QIAstat-Dx BCID GPF Plus AMR Panel

{0} FDA U.S. FOOD & DRUG ADMINISTRATION 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY # I Background Information: A 510(k) Number K254194 B Applicant QIAGEN GmbH C Proprietary and Established Names QIAstat-Dx BCID GPF Plus AMR Panel D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | PAM | Class II | 21 CFR 866.3365 - Multiplex Nucleic Acid Assay For Identification Of Microorganisms And Resistance Markers From Positive Blood Cultures | MI - Microbiology | | PEO | Class II | 21 CFR 866.3365 - Multiplex Nucleic Acid Assay For Identification Of Microorganisms And Resistance Markers From Positive Blood Cultures | MI - Microbiology | | PEN | Class II | 21 CFR 866.3365 - Multiplex Nucleic Acid Assay For Identification Of Microorganisms And Resistance Markers From Positive Blood Cultures | MI - Microbiology | # II Submission/Device Overview: A Purpose for Submission: To obtain substantial equivalence determination for the QIAstat-Dx BCID GPF Plus AMR Panel B Measurand: Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} Nucleic acid sequences from the following gram-positive bacteria and fungi and antimicrobial resistance markers: - Bacillus cereus group - Corynebacterium - Enterococcus faecalis - Enterococcus faecium - Listeria monocytogenes - Micrococcus spp. - Staphylococcus aureus - Staphylococcus epidermidis - Staphylococcus lugdunensis - S. capitis/S. hominis - Streptococcus agalactiae - Streptococcus anginosus group - Streptococcus pneumoniae - Streptococcus pyogenes - Cryptococcus neoformans/gattii - Fusarium - Candida auris - Pan Gram-Negative - Pan Candida Group 1 - Pan Candida Group 2 - aac(6') aph(2') - cfr - erm(A) - erm(C) - mecA - mecC - tet(K) - tet(M) - vanA - vanB ### C Type of Test: A multiplexed nucleic acid test intended for use with the QIAstat-Dx Analyzer 2.0 for the qualitative in vitro detection and identification of nucleic acids from gram-positive bacteria and fungi in a positive blood culture media sample. ### III Intended Use/Indications for Use: #### A Intended Use(s): See Indications for Use below. #### B Indication(s) for Use: The QIAstat-Dx BCID GPF Plus AMR Panel is a qualitative multiplexed nucleic acid real-time PCR-based in vitro diagnostic test intended for use with the QIAstat-Dx Analyzer 2.0. The K254194 - Page 2 of 56 {2} QIAstat-Dx BCID GPF Plus AMR Panel is capable of simultaneous detection and identification of multiple gram-positive bacterial and fungal pathogens and selected genetic determinants associated with antimicrobial resistance. Additionally, the QIAstat-Dx BCID GPF Plus AMR Panel is capable of detecting a wide variety of gram-negative bacteria (Pan Gram Negative assay). The QIAstat-Dx BCID GPF Plus AMR Panel is intended for use in positive blood culture samples from patients with blood stream infections identified via Gram stain to contain gram-positive bacteria or fungi. The following gram-positive bacteria are identified and differentiated using the QIAstat-Dx BCID GPF Plus AMR Panel: - Bacillus cereus group - Corynebacterium - Enterococcus faecalis - Enterococcus faecium - Listeria monocytogenes - Micrococcus spp. - Staphylococcus aureus - Staphylococcus epidermidis - Staphylococcus lugdunensis - S. capitis/S. hominis - Streptococcus agalactiae - Streptococcus anginosus group - Streptococcus pneumoniae - Streptococcus pyogenes The following fungi are identified and differentiated using the QIAstat-Dx BCID GPF Plus AMR Panel: - Cryptococcus neoformans/gattii - Fusarium - Candida auris The QIAstat-Dx BCID GPF Plus AMR Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include: - A broad Pan Gram-Negative assay - Candida Group 1: (detects but does not differentiate 6 species) - Candida albicans - Candida tropicalis - Candida dubliniensis - Candida famata - Candida guilliermondii - Candida kefyr - Candida Group 2: (detects but does not differentiate 4 species) - Candida glabrata K254194 - Page 3 of 56 {3} ○ Candida krusei ○ Candida parapsilosis ○ Candida lusitaniae The QIAstat-Dx BCID GPF Plus AMR Panel contains assays for the detection of the following genetic determinants associated with resistance to antimicrobial agents: - aac(6') aph(2') - cfr - erm(A) - erm(C) - mecA - mecC - tet(K) - tet(M) - vanA - vanB The QIAstat-Dx BCID GPF Plus AMR Panel contains assays for the detection of genetic determinants associated with resistance to gentamicin/tobramycin/kanamycin (aac(6')-aph(2')), PhLOPSA multi-class resistance phenotype (cfr), erythromycin (ermA and