V Rx Only. Staph ID/R Blood Culture Panel. Intended Use

Transcription

1 V Rx Only Staph ID/R Blood Culture Panel Intended Use The Great Basin Staph ID/R Blood Culture Panel is a qualitative, multiplex, nucleic acid-based in vitro diagnostic assay intended for the simultaneous identification of nucleic acid from Staphylococcus aureus, Staphylococcus lugdunensis and various Staphylococcus species to the genus level and the detection of the meca gene for methicillin resistance directly from patient positive blood culture specimens. The test utilizes automated hot-start enabled polymerase chain reaction (PCR) for the amplification of specific DNA targets detected by hybridization probes immobilized on a silicon chip surface. The assay is performed directly on positive blood culture specimens identified as positive by continuous monitoring blood culture system that demonstrates the presence of organisms as determined by Gram stain to contain gram-positive cocci in clusters (GPCC) or gram-positive cocci in singles (GPC). The test may be performed using blood culture bottles. The Staph ID/R Blood Culture Panel identifies Staphylococcus aureus (SA), and Staphylococcus lugdunensis, and detects other Staphylococcus species without identification to species level. The Staph ID/R Blood Culture Panel is indicated for use in conjunction with other clinical or laboratory findings to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor these infections. Subculturing positive blood cultures is necessary to recover viable organisms for further identification, susceptibility testing, or epidemiological typing to identify organisms in the blood culture that are not detected by the Great Basin Staph ID/R Blood Culture Panel. If detected, meca may or may not be associated with Staphylococcus spp. detected or the agent responsible for the disease. Negative results for meca antimicrobial resistance gene assays do not always indicate susceptibility, as other mechanisms of resistance to methicillin exist. Summary and Explanation Each year, nearly 2 million patients in the United States (U.S.) acquire a nosocomial infection and about 90,000 of those infections are fatal. 3 In the most recent National Nosocomial Infection Surveillance System (NNIS, 2003), high rates of resistant bacterial infections were reported by nationwide hospital intensive care units (ICUs). In the U.S., sepsis is the leading cause of death in non-coronary ICUs and the 10 th leading cause of death overall, with the cost of care exceeding US17 billion annually. 3 Staphylococci bacteria are major causes of nosocomial and community acquired infections, leading to serious illnesses associated with significant rates of morbidity and mortality. The incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections rose 11% (to 59.5%) and resistant coagulase-negative Staphylococcus (CoNS), 1% (to 89.1%) over the previous surveillance period. 3,5 The increase in the number of Staphylococcus strains possessing resistance to methicillin has become a serious concern as therapeutic options for patient management have become fewer, and associated mortality is increasing. 1,2,3 For hospitalized patients, MRSA bloodstream infections are now associated with greater lengths of stay, higher mortality, and increased costs. 1 Coagulase-negative staphylococci are regarded as opportunistic pathogens frequently isolated from blood cultures and are associated with hospital-acquired bacteremia, particularly intravascular-related infections. 7,8 Patients requiring critical care, or who are immunocompromised are at higher risk for developing CoNS bloodstream HAIs. 7 Residing as normal skin and environmental flora, CoNS have been isolated from bloodstream infections at varying degrees of prevalence and infections associated with sepsis, endocarditis, osteomyelitis, meningitis, pneumonia, implantable devices and others. The most commonly occurring isolates include: Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, and Staphylococcus warneri 7,8. Conventional microbiological culture techniques for identification and antimicrobial susceptibility methods for MRSA, SA and CoNS require pure, viable colonies and results may not be available for hours. Identification of the mechanisms that confer this resistance, like the meca gene, will provide the physician with important information for making appropriate therapeutic and patient management decisions in a timely manner. Identification of MRSA, SA, MR-CoNS (methicillin-resistant coagulase-negative Staphylococcus), and CoNS from Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 1 of 33

2 positive blood culture bottles will also assist surveillance systems to assess the burden of methicillin-resistant infections encountered in healthcare settings with the objectives to develop and implement effective infection control strategies and programs. Principle of the Assay The Great Basin System utilizes automated hot-start enabled polymerase chain reaction (PCR) amplification technology to amplify specific nucleic acid sequences that are detected using species specific Staphylococcal DNA hybridization probes immobilized on a modified silicon chip surface. D Target genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of the PCR reaction. During the PCR process, double-stranded DNA is separated and target nucleic acid sequences are amplified by thermal cycling. Biotin-labeled primers direct amplification of specific nucleic acid sequences within a variable region of the tuf gene for identification of coagulase-negative Staphylococcus species, within a conserved region of the thermonuclease (nuc) gene for specific identification of Staphylococcus aureus, and the meca gene for detecting oxacillin/methicillin resistance. Following the PCR process, biotin-labeled, amplified target DNA sequences are hybridized to an array of probes immobilized on the silicon chip surface, then incubated with anti-biotin antibody, conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is removed by washing, and tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex. The resulting signal is detected by the automated Optical Reader. The single-use test cartridge contains blister packs, fluidic channels, processing chambers, and the assay chip coated with an array of sequence-specific detection probes. All reagents are contained within the integrated blister packs with the exception of the amplification enzymes that are lyophilized and placed into the Amplification Chamber. A positive blood culture specimen is placed into a sample port of the test cartridge for processing. Multiple fluidic channels move reagents from integrated blister packs to chambers where reagent mixing and sample processing occur. A waste chamber, self-contained and segregated within the test cartridge, collects and stores reagent waste. For a full description of the system, see the PA500 System Operator Manual. Reagents and Materials Provided Staph ID/R Blood Culture Panel Test Cartridge Kit (10 Assay Cartridges per Kit) Cat. No. GBSIDR-10 Refrigerate at 2 to 8 C. Each Test Cartridge includes: Blister Pack 1 Dilution Buffer Tris buffer, salts, surfactant, BSA (bovine serum-albumin), antibacterial agents Blister Pack 2 Extraction Buffer Enzymes, salts Blister Pack 3 Wash Solution Saline Sodium Citrate (SSC) buffer, surfactant, preservative Blister Pack 4 Hybridization Buffer SSC buffer, surfactant, preservative, Blister Pack 5 Conjugate Sodium citrate buffer, salts, fetal bovine serum (FBS), peroxidase conjugated monoclonal mouse antibody, preservative Blister Pack 6 Substrate Tetramethylbenzidine (TMB) Chamber 1 (CC1) Stir bar, SPC Lyophilized Bacillus subtilis Chamber 2 (CC2) Stir bar Chamber 3 (Amp) Amplification Reagents Tris buffer salts, sucrose, surfactant, nucleotides, primers, DNA polymerase Chamber 3 (Detect) Silicon chip with immobilized DNA probes Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 2 of 33

