | | 95% CI | 95% CI | 95% CI | | | (94.8 – 100) | (87.5 – 99.6) | (97.2 – 100) | 1 One false negative sample (tested 1 hour and 15 minutes from the time of Gram stain) was a mixed culture comprised of E. faecium, methicillin resistant Staphylococcus aureus and Klebsiella pneumoniae. Repeat testing one week later was weak red positive. 2 Includes 1 mixed culture comprised of E. faecalis, E. gallinarum and Serratia marcescens 3 Other organisms included: Streptococcus species (unspecified Alpha hemolytic streptococci, unspecified Beta hemolytic streptococci, S. agalactiae, S. anginosus, S. constellatus, S. gallolyticus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. pyogenes, S. salivarius, S. sanguinis, S. viridans), Staphylococcus species (S. aureus, S. capitis, S. epidermidis, S. hominis, and other unspecified coagulase negative staphylococci), Lactococcus lactis, Lactococcus raffinolactis, Leuconostoc spp, Granulicatella adiacens, unspecified anaerobic gram positive cocci, and gram negative rods in mixed culture with gram positive cocci in pairs and chains. In the clinical studies, bottles were stored at room temperature after Gram stain {11} and before Enterococcus QuickFISH testing. The time between Gram stain and preparation of the Enterococcus QuickFISH slides varied from less than 2 hours to greater than 48 hours. There was only one test discrepancy in the study (1/244). This sample was from a mixed culture and was tested within 2 hours of Gram stain. ## Performance Data for Enterococcus QuickFISH BC vs. Reference Identification Methods by Blood Culture Bottle Types (all samples) | Bottle Type | Positive Percent Agreement E. faecalis | Positive Percent Agreement other enterococci | Negative Percent Agreement | | --- | --- | --- | --- | | Total BACTEC (BACTEC Plus Aerobic and BACTEC Lytic/10 Anaerobic) | 100% (42/42) 95% CI (91.6 – 100) | 95.8% (23/24) 95% CI (79.8 – 99.3) | 100% (74/74) 95% CI (95.1 – 100) | | Total BacT/ALERT (BacT/ALERT SA Aerobic and BacT/ALERT SN Anaerobic | 100% (23/23) 95% CI (85.7 – 100) | 100% (13/13) 95% CI (77.2 – 100) | 100% (45/45) 95% CI (92.1 – 100) | 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: Enterococcus faecalis: multiple bright green fluorescent cocci in multiple fields of view. Selected other enterococci: multiple bright red fluorescent cocci in multiple fields of view. Non-enterococci and species of enterococci not identified by this assay appear non-fluorescent. 12 {12} 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. 13