ermC), methicillin (mecA and mecC), tetracycline (tetK and tetM) and vancomycin (vanA and vanB). The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for certain antimicrobial resistance assays do not indicate susceptibility, as multiple mechanisms of resistance exist. The test is performed on blood culture samples identified as positive by a continuous monitoring blood culture system and gram-positive bacteria or fungi as determined by Gram stain. The QIAstat-Dx BCID GPF Plus AMR Panel is intended as an aid in diagnosis of bloodstream infections identifying specific pathogens and genetic determinants associated with resistance. Results should be used in conjunction with other clinical or laboratory information, e.g., Gram stain results. Sub-culturing of positive blood cultures is necessary for identification of organisms not detected by QIAstat-Dx BCID GPF Plus AMR Panel and susceptibility testing. Results should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results do not preclude the presence of pathogens that are not detected by this test. Positive QIAstat-Dx BCID GPF Plus AMR results do not rule out infection or co-infection with organisms not included in the QIAstat-Dx BCID GPF Plus AMR Panel. ### C Special Conditions for Use Statement(s): Rx - For Prescription Use Only For in vitro diagnostic use only ### D Special Instrument Requirements: QIAstat-Dx Analyzer 2.0 K254194 - Page 4 of 56 {4} # IV Device/System Characteristics: # A Device Description: The QIAstat-Dx BCID GPF Plus AMR Panel is an automated test for the detection and identification of nucleic acids from gram-positive bacteria, fungi, and resistance markers in a positive blood culture sample. The test is performed directly on blood culture media using blood culture bottles identified as positive by a continuous monitoring blood culture system, and which contain gram-positive bacteria or fungi, as determined by Gram stain. The QIAstat-Dx BCID GPF Plus AMR Panel test system consists of: - The QIAstat-Dx BCID GPF Plus AMR Panel cartridge kit containing six (6) individually packed, single use cartridges, which are pre-packaged with wet and dry chemistry reagents necessary to perform sample preparation, nucleic acid amplification, and assay-specific PCR detection, and six (6) individually packed, single use transfer pipettes. - The QIAstat-Dx Analyzer 2.0 consisting of one (1) Operational Module PRO and up to four (4) Analytical Modules with software version 1.6 or higher. The QIAstat-Dx Application Software is embedded in the QIAstat-Dx Analyzer 2.0. The Assay Definition File (ADF) is panel-specific software installed on the Operational Module PRO. Calibrators and Controls: Negative and positive external controls are recommended but not provided with the QIAstat-Dx BCID GPF Plus AMR Panel. An internal control ("IC") within the cartridge which provides a full process control covering lysis, nucleic acid purification, and DNA amplification. The IC is Schizosaccharomyces pombe and is located in the IC cavity and mixed with the sample during sample preparation (See "Principle of Operation"). The QIAstat-Dx Analyzer 2.0 is provided factory calibrated and does not require user calibration. The QIAstat-Dx Analyzer 2.0 includes self-check controls to verify the performance of all sensors and actuators and alert the user in case of failure. # B Principle of Operation: The QIAstat-Dx BCID GPF Plus AMR Panel is for use with the QIAstat-Dx Analyzer 2.0. Samples grown in BACT/ALERT and BACTEC bottles are diluted in saline prior to loading the cartridge into the instrument, while VersaTrek bottles do not require dilution. Following pre-dilution, the sample is loaded into the Lysis Chamber of the cartridge with a transfer pipette, and the cartridge is inserted into the Analytical Module of the analyzer. All reagents are pre-loaded and self-contained in the cartridge. The QIAstat-Dx Analyzer 2.0 runs the predefined assay protocols, and multiple steps are automatically performed by the analyzer. Resuspension of Internal Control (IC) and Proteinase K: Following insertion of the cartridge, the IC and Proteinase K is resuspended with the buffer located in Reservoir 1. The buffer is added to the interconnected IC cavity and Proteinase K cavity and transferred repeatedly between the transfer chamber and the cavities to ensure resuspension. K254194 - Page 5 of 56 {5} Cell Lysis: Lysis of the sample and IC occurs by a combination of chemical and mechanical processes using a rotor inside the lysis chamber in the