3 H F D H H Materials Required but Not Provided PA500 Portrait Analyzer System and Operator Manual Calibrated, 50 µl, fixed-volume pipette 200µL sterile, barrier filter pipette tips Storage and Stability Precautions Upon receipt, the Staph ID/R Blood Culture Panel Test Cartridge Kit should be stored at 2-8 C. Test cartridge should be stored in its original pouch, unopened, prior to use. Use test cartridge within 30 minutes after opening the pouch, and within 10 minutes once the blood culture specimen is loaded into cartridge sample port. Do not use reagent test cartridges that have passed the expiration date. For in vitro diagnostic use only. All patient specimens should be handled as though they are capable of transmitting disease. Use universal precautions specified in the OSHA Bloodborne Pathogen Rule, December Observe established precautions against microbiological hazard through all procedures. Use of protective gloves during assaying and washing hands thoroughly after assay performance is recommended. Completed test cartridges should be discarded in biohazard containers in accordance with Federal, State, and Local regulations. Each laboratory should follow their established institutional guidelines for laboratory safety. Each test cartridge is intended for a single-specimen use. Test cartridges should be stored at 2-8 C. Do not open the test cartridge pouch until ready to use. Do not use punctured or damaged test cartridges. Do not use test cartridges past the expiration date printed on the label. After loading the blood culture specimen into the test cartridge sample port, the test cartridge should be processed in the PA500 Analyzer within 30 minutes. Blood culture specimens should be gently mixed prior to assaying, by swirling and/or inversion. The Staph ID/R Blood Culture Panel test cartridges are for use only with the PA500 Analyzer. Only positive blood culture specimens, found to contain gram-positive cocci in clusters or singles, should be assayed by the Staph ID/R Blood Culture Panel. Specimen Collection and Storage Blood cultures should be collected, processed, and stored following standard laboratory procedures. A Gram stain should be performed from a positive blood culture following standard laboratory practice. Specimens for use with the Staph ID/R Blood Culture Panel test cartridges are those positive blood culture specimens found to contain Gram-positive cocci in singles, or clusters (GPC or GPCC). Positive blood culture specimens that can be tested within 18 hours can be maintained at Room Temperature (15-30 C). Positive blood culture specimens may be stored up to 72 hours at 2-8 C before testing. For positive blood culture bottles revealing Gram positive cocci in clusters (GPCC) or singles (GPC) by gram stain, remove an aliquot of at least 50µl of well-mixed culture and transfer to a sterile, labeled collection tube. Positive blood culture results may critically impact appropriate patient management and care, therefore it is vital that blood culture specimens be processed and positive results reported to healthcare providers in a timely manner, according to your established laboratory policies and institutional guidelines Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 3 of 33

4 Assay Procedure 1. Open Staph ID/R Blood Culture Panel cartridge pouch and remove test cartridge. Test cartridges can be used immediately upon removing from 2-8 C storage. 2. Pipette 50µL of well-mixed GPC or GPCC positive blood culture into the test cartridge, ensuring the pipette tip is secured in the cartridge sample port and pushing the pipette plunger to the first stop only. NOTE: Sample-loaded test cartridges should be processed on the PA500 Analyzer within 30 minutes of adding sample to the test cartridge. 3. Snap the test cartridge sample port closed. Starting the Test 1. Push the test cartridge into the PA500 Analyzer until it stops, and close the door. 2. On the PA500 Analyzer User Interface screen, log in to the Analyzer Program by entering the user ID and password. 3. Scan the cartridge barcode or enter the test cartridge lot number and specimen ID. The specimen ID is associated with the test results and is shown in the results report. 4. Click Start Test on the User Interface screen. The assay will start immediately and a solid blue running light will appear on the PA500 Analyzer front panel to indicate a test is in process. When the test is finished, the blue running light will blink continuously. 5. Results will be displayed on the User Interface screen and a report will be generated. 6. Click End Session. Open the door, remove the used test cartridge, and discard into the biohazard trash in accordance with your laboratory s established biohazardous waste disposal procedures. View Results For detailed instructions for how to view results, see the PA500 Portrait Analyzer System Operator Manual. Interpretation of Results Results are interpolated by the Staph ID/R Blood Culture Panel from measured optical signals and embedded calculation algorithms. Possible results include: Species Probes Detected Final Calls Generated for Staph ID/R Blood Culture Panel Staphylococcus aureus Staphylococcus lugdunensis 2 nd Staph detected Staphylococcus species Reported Identification Call Not Detected Not Detected Not Detected Detected Staphylococcus species OTHER than S. aureus or S. lugdunensis Not Detected Not Detected Detected Detected Staphylococcus species OTHER than S. aureus or S. lugdunensis Detected Not Detected Detected Detected Staphylococcus aureus in mixed Staph infection (NOT S. lugdunensis ) Not Detected Detected Detected Detected Staphylococcus lugdunensis in mixed Staph infection (NOT S. aureus ) Detected Not Detected Not Detected Detected Staphylococcus aureus Not Detected Detected Not Detected Detected Staphylococcus lugdunensis Detected Detected N/A Detected Staphylococcus aureus ; Staphylococcus lugdunensis meca Detected/ Not Detected Detected/ Not Detected Detected/ Not Detected Detected/ Not Detected Detected/ Not Detected Detected/ Not Detected Detected/ Not Detected Not Detected Not Detected Not Detected Not Detected Negative - No Staphylococcus species detected N/A Not Detected Not Detected Not Detected Not Detected Invalid - internal control failed N/A N/A N/A N/A N/A Test Incomplete* N/A * Test run prematurely aborted Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 4 of 33