presence of silica beads and a buffer that acts as a chemical agent in aiding the mechanical process. The fast movement of the rotor in the presence of the silica beads results in sample agitation, which creates turbulence and shear forces that lyses the cell wall. Following mechanical lysis, the primary lysate is transferred to the purification chamber through a frit of 80 µm pore size. The second lysis buffer (from Reservoir 2) is added to the primary lysate to complete chemical lysis. Nucleic Acid Purification: Buffer containing a high chaotropic salt concentration (from Reservoir 4) is added to the lysate in the purification chamber and allowed to bind to the membrane, and the remaining unwanted cellular components of the lysate are washed away with washing buffer from Reservoirs 5 and 6. Once completed, the nucleic acids are released from the membrane using an elution buffer from Reservoir 8. Rehydration of Master Mix: The eluate is delivered to the reservoir of the Dry Chemistry Container (DCC) to rehydrate the Master Mix and is mixed. Aliquoting and PCR: Aliquots (15 µL) of mixed eluate/Master Mix are transferred to each of eight (8) Reaction Chambers containing the target-specific primers and probes. Within each Reaction Chamber, multiplex, real-time PCR is performed. Increase in fluorescence (indicative of detection of each target analyte) is detected directly within each Reaction Chamber. Reporting of Results: Test results are generated in approximately 75 minutes. Detected targets are reported as “Positive/Detected”. Targets that are not detected are reported as “Negative/Not Detected”. The results screen displays a message indicating that the IC “Passed” when the test is run successfully, and a message of “Failed” indicates that the internal control was not amplified. If the IC fails, detected targets are reported as “Positive with Warning” (“POSITIVE*”), and negative results are reported as “Invalid”, and testing should be repeated. # V Substantial Equivalence Information: # A Predicate Device Name(s): ePlex Blood Culture Identification Gram Positive (BCID-GP) Panel, ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel # B Predicate 510(k) Number(s): K181663, K182690 # C Comparison with Predicate(s): | Device & Predicate Device(s): | K251440 | K181663 | K182690 | | --- | --- | --- | --- | | Device Trade Name | QIAstat-Dx BCID GPF Plus AMR Panel | ePlex BCID-GP Panel | ePlex BCID-FP Panel | | General Device Characteristic Similarities | | | | K254194 - Page 6 of 56 {6} | Intended Use/Indications For Use | The QIAstat-Dx BCID GPF Plus AMR Panel is a qualitative multiplexed nucleic acid real-time PCR-based in vitro diagnostic test intended for use with the QIAstat-Dx Analyzer 2.0. The QIAstat-Dx BCID GPF Plus AMR Panel is capable of simultaneous detection and identification of multiple gram-positive bacterial and fungal pathogens and selected genetic determinants associated with antimicrobial resistance. Additionally, the QIAstat-Dx BCID GPF Plus AMR Panel is capable of detecting a wide variety of gram-negative bacteria (Pan Gram Negative assay). The QIAstat-Dx BCID GPF Plus AMR Panel is intended for use in positive blood culture samples from patients with blood infections identified via Gram stain to contain gram-positive bacteria or fungi. The following gram-positive bacteria are identified and differentiated using the QIAstat-Dx BCID GPF Plus AMR Panel: [See list of organism targets below] The following fungi are identified and differentiated using the QIAstat-Dx BCID GPF Plus AMR Panel: [See list of organism targets below] The QIAstat-Dx BCID GPF Plus AMR Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include: • A broad Pan Gram-Negative assay • Candida Group 1 [See list of group targets below] | The GenMark ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-positive bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GP Panel is capable of detecting a wide variety of gram-negative bacteria (Pan Gram-Negative assay) and several Candida species (Pan Candida assay). The ePlex BCID-GP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-positive organism. The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GP Panel: [See list of organism targets below] The ePlex BCID-GP Panel contains assays for the detection of genetic determinants associated with resistance to methicillin (mecA and mecC) and vancomycin (vanA and vanB) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. The ePlex BCID-GP Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or | The GenMark ePlex Blood Culture Identification Fungal Pathogen (BCID-FP) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous detection and identification of multiple potentially pathogenic fungal organisms in positive blood culture. The ePlex BCID-FP Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain fungal organism. The following fungal organisms are identified using the ePlex BCID-FP Panel: [See list of organism targets below] The detection and identification of specific fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-FP Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-FP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-FP Panel, susceptibility testing and | | --- | --- | --- | --- | K254194 - Page 7 of 56 {7} | | • Candida Group 2 [See list of group targets below] The QIAstat-Dx BCID GPF Plus AMR Panel contains assays for the detection of the following genetic determinants associated with resistance to antimicrobial agents: [See list of gene targets below] The QIAstat-Dx BCID GPF Plus AMR Panel contains assays for the detection of genetic determinants associated with resistance to gentamicin/tobramycin/kanamycin (aac(6')-aph(2')), PhLOPSA multi-class resistance phenotype (cfr), erythromycin (ermA and ermC), methicillin (mecA and mecC), tetracycline (tetK and tetM) and vancomycin (vanA and vanB). The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for certain antimicrobial resistance assays do not indicate susceptibility, as multiple mechanisms of resistance exist. The test is performed on blood culture samples identified as positive by a continuous monitoring blood culture system and as gram-positive bacteria or fungi as determined by Gram stain. The QIAstat-Dx BCID GPF Plus AMR Panel is intended as an aid in diagnosis of bloodstream infections identifying specific pathogens and genetic determinants associated with resistance. Results should be used in conjunction with other clinical and laboratory information, e.g., Gram stain results. Sub-culturing of positive blood cultures is necessary for identification of organisms not detected by QIAstat-Dx BCID GPF Plus AMR Panel and susceptibility testing. Results | organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include a broad Pan Gram-Negative assay as well as a Pan Candida assay, which is designed to detect four of the most prevalent Candida species: Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GP Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-GP Panel and for susceptibility testing, differentiation of mixed growth and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of blood stream infection. | differentiation of mixed growth) and clinical presentation must be taken into consideration in the final diagnosis of bloodstream infection. | | --- | --- | --- | --- | K254194 - Page 8 of 56 {8} | | should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results do not preclude the presence of pathogens that are not detected by this test. Positive QIAstat-Dx BCID GPF Plus AMR results do not rule out infection or co-infection with organisms not included in the QIAstat-Dx BCID GPF Plus AMR Panel. | | | | --- | --- | --- | --- | | Measurand | Target DNA of gram-positive bacteria, fungi, and resistance markers | Target DNA of gram-positive bacteria and resistance markers | Target DNA of fungi | | Specimen Type | Positive blood culture (PBC) bottles | Same | Same | | Media Types | • BACTEC Plus Aerobic • BACTEC Plus Anaerobic • BACTEC Standard Aerobic • BACTEC Standard Anaerobic • BACTEC Peds Plus • BACTEC Lytic Anaerobic • BACT/ALERT SA Standard Aerobic • BACT/ALERT SN Standard Anaerobic • BACT/ALERT FA PLUS Aerobic • BACT/ALERT FN PLUS Anaerobic • BACT/ALERT PF PLUS Aerobic • Versatrek Redox 1 EZ Draw Aerobic • Versatrek Redox 2 EZ Draw Anaerobic | Same | Same plus: • BACTEC Myco • BACT/ALERT MP Mycobacteria | | Test Design | Test cartridge | Same | Same | | Test Technology | Multiplex, nucleic acid amplification test (NAAT) | Same | Same | | Sample Processing and Extraction | Automated in instrument | Same | Same | | Quality Controls | Internal processing control. External controls are recommended. | Internal control | Internal control | | Results Output | Positive/Detected Negative/Not Detected | Same | Same | | **General Device Characteristic Differences** | | | | | Organisms Detected | Gram-positive: • *Bacillus cereus* group • *Corynebacterium* | • *Bacillus cereus* group • *Bacillus subtilis* group • *Corynebacterium* | • *Candida albicans* • *Candida