5 1. If a sample is positive for S. aureus or S. lugdunensis, then the presence of an additional Staphylococcus species (aka 2 nd Staph organism) is evaluated. 2. The meca status will be reported if detected in the presence of all Staphylococcus. If Staphylococcus is not detected then the meca capture probe spot is not evaluated. 3. For Negative No Staphylococcus species detected reported calls, the SPC internal control must be present. Reasons to Repeat the Assay If an invalid result is obtained once, repeat the test using a new test cartridge (do not re-use test cartridges). If invalid results are obtained a second time, contact Great Basin Customer Support at Warning/Failure Messages Integrated into the PA500 Analyzer System is a series of logic points for ensuring successful assay completion. Warning/Failure messages will appear on the User Interface during a run, and require user intervention before the test may continue. These logic points and messages are fully described in the Portrait PA500 Analyzer System Operator Manual. Quality Control Staph ID/R Blood Culture Panel Quality Control procedures are designed to monitor reagent performance, and to indicate correct performance of each test cartridge. Each test cartridge has Integrated Control Features that are automatically performed with every blood culture specimen assay. Built-in Quality Controls Each test includes a Sample Process Control (SPC). Sample Process Control (SPC) - Ensures the sample was correctly processed. The SPC contains Bacillus subtilis in the form of a lyophilized pellet that is included in each test cartridge to verify adequate processing of Staph ID/R Blood Culture Panel sample. The SPC verifies that lysis of the specimen has occurred if the organisms are present and verifies that specimen processing is adequate. Additionally, SPC detects inhibition of PCR reactions, ensuring the PCR reaction conditions are appropriate and that the amplification reagents are functional. The SPC should be positive in a negative sample and can be either negative or positive in a positive sample. External Controls External positive and negative controls are intended to monitor for correct procedural technique and reagent integrity. The External Positive Control is intended to monitor for substantial reagent failure. The External Negative Control is intended to confirm non-reactivity. External control testing should be performed in conformance with local, state, and federal regulations or accreditation organizations as applicable and should follow the user s laboratory standard quality control procedures. 1. External Positive Controls are not provided by Great Basin. Commercially available Staphylococcus strains are recommended to use as a control (examples of recommended strains are listed below). Staphylococcus suspensions in blood culture media may be used as Positive Controls. Recommended Strains Staphylococcus aureus, meca present ATCC: Staphylococcus epidermidis, meca absent ATCC: Staphylococcus lugdunensis, meca absent ATCC: Staphylococcus epidermidis, meca present ATCC: Negative blood culture control 2. A 50 µl aliquot of pure blood culture media is recommended for use as an external Negative Control Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 5 of 33

6 An Invalid result for any control invalidates the assay result. Good laboratory practice recommends the use of control samples. The Staph ID/R Blood Culture Panel should not be used in patient testing if the appropriate controls do not produce the expected results. Limitations 1. The performance of the Staph ID/R Blood Culture Panel was validated using the procedures provided in this package insert only. Modifications to these procedures may alter the performance of the test. 2. Negative results for Staphylococcus species or meca should not be used as the sole basis for diagnosis, treatment or management decisions. Results from the Staph ID/R Blood Culture Panel should be interpreted by the clinician in conjunction with other laboratory and clinical data. 3. Antimicrobial resistance can occur via multiple mechanisms. A meca absent result for the Staph ID/R Blood Culture Panel antimicrobial resistance gene assay does not indicate antimicrobial susceptibility. Subculturing and standard susceptibility testing of isolates is required to determine antimicrobial susceptibility. 4. The Staph ID/R Blood Culture Panel may generate a S. aureus, meca absent result when testing borderline oxacillin resistant SA (BORSA). The mechanism of oxacillin resistance in BORSA strains may be due to other factors (e.g. increased production of β- lactamase) rather than the presence of the meca gene. BORSA with oxacillin MICs of 4-8 µg/ml are considered borderline resistant but will be reported as meca negative by the Staph ID/R Blood Culture Panel. 5. The Staph ID/R Blood Culture Panel may generate a S. aureus, meca absent result when testing modified SA (MOD-SA). The mechanism of oxacillin resistance in MOD-SA strains is due to other factors (e.g., changes in affinity of penicillin binding proteins for Oxacillin) rather than presence of the meca gene. MOD-SA with oxacillin MICs of 4-8 µg/ml are considered borderline resistant but, will be reported as meca negative by the Staph ID/R Blood Culture Panel. 6. The Staph ID/R Blood Culture Panel will generate a S. aureus, meca absent when testing a strain containing a meca homologue known as mecc, such as SA LGA The Staph ID/R Blood Culture Panel will generate a S. aureus meca present result when testing a mixed infection blood culture specimen containing both meca present coagulase negative Staphylococcus (MR- CoNS) and meca absent SA. 8. This test is for use only with positive blood culture bottle specimens containing Gram-positive cocci; performance characteristics with other clinical specimen types have not been established. 9. Clinical performance characteristics of the Staph ID/R Blood Culture Panel have only been determined with positive blood culture samples using the BD BACTEC Medium that demonstrated the presence of organisms by Gram stain evaluation. Other blood culture bottle types were evaluated analytically only (see page 31). 10. For prescription use only. 11. Erroneous test results may occur from improper specimen collection, handling or storage, presence of inhibitors, technical errors, sample mix-up, or because the number of organisms in the specimen is below the analytical sensitivity of the test. Careful compliance with the instructions provided in this insert is necessary to avoid erroneous results. 12. Positive test results do not necessarily indicate the presence of viable organisms. A positive result is however presumptive for the Staphylococcus species identified, and if detected, the presence of Staphylococcus spp, containing meca. 13. It is difficult to determine the clinical significance of the results when testing a mixed infection blood culture containing meca negative S. aureus (SA) and meca positive coagulase-negative Staphylococcus species (MR- CoNS), as the meca target DNA cannot be attributed to either of the species. Subculture and further characterization of each isolate may be necessary to determine diagnosis, treatment, or other clinical management procedures Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 6 of 33

7 14. A positive result from the Staph ID/R Blood Culture Panel does not exclude the possibility of a polymicrobial Staphylococcal infection. 15. Mutations or polymorphisms in primer or probe binding regions may affect detection of new or unknown variants resulting in a false negative result with the Staph ID/R Blood Culture Panel. Genetic rearrangements, translocations, or deletions in some variants may result in false positive results. 16. For samples with the result meca absent further testing is recommended using an FDA-cleared phenotypic antimicrobial susceptibility testing method on isolated colonies recovered from the blood culture bottle. 17. In a mixed culture containing more than one Staphylococcus strain, results can be false negative or variable depending on the concentration of Staphylococcus species present, particularly if one of the Staphylococcus strains is close to the LOD of the assay. 18. This test has not been validated for testing samples other than positive blood culture samples that demonstrate the presence of Gram positive cocci by Gram stain evaluation. 19. The effect of interfering substances has only been evaluated for those listed in the labeling. Interference by substances other than those described in the Interference section below could lead to erroneous results. 20. Cross-reactivity with organisms other than those listed in the Interpretation of Results section above or the Analytical Specificity section below may lead to erroneous results. Performance Data Analytical Studies Analytical Sensitivity The limit of detection (LoD) of the Staph ID/R Blood Culture Panel to Staphylococcus with or without meca was assessed and confirmed with twenty-two (22) different strains. For these studies, overnight incubations of each bacterial stock in Tryptic Soy Broth (TSB) were measured by optical density to estimate the inoculum, serially diluted and spiked into a BACTEC Plus Aerobic/F blood bottle containing negative blood. Spiked bottles were incubated until alarm positivity in a BACTEC Blood Culture System. Alarm positive blood cultures were gram stained, diluted, plated on agar plates and colonies were counted the following day. The enumerated blood culture bottles were diluted to a 1x targeted sample input CFU/mL range in BACTEC Plus Aerobic/F media containing negative blood, tested on device and plated on agar plates to enumerate the actual sample input CFU/mL. The estimations of the sample input CFU/mL were revised to reflect the correct CFU/mL by this colony counting method. The LoDs for six (6) S. aureus strains ± meca are reported as x10 5 CFU/mL; the LoD for each S. aureus strain is listed in Table 1. Table 1. Performance of the Staph ID/R Blood Culture Panel on a minimum of 20 replicates of six (6) Species ATCC # Sample Input Correct Staph ID/R Blood (CFU/mL) Culture Panel Results BAA E+05 23/23 S. aureus, meca + BAA E+05 20/20 BAA E+05 22/ E+05 21/21 S. aureus, meca E+05 22/ E+05 20/20* *This set of test runs also contained 1 "Invalid" run Staphylococcus aureus strains ± meca for establishing LoD Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 7 of 33

8 The LoDs for six (6) S. epidermidis strains ± meca are reported as x10 5 CFU/mL; the LoD for each S. epidermidis strain is listed in Table 2. Table 2. Performance of the Staph ID/R Blood Culture Panel on a minimum of 20 replicates of six (6) Staphylococcus epidermidis strains ± meca for establishing LoD. Sample Input Correct Staph ID/R Blood Species ATCC # (CFU/mL) Culture Panel Results E+05 20/ E+05 22/22 S. epidermidis, meca E+05 27/ E+05 23/23 S. epidermidis, meca- The LoDs for three (3) S. lugdunensis strains without meca are reported as x10 5 CFU/mL; the LoD for each S. lugdunensis strain is listed in Table 3. Table 3. Performance of the Staph ID/R Blood Culture Panel on a minimum of 20 replicates of three (3) Staphylococcus lugdunensis strains ± meca for establishing LoD. LoDs for seven (7) Staphylococcus species ± meca, excluding S. aureus, S. epidermidis, and S. lugdunensis, are reported as x10 5 CFU/mL. The species were selected for inclusion in the LoD studies based on tuf sequence variation from in silico analysis of the target sequence. The LoD for each Staphylococcus strain is listed in Table 4. Table 4. Performance of the Staph ID/R Blood Culture Panel on a minimum of 20 replicates of seven (7) Staphylococcus strains ± meca for establishing LoD. Analytical Reactivity (Inclusivity) E+05 23/ E+05 22/23 Species ATCC # Sample Input Correct Staph ID/R Blood (CFU/mL) Culture Panel Results E+05 22/23 S. lugdunensis, meca E+05 23/ E+05 23/23 Species ATCC # Sample Input Correct Staph ID/R Blood (CFU/mL) Culture Panel Results S. capitis, meca E+05 20/20* S. haemolyticus, meca + BAA E+05 19/20 S. hominis, meca E+05 23/23* S. pasteuri, meca E+05 21/22 S. sciuri, meca E+05 21/21** S. simulans, meca E+05 23/23 S. warneri, meca E+05 22/22 *This set of test runs also contained 1 "Invalid" run **This set of test runs also contained 2 "Invalid" runs The analytical reactivity of the Staph ID/R Blood Culture Panel was tested against an additional 48 well characterized S. aureus strains from ATCC representing USA and SCCmecA I-VI, XI types representative of temporal and geographical diversity. In addition, 104 untyped strains, representing S. aureus, S. epidermidis, S. lugdunensis, and other various Staphylococcus species were tested in the Staph ID/R Blood Culture Panel Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 8 of 33

9 The Staph ID/R Blood Culture Panel correctly detected all of the additional Staphylococcus strains, meca present or absent (Table 5). Table 5. Analytical Reactivity (Inclusivity) Panel. Staphylococcus strains and results for inclusivity by the Staph ID/R Blood Culture Panel. Staphylococcus species; ATCC, CCUG, NRS, Clinical # SCCmec, PFGE type / source Sample Input (CFU/mL) Correct Staph ID/R Blood Culture Panel Result S. aureus BAA-38 I, Denmark 1.0x10 6 2/2 S. aureus BAA-44 I, Iberian 9.2x10 5 2/2 S. aureus II, Japan 1.1x10 6 2/2 S. aureus BAA-41 II, USA x10 6 2/2 S. aureus BAA-1681 II, USA x10 6 2/2 S. aureus BAA-1682 II, USA x10 6 2/2 S. aureus BAA-1761 II, USA x10 6 2/2 S. aureus NRS660 II, USA x10 6 2/2 S. aureus BAA-1720 II, USA x10 6 2/2 S. aureus BAA-1750 II, USA x10 5 2/2 S. aureus BAA-1760 II, USA x10 6 2/2 S. aureus NRS651 II, USA x10 6 2/2 S. aureus BAA-39 III, Hungary 6.4x10 6 2/2 S. aureus III, ST x10 6 2/ Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 9 of 33

10 S. aureus BAA-1680 IV, USA x10 6 2/2 S. aureus BAA-1717 IV, USA x10 6 2/2 S. aureus NRS643 IV, USA x10 6 2/2 S. aureus NRS662 IV, USA x10 6 2/2 S. aureus NRS688 IV, USA x10 6 2/2 S. aureus NRS716 IV, USA x10 6 2/2 S. aureus BAA-1683 IV, USA x10 6 2/2 S. aureus BAA-1696 IV, USA x10 6 2/2 S. aureus BAA-1707 IV, USA x10 6 2/2 S. aureus BAA-1752 IV, USA x10 6 2/2 S. aureus BAA-1684 IV, USA x10 6 2/2 S. aureus BAA-1689 IV, USA x10 6 2/2 S. aureus BAA-1763 IV, USA x10 6 2/2 S. aureus NRS685 IV, USA x10 6 2/2 S. aureus BAA-1754 IV, USA x10 6 2/2 S. aureus BAA-1755 IV, USA x10 6 2/2 S. aureus BAA-1758 IV, USA x10 5 2/2 S. aureus BAA-1768 IV, USA x10 5 2/2 S. aureus NRS692 IV, USA x10 6 2/2 S. aureus NRS675 IV, USA x10 6 2/2 S. aureus BAA-1747 IV, USA x10 6 2/2 S. aureus NRS483 IV, USA x10 6 2/2 S. aureus NRS730 IV, USA x10 6 2/2 S. aureus BAA-1764 IV, USA x10 5 2/2 S. aureus NRS484 IV, USA x10 6 2/2 S. aureus BAA-1766 V, USA x10 6 2/2 S. aureus BAA-2094 V, WA-MRSA 1.0x10 6 2/2 S. aureus BAA-42 VI, USA x10 6 2/2 S. aureus (mecc ) BAA-2313 XI, CC x10 6 2/2 S. aureus BAA-1751 Untyped, USA x10 6 2/2 S. aureus BAA-1771 Untyped, USA x10 6 2/2 S. aureus BAA-1718 NA, USA x10 6 2/2 S. aureus BAA-1749 NA, USA x10 6 2/2 S. aureus BAA-1765 NA, USA x10 6 2/2 S. aureus BAA-40 Untyped, Lisbon 6.5x10 6 2/2 S. aureus BAA-1685 Untyped, ATCC 4.8x10 6 2/2 S. aureus BAA-1708 Untyped, ATCC 2.4x10 6 2/2 S. aureus BAA-1721 Untyped, UK 9.0x10 5 2/2 S. aureus 6538 Untyped, ATCC 3.1x10 6 2/2 S. aureus Untyped, ATCC 1.4x10 6 2/2 S. aureus Untyped, ATCC 2.1x10 6 2/2 S. aureus Untyped, ATCC 9.0x10 5 2/ Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 10 of 33