auris* | K254194 - Page 9 of 56 {9} | | • Enterococcus faecalis • Enterococcus faecium • Listeria monocytogenes • Micrococcus spp. • Staphylococcus aureus • Staphylococcus epidermidis • Staphylococcus lugdunensis • S. capitis/S. hominis • Streptococcus agalactiae • Streptococcus anginosus group • Streptococcus pneumoniae • Streptococcus pyogenes Fungi: • Cryptococcus neoformans/gattii • Fusarium • Candida auris Pan Gram-negative Candida Group 1: • Candida albicans • Candida tropicalis • Candida dubliniensis • Candida famata • Candida guilliermondii • Candida kefyr Candida Group 2: • Candida glabrata • Candida krusei • Candida parapsilosis • Candida lusitaniae | • Cutibacterium acnes (Propionibacterium acnes) • Enterococcus • Enterococcus faecalis • Enterococcus faecium • Lactobacillus • Listeria • Listeria monocytogenes • Micrococcus • Staphylococcus • Staphylococcus aureus • Staphylococcus epidermidis • Staphylococcus lugdunensis • Streptococcus • Streptococcus agalactiae (GBS) • Streptococcus anginosus group • Streptococcus pneumoniae • Streptococcus pyogenes (GAS) • Pan Gram-negative • Pan Candida | • Candida dubliniensis • Candida famata • Candida glabrata • Candida guilliermondii • Candida kefyr • Candida krusei • Candida lusitaniae • Candida parapsilosis • Candida tropicalis • Cryptococcus gattii • Cryptococcus neoformans • Fusarium • Rhodotorula. | | --- | --- | --- | --- | | Resistance Markers Detected | • aac(6') aph(2') • cfr • erm(A) • erm(C) • mecA • mecC • tet(K) • tet(M) • vanA • vanB | • mecA • mecC • vanA • vanB | N/A | | Equipment Required | QIAstat-Dx Analyzer 2.0 with QIAstat-Dx Application Software and QIAstat-Dx BCID GPF Plus AMR Panel Assay Definition File | GenMark ePlex instrument with GenMark ePlex System Software and GenMark ePlex BCID-GP Panel Software | GenMark ePlex instrument with GenMark ePlex System Software and GenMark ePlex BCID-FP Panel Software | # VI Standards/Guidance Documents Referenced: | Document # | Title | | --- | --- | | ISO 14971 Third Edition 2019-12 | Medical Devices - Application of Risk Management to Medical Devices | K254194 - Page 10 of 56 {10} | IEC 62304 Edition 1.1 2015-06 CONSOLIDATED VERSION | Medical device software - Software life cycle processes | | --- | --- | | IEC 81001-5-1 Edition 1.0 2021-12 | Health software and health IT systems safety, effectiveness and security - Part 5-1: Security — Activities in the product life cycle | | IEC 61010-1 Edition 3.1 2017-01 CONSOLIDATED VERSION | Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use - Part 1: General Requirements, Including Corrigendum 1. (2017-01) | | IEC 60601-1-2 Edition 4.1 2020-09 CONSOLIDATED VERSION | Medial Electrical Equipment - Part 1-2: General Requirements for Basic Safety and Essential Performance - Collateral Standard: Electromagnetic Disturbances - Requirements and Tests (2014-02) | | IEC 62366-1 Edition 1.1 2020-06 CONSOLIDATED VERSION | Medical devices - Part 1: Application of usability engineering to medical devices | ## VII Performance Characteristics (if/when applicable): ### A Analytical Performance: #### 1. Precision/Reproducibility: A precision/reproducibility study was conducted to assess the performance of the QIAstat-Dx BCID GPF Plus AMR Panel. Two positive samples (1 low “bottle positive”, 1 high “bottle positive” + 24 hours) and one negative sample were prepared. Positive samples were prepared by combining representative, on-panel isolates covering a range of organisms. Stock isolates were inoculated with whole blood into blood culture bottles and incubated in a blood culture monitoring system until “positive ring” (plus 24 hours for high positive samples). Positive samples were then prepared by combining equal volumes of each culture, with different cartridge reaction chambers detecting different targets within each pool. The negative sample was prepared by inoculating a blood culture bottle with whole blood and an off-panel organism and incubating until “positive ring”. Table 1 shows the sample composition and testing strategy for precision/reproducibility studies. Studies were conducted in BD BACTEC Plus Aerobic bottles. Table 1. Sample Composition and Test Strategy for Reproducibility Studies | Sample | Target(s) | Organism | Source ID | | --- | --- | --- | --- | | ‘Positive’ or ‘High Positive’ | S. aureus mecC | S. aureus | ATCC BAA 2313 | | | E. faecalis VanB Aac(6') aph(2') | E. faecalis | ATCC 51299 | | | L. monocytogenes | L. monocytogenes | NCTC 5105 | | | S. anginosus group | S. anginosus | CCUG 