11 S. aureus Untyped, ATCC 3.1x10 6 2/2 S. aureus Untyped, ATCC 4.1x10 6 2/2 S. aureus Untyped, ATCC 2.4x10 6 2/2 S. aureus Untyped, ATCC 4.5x10 6 2/2 S. aureus Untyped, ATCC 4.0x10 6 2/2 S. aureus Untyped, ATCC 3.0x10 6 2/2 S. aureus Untyped, ATCC 3.2x10 6 2/2 S. aureus Untyped, ATCC 3.6x10 6 2/2 S. aureus BORSA MCW1 Untyped, Wisconsin, Ledeboer Lab 1.9x10 6 2/2 S. aureus BORSA MCW2 Untyped, Wisconsin, Ledeboer Lab 3.6x10 6 2/2 S. aureus BORSA Untyped, New Jersey, Kreiswirth Lab 2.9x10 6 2/2 S. aureus BORSA Untyped, New Jersey, Kreiswirth Lab 3.4x10 6 2/2 S. aureus Empty Mec Cassette 45 Untyped, Iowa, Diekema lab 6.3x10 5 2/2 S. aureus Empty Mec Cassette 46 Untyped, Iowa, Diekema lab 2.7x10 6 2/2 S. aureus Empty Mec Cassette 50 Untyped, Iowa, Diekema lab 3.7x10 6 2/2 S. aureus Empty Mec Cassette 51 Untyped, Iowa, Diekema lab 2.2x10 6 2/2 S. auricularis ATCC 3.2x10 5 2/2 S. auricularis ATCC 3.2x10 5 2/2 S. capitis subsp. capitis ATCC 7.2x10 5 2/2 S. capitis subsp. ureolyticus ATCC 2.2x10 6 2/2 S. caprae ATCC 9.2x10 5 2/2 S. caprae ATCC 1.3x10 6 2/2 S. chromogenes ATCC 4.8x10 6 2/2 S. chohnii subsp. cohnii ATCC 4.3x10 6 2/2 S. chohnii subsp. urealyticus ATCC 9.3x10 5 2/2 S. condimenti 4753 ATCC 1.2x10 6 2/2 S. carnosus ATCC 4.9x10 6 2/2 S. delphini ATCC 1.7x10 5 2/2 S. epidermidis ATCC 1.2x10 6 2/2 S. epidermidis ATCC 1.8x10 6 2/2 S. epidermidis ATCC 4.6x10 6 2/2 S. epidermidis ATCC 1.2x10 6 2/2 S. epidermidis ATCC 2.0x10 6 2/2 S. epidermidis ATCC 2.7x10 6 2/ Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 11 of 33

12 S. epidermidis ATCC 1.1x10 6 2/2 S. epidermidis ATCC 6.6x10 5 2/2 S. epidermidis ATCC 3.9x10 6 2/2 S. epidermidis ATCC 9.4x10 5 2/2 S. epidermidis ATCC 8.5x10 5 2/2 S. epidermidis ATCC 3.5x10 5 2/2 S. equorum ATCC 4.2x10 6 2/2 S. felis ATCC 1.1x10 6 2/2 S. fleuretti BAA-274 ATCC 1.3x10 6 2/2 S. gallinarum ATCC 2.2x10 6 2/2 S. gallinarum ATCC 2.5x10 6 2/2 S. haemolyticus BAA-1693 ATCC 3.5x10 6 2/2 S. haemolyticus ATCC 1.2x10 6 2/2 S. haemolyticus ATCC 4.8x10 6 2/2 S. haemolyticus ATCC 3.9x10 6 2/2 S. haemolyticus ATCC 3.5x10 6 2/2 S. hominis ATCC 3.2x10 6 2/2 S. hominis ATCC 1.6x10 6 2/2 S. hominis ATCC 3.0x10 6 2/2 S. hominis ATCC 2.4x10 6 2/2 S. hominis subsp. novobiosepticus ATCC 2.2x10 6 2/2 S. intermedius ATCC 1.1x10 6 2/2 S. intermedius ATCC 1.0x10 6 2/2 S. intermedius ATCC 2.5x10 6 2/2 S. kloosii ATCC 2.4x10 6 2/2 S. lentus ATCC 6.8x10 5 2/2 S. lugdunensis 4436 CCM, Czech 2.9x10 6 2/2 S. lugdunensis 7990 ATCC, NCTC 3.0x10 6 2/2 S. lugdunensis ATCC 2.0x10 6 2/2 S. lugdunensis CCUG, Sweden 4.3x10 6 2/2 S. lugdunensis ATCC 8.2x10 5 2/2 S. lugdunensis ATCC 3.0x10 6 2/2 S. lutrae ATCC 8.4x10 5 2/2 S. massiliensis 7895 ATCC 1.8x10 6 2/2 S. muscae ATCC 1.4x10 6 2/2 S. nepalensis ATCC 3.3x10 6 2/2 S. pasteuri ATCC 5.1x10 6 2/2 S. pettenkoferi 36 Indianapolis, Denys Lab 2.0x10 6 2/2 S. piscifermentans ATCC 2.4x10 6 2/2 S. pulvereri ATCC 1.2x10 6 2/2 S. pseudintermidus ATCC 1.7x10 6 2/2 S. saccharolyticus ATCC 7.3x10 5 2/ Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 12 of 33

13 S. saprophyticus BAA-750 ATCC 2.1x10 6 2/2 S. saprophyticus ATCC 1.7x10 6 2/2 S. schleiferi subsp. coagulans ATCC 1.7x10 6 2/2 S. schleiferi subsp. schleiferi ATCC 1.3x10 6 2/2 S. sciuri ATCC 5.5x10 5 2/2 S. sciuri ATCC 1.1x10 6 2/2 S. sciuri ATCC 2.9x10 6 2/2 S. sciuri subsp. carnaticus ATCC 2.1x10 6 2/2 S. sciuri subsp. rodentium ATCC 1.8x10 6 2/2 S. simulans ATCC 5.3x10 5 2/2 S. simulans ATCC 5.7x10 5 2/2 S. simiae 7213 ATCC 1.2x10 6 2/2 S. succinus subsp. succinus ATCC 1.6x10 5 2/2 S. vitulinus ATCC 1.1x10 6 2/2 S. warneri ATCC 2.4x10 6 2/2 S. warneri ATCC 1.1x10 6 2/2 S. warneri ATCC 4.2x10 6 2/2 S. warneri ATCC 1.4x10 6 2/2 S. xylosus ATCC 1.7x10 6 2/2 S. xylosus ATCC 3.5x10 6 2/2 In addition to these studies, a subset of eight (8) S. aureus strains representing SCCmecA subtypes I-V, one (1) mecc strain, four (4) Borderline Oxacillin Resistant S. aureus (BORSA), four (4) Empty Cassette S. aureus variants, and multiple S. epidermidis and S. lugdunensis strains were selected to be part of a Challenge Panel. The challenge panel was tested for oxacillin MIC using BD Phoenix ID. The results from the MIC determination and card results are listed in Table Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 13 of 33

14 Table 6. Analytical Reactivity (Inclusivity) Challenge Panel. Staphylococcus strains tested for oxacillin MIC and for inclusivity by the Staph ID/R Blood Culture Panel. Staphylococcus Oxacillin Strain SCCmec Type PFGE/Type Strain Significance species MIC (µg/ml) Staph ID/R Blood Culture Panel Result S. aureus BAA-38 I Unknown MRSA >2 S. aureus, meca Present S. aureus II Genome sequenced MRSA >2 S. aureus, meca Present S. aureus BAA-1682 II USA100 MRSA >2 S. aureus, meca Present S. aureus BAA-1681 II USA100 MRSA 2 S. aureus, meca Present S. aureus III ST239 MRSA >2 S. aureus, meca Present S. aureus BAA-1680 IV USA300 MRSA >2 S. aureus, meca Present S. aureus BAA-1684 IV USA500 MRSA >2 S. aureus, meca Present S. aureus BAA-2094 V WA-MRSA MRSA 1 S. aureus, meca Present S. aureus BAA-2313 XI (mecc ) CC130 mecc - MRSA 2 S. aureus, meca Absent S. aureus NA Unknown Empty Cassette 0.5 S. aureus, meca Absent S. aureus NA Unknown Empty Cassette 0.5 S. aureus, meca Absent S. aureus NA Unknown Empty Cassette 0.25 S. aureus, meca Absent S. aureus NA Unknown Empty Cassette 0.25 S. aureus, meca Absent S. aureus NA Unknown BORSA >2 S. aureus, meca Absent S. aureus NA Unknown BORSA >2 S. aureus, meca Absent S. aureus NA Unknown BORSA 1 S. aureus, meca Absent S. aureus MCW1 NA Unknown BORSA 0.5 S. aureus, meca Absent S. aureus MCW2 NA Unknown BORSA 0.25 S. aureus, meca Absent S. aureus NA serotype 3 MSSA 0.5 S. aureus, meca Absent S. aureus NA Unknown MSSA 0.5 S. aureus, meca Absent S. aureus NA Unknown MSSA 0.25 S. aureus, meca Absent S. aureus 6538 NA Unknown MSSA 0.25 S. aureus, meca Absent S. aureus NA Unknown MSSA 0.25 S. aureus, meca Absent S. epidermidis NA Genome sequenced MRSE >1 S. epidermidis NA Unknown MRSE >1 S. epidermidis NA Unknown MRSE >1 S. epidermidis NA Unknown MRSE >1 S. epidermidis NA Unknown MRSE >1 S. epidermidis NA Unknown MRSE >1 S. epidermidis NA Unknown MRSE >1 S. epidermidis NA Unknown MRSE 1 S. epidermidis NA Unknown MSSE 0.25 S. epidermidis NA Unknown MSSE 0.25 S. hominis NA Unknown MR Staph ssp >1 Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Absent Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Absent Staphylococcus species OTHER than S. aureus or S. lugdunensis, meca Present S. lugdunensis NA Unknown MS - Lug 0.25 S. lugdunensis, meca Absent S. lugdunensis NA Unknown MS - Lug 0.25 S. lugdunensis, meca Absent S. lugdunensis NCTC 7990 NA Unknown MS - Lug 0.25 S. lugdunensis, meca Absent S. lugdunensis NA Unknown MS - Lug 0.25 S. lugdunensis, meca Absent Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 14 of 33

15 All strains showed expected oxacillin MIC results, including the BORSA strains, which showed a range of oxacillin resistance ( µg/ml) as expected for strains resistant by alternative mechanisms other than meca. In addition, all empty cassette strains, which lack meca, were sensitive to oxacillin ( µg/ml). Samples were correctly detected as meca Present or Absent in the Staph ID/R Blood Culture Panel. The Staph ID/R did not detect meca for mecc strain BAA-2313, empty cassette and BORSA strains as expected. Analytical Specificity (Exclusivity) Studies were performed to assess the potential cross-reactivity of the Staph ID/R Blood Culture Panel with 116 offpanel microflora (bacterial, yeast, and mycoplasma strains). BACTEC Plus Aerobic/F or Anaerobic/F media (for anaerobic strains) containing negative blood were inoculated with isolates and incubated in a BACTEC Blood Culture System until alarm positivity. Alarm positive samples were incubated for additional time in the Blood Culture System consistent with specimen stability studies to obtain a target microorganism bottle load 10 8 CFU/mL. The positive alarm bottles were Gram stained, diluted, plated and counted to confirm all organisms were tested at 1x10 8 CFU/mL or higher. For two (2) organisms, alarm positive bottles samples were substituted with genomic DNA at a final concentration of 10 8 copies/ml. Genomic DNA was spiked into a matrix of negative blood and BACTEC Plus Aerobic/F media. A minimum of two (2) replicates was tested in the Staph ID/R Blood Culture Panel for each of the bacterial, fungal and mycoplasma strains evaluated and these data are summarized in Table 7. Table 7. Analytical Specificity (Exclusivity) Panel. Non-Staphylococcus microorganisms or DNA from microorganisms tested for exclusivity by the Staph ID/R Blood Culture Panel. Exclusivity Species Strain (ATCC, CCUG, Clinical) Sample Input in CFU/mL or copies/ml (gdna) Correct Staph ID/R Blood Panel Result Gram Positive Bacteria Actinomyces odontolyticus x10 9 2/2 (1) Abiotrophia defectiva x10 8 2/2 Aerococcus urinae x10 8 2/2 Arcanobacterium haemolyticum BAA x /11 (2) Bacillus cereus x10 9 2/2 Corynebacterium diphtheriae x10 9 2/2 Corynebacterium jeikeium x10 8 2/2 Enterococcus avium x10 9 2/2 Enterococcus casseliflavus x10 9 2/2 Enterococcus durans x10 8 2/2 Enterococcus faecalis x10 9 2/2 Enterococcus faecalis x10 9 2/2 Enterococcus faecalis, van A 1MC 3.1x10 8 2/2 Enterococcus faecalis, van B x10 9 2/2 Enterococcus faecium x10 8 2/2 Enterococcus faecium x10 8 2/2 Enterococcus faecium, van A x10 8 2/2 Enterococcus gallinarum x10 8 2/2 Enterococcus gallinarum x10 8 2/2 Enterococcus hirae x10 8 2/2 Enterococcus raffinosus x10 8 2/2 Gemella morbillorum x10 9 2/2 Globicatella sanguinis x10 8 2/ Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 15 of 33

16 Kocuria kristinae BAA x10 9 2/2 Kocuria rosea x10 8 2/2 Kytococcus schroeteri (oxacillin resistant) BAA x10 8 2/2 Lactobacillus acidophilus x10 8 2/2 Lactococcus lactis x10 8 2/2 Lactococcus lactis x10 9 2/2 Leuconostoc mesenteroides subsp. mesenteroides x10 9 2/2 Leuconostoc mesenteroides subsp. mesenteroides x10 8 2/2 Leuconostoc pseudomesenteroides x10 9 2/2 Listeria grayi x10 9 2/2 Listeria innocua x10 8 2/2 Listeria ivanovii x10 9 2/2 Listeria monocytogenes x10 8 2/2 Listeria seeligeri x10 9 2/2 Macrococcus caseolyticus x /13 (1,2) Micrococcus luteus x10 8 2/2 Micrococcus lylae x10 8 2/2 Mycobacterium avium x10 8 (gdna) 2/2 Pediococcus damnosus x10 9 2/2 Pediococcus pentosaceus x10 8 2/2 Peptostreptococcus anaerobius x10 8 2/2 Planococcus citreus x10 8 2/2 Planococcus kocurii x10 8 2/2 Propionibacterium acnes x10 8 2/2 Rhodococcus equi x10 8 2/2 Rothia dentocariosa BAA x10 8 2/2 Rothia mucilaginosa x10 8 2/2 Streptococcus agalactiae BAA x10 8 4/4 Streptococcus agalactiae x10 8 2/2 Streptococcus angunosis NCTC x10 8 2/2 Streptococcus constellatus x10 9 2/2 Streptococcus dysagalactiae x10 8 2/2 Streptococcus equi x10 8 2/2 Streptococcus gallolyticus x10 9 2/2 Streptococcus gallolyticus x10 9 2/2 Streptococcus mitis x10 8 2/2 Streptococcus mutans x10 8 2/2 Streptococcus mutans x /13 (2,3) Streptococcus parasanguinis x10 8 2/2 Streptococcus pneumoniae ARUP 1.6x10 8 2/2 Streptococcus pyogenes x10 8 2/2 Streptococcus pyogenes x10 8 2/2 Streptococcus pyogenes x10 9 2/2 Streptococcus sanguinis x10 8 2/2 Streptococcus thoraltensis x10 9 2/2 Streptococcus uberis x10 8 2/2 (1) Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 16 of 33

17 Gram Negative Bacteria Acinetobacter baumannii x10 8 2/2 Acinetobacter calcoaceticus x10 8 2/2 Acinetobacter haemolyticus x10 8 2/2 Acinetobacter lwoffi x10 9 2/2 Bacteriodes fragilis x10 8 2/2 Bordetella pertussis x10 9 2/2 Burkholderia cepacia x10 8 2/2 Citrobacter amalonaticus x10 8 2/2 Citrobacter freundii x10 9 2/2 Citrobacter koseri x10 9 2/2 Enterobacter aerogenes x10 9 2/2 Enterobacter cloacae x10 9 2/2 Escherichia coli BAA x10 9 2/2 (1) Escherichia coli x10 9 2/2 Fusobacterium nucleatum x10 8 2/2 Haemophilus haemolyticus x10 9 2/2 Hafnia alvei x10 9 2/2 Klebsiella oxytoca x10 9 2/2 Klebsiella pneumoniae x10 9 2/2 Klebsiella pneumoniae BAA x10 9 2/2 Kluyvera intermedia x10 8 2/2 (1) Moraxella catarrhalis x10 9 2/2 (1) Morganella morganii x10 9 2/2 Neisseria gonorrhoeae x10 9 2/2 Neisseria meningitidis x10 9 2/2 (1) Neisseria subflava x10 7 2/2 (1) Oligella urethralis x10 8 2/2 Proteus mirabilis x /13 (1,4) Proteus vulgaris x10 8 2/2 Providencia rettgeri x10 8 2/2 Providencia rustigianii x10 8 2/2 Pseudomonas aeruginosa x10 9 2/2 Pseudomonas putida x10 9 2/2 Salmonella enterica x10 9 2/2 Salmonella typhimurium x10 9 2/2 (1) Serratia liquefaciens x /15 (2,5) Serratia marcescens x10 8 2/2 Shigella sonnei x10 8 2/2 Stenotrophomonas maltophilia x10 9 2/2 Yersinia enterocolitica x10 9 2/ Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 17 of 33

18 Yeast Candida albicans x10 9 2/2 Candida glabrata x10 9 2/2 Candida krusei x10 8 2/2 Candida parapsilosis x10 8 2/2 Candida tropicalis ARUP 2 1.5x10 9 2/2 Cryptococcus neoformans x10 9 2/2 Mycoplasma Mycoplasma pneumoniae x10 8 (gdna) 2/2 (6) (1) This set of test runs also contained 1 "Invalid" run (2) This set of test runs contained 1 false positive result (3) This set of test runs also contained 10 "Invalid" runs (4) This set of test runs contained 2 false positive results (5) This set of test runs also contained 5 "Invalid" runs (6) This set of test runs also contained 2 "Invalid" runs The vast majority of strains tested Staphylococcus NEGATIVE, indicating no cross-reactivity or interference with internal controls. The exceptions were twenty-seven (27) invalid calls and six (6) Staphylococcus POSITIVE calls, all noted in Table 7. One (1) invalid call out of two (2) tests was observed for a single strain of the following species: Actinomyces odontolyticus, Escherichia coli, Kluyvera intermedia, Moraxella catarrhalis, Neisseria meningitides, Neisseria subflava, Proteus mirabilis, Salmonella typhimurium, and Streptococcus uberis. Each invalid case resolved upon re-testing as Staphylococcus NEGATIVE. Two (2) invalid calls out of two (2) tests were observed for Mycoplasma pneumoniae upon initial testing. Two (2) valid calls out of two (2) tests were obtained upon re-testing as Staphylococcus NEGATIVE. One (1) Staphylococcus POSITIVE call out of two (2) tests was observed for a single strain of the following species: Arcanobacterium haemolyticum, Macrococcus caseolyticus, Streptococcus mutans, Proteus mirabilis, Serratia liquefaciens. Each positive result resolved upon re-testing as Staphylococcus NEGATIVE with a minimum of six (6) repeat tests, indicating the positive results previously obtained were likely a single contamination event in one card. One or more invalid calls were observed upon re-testing for the following species: Macrococcus caseolyticus, Streptococcus mutans and Serratia liquefaciens as noted in Table 7. Microbial Interference Off-Panel Microbial Interference: As a follow up to the previous exclusivity and inclusivity studies, the Staph ID/R Blood Culture Panel was further evaluated for the ability to detect low level Staphylococcus species in the presence of fourteen (14) off-panel microorganism strains that should not be detected. The off-panel strains represent Gram Positive, Gram Negative, Yeast and likely skin contaminants. BACTEC Plus Aerobic/F or Anaerobic/F Bottles containing blood were inoculated with competing off-panel microorganisms. The off-panel strains were grown to high concentrations by incubating 8 hours past bottle ring On-board, consistent with incubation time and temperatures tested in the specimen stability study. The bottle contents were confirmed by Gram stain and serial dilutions plated on agar and counted the following day to confirm a concentration of >10 8 CFU/mL. Bottles were stored at 4 C and tested within 72 hours in combination with Staphylococcus TSB cultures at approximately 1-2.5x10 6 CFU/mL for each strain. The concentration of the Staphylococcus strains were verified by plating serial dilutions on agar and performing colony counts the following day Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 18 of 33

19 The studies assessed the detection of 5 ATCC Staphylococcus test strains at the indicated concentrations: S. aureus (meca+) BAA-1682 ( x10 6 CFU/mL) S. aureus (meca-) ( x10 6 CFU/mL), S. epidermidis (meca+) ( x10 6 CFU/mL), S. epidermidis (meca-) strain ( x10 6 CFU/mL), and S. lugdunensis (meca-) strain (1-1.2x10 6 CFU/mL). A minimum of two (2) replicate Staph ID/R Blood Culture Panels were performed for each combination of competing and test organisms. The data for off-panel competing organisms are summarized in Table 8. Table 8. Microbial Interference Panel (Off-panel): Non-Staphylococcus microbial strains tested for microbial interference in detecting five (5) Staphylococcus strains by the Staph ID/R Blood Culture Panel. "Off-Panel" Microorganisms Species, Sample Input 10 8 CFU/mL; ATCC/NCTC strain # S. aureus BAA x10 6 Species; ATCC Strain #; Sample Input (CFU/mL) S. aureus x10 6 S. epidermidis x10 6 S. epidermidis x10 6 S. lugdunensis x10 6 Gram Positive Bacteria Corynebacterium jeikeium /2 2/2 2/2 2/2 2/2 Enterococcus faecalis /2 2/2 2/2" 2/2 2/2 Enterococcus faecium /2 2/2 2/2" 2/2 2/2 Listeria monocytogenes /2 2/2 2/2" 2/2" 2/2 Micrococcus luteus /2 2/2 2/2 2/2 2/2 Propionibacterium acnes /2 2/2 2/2 2/2 2/2 Streptoccocus agalactiae /2 2/2 2/2* 2/2 2/2 Streptococcus anginosus /2 2/2 2/2 2/2 2/2" Streptococcus pneumoniae /2 2/2 2/2 2/2 2/2 Streptococcus pyogenes /2 2/2 2/2" 2/2 2/2 Gram Negative Bacteria Escherichia coli /2 2/2 2/2 2/2" 2/2* Klebsiella pneumoniae /2 2/2 2/2 2/2 2/2 Pseudomonas aeruginosa /2 2/2 2/2 2/2 2/2 Yeast Candida albicans /2 2/2 2/2 2/2 2/2 *This set of test runs also contained 1 "Invalid" run "This set of test runs initially miscalled, but called correctly with a higher CFU/mL input of the low level target For the valid runs tested, the potentially interfering off-panel microorganisms did not interfere with the detection of the Staphylococcus strains, resulting in POSITIVE calls as expected. In some cases, a miscall was observed, and the low-target Staphylococcus strains were re-tested at a higher concentration and resulted in a positive result as expected. The re-tested concentrations are included in the table and were within the 2-3X LoD range for each species. No interference was observed for S. aureus with all species tested, there were six (6) cases of interference with S. epidermidis at initial concentrations when tested against E. faecalis, E. faecium, L. monocytogenes, S. pyogenes and E. coli. Higher concentrations of S. epidermidis resolved the interference (within 2-3X LoD). A single case of interference was observed with S. lugdunensis and S. anginosus, but resolved at higher concentrations of S. lugdunensis (within 2-3X LoD). Staphylococcus Microbial Interference: Twelve (12) Staphylococcus species expected to be co-detected with the low level Staphylococcus species. BACTEC Plus Aerobic/F bottles containing blood were inoculated with Staphylococcus isolates. The bacteria were grown to high concentrations by incubating 8 hours past bottle ring On-board, consistent with incubation time and temperatures tested in the specimen stability study. The bottle contents were confirmed by Gram stain and serial dilutions plated on agar and counted the following day to confirm a concentration of >10 8 CFU/mL for competing Staphylococcus strains. Bottles were stored at 4 C and tested within 72 hours in combination with Staphylococcus TSB cultures at approximately 1-2.5x10 6 CFU/mL for each strain. The concentration of the Staphylococcus strains were verified by plating serial dilutions on agar and performing colony counts the following day Staph ID/R Blood Culture Panel Package Insert 09/2016 Page 19 of 33