27298 | | | C. neoformans/gattii | C. gattii | ATCC MYA 4071 | | Mix 4 (Bottle positive) or 5 (Bottle positive + 24h) | Candida spp. Group 2 | C. glabrata | ATCC 15126 | | | Corynebacterium | Corynebacterium striatum | ATCC 43735 | | | S. aureus tetK ermC mecA | S. aureus | ATCC 1556 | | | B. cereus group | B. cereus | ATCC 21769 | | Mix 6 (Bottle positive) or 7 | Fusarium | Fusarium proliferatum | NCPF 7484 | | | Pan Gram Negative | Acinetobacter baumannii | NCTC 13302 | K254194 - Page 11 of 56 {11} | (Bottle positive + 24h) | S. epidermidis Aac(6') aph(2') cfr mecA | S. epidermidis | PA212221 | | --- | --- | --- | --- | | 'Negative' | N/A | Streptococcus peroris | CCUG 39814 | ### Site-to-Site Variability A site-to-site study was conducted at three external sites using six to seven QIAstat-Dx Analyzers per site. Runs were performed at each site over the course of six non-consecutive days, with two operators each performing three replicates per sample each day, resulting in 36 replicates of each sample per site (3 replicates × 1 run × 2 operators × 6 days × 3 sites = 108 replicates at each concentration). A single lot of the candidate device was used for all runs in this study. A run was considered valid if no error code was detected by the software. Valid runs can be “Positive” (any target positive with passed Internal Control or “IC”) or “Negative” (all targets negative with passed IC). Any run that caused an error code was repeated to obtain a valid result. Results where there was a failed IC and at least one positive (“Positive with warning”) were considered invalid for analytical performance studies and were repeated to obtain a valid result. Acceptance criteria are ≥ 95% agreement of positive and high positive samples and 100% agreement of negative samples with expected results. For all positive samples, tested at all three (3) test sites, 100% detection was obtained for all expected targets (108/108 detected positive hits). Results are summarized in Table 2 below. Table 2. Results of Site-to-Site Reproducibility Study | Organism | Target Type | Agreement with Expected Results | | | | 95% C.I. | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Site 1 | Site 2 | Site 3 | Overall | Lower | Upper | | *B. cereus* group | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (37/37) | 100% (37/37) | 100% (36/36) | 100% (110/110) | 96.7% | 100% | | *Candida* spp. Group 2 | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (37/37) | 100% (37/37) | 100% (36/36) | 100% (110/110) | 96.7% | 100% | | *Corynebacterium* | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (37/37) | 100% (37/37) | 100% (36/36) | 100% (110/110) | 96.7% | 100% | | *C. neoformans/ gattii* | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | *E. faecalis* | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | *Fusarium* | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | K254194 - Page 12 of 56 {12} | Organism | Target Type | Agreement with Expected Results | | | | 95% C.I. | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Site 1 | Site 2 | Site 3 | Overall | Lower | Upper | | L. monocytogenes | Ring Positive | 100%(36/36) | 100%(36/36) | 100%(36/36) | 100%(108/108) | 96.6% | 100% | | | Ring Positive+24 Hours | 100%(36/36) | 100%(36/36) | 100%(36/36) | 100%(108/108) | 96.6% | 100% | | Pan Gram Negative | Ring Positive | 100%(36/36) | 100%(36/36) | 100%(36/36) | 100%(108/108) | 96.6% | 100% | | | Ring Positive+24 Hours | 100%(36/36) | 100%(36/36) | 100%(36/36) | 100%(108/108) | 96.6% | 100% | K254194 - Page 13 of 56 {13} | Organism | Target Type | Agreement with Expected Results | | | | 95% C.I. | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Site 1 | Site 2 | Site 3 | Overall | Lower | Upper | | mecC | Ring Positive +24 Hours | 100% (37/37) | 100% (37/37) | 100% (36/36) | 100% (110/110) | 96.7% | 100% | | | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | tetK | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (37/37) | 100% (37/37) | 100% (36/36) | 100% (110/110) | 96.7% | 100% | | VanB | Ring Positive | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | | | Ring Positive +24 Hours | 100% (36/36) | 100% (36/36) | 100% (36/36) | 100% (108/108) | 96.6% | 100% | # *Lot-to-Lot Variability* A lot-to-lot study was conducted at one site using one operator on three or four QIAstat-Dx Analyzers per day over the course of 16 consecutive days. Three cartridge lots were used in the study generating a total of 90 replicates per sample. For all positive samples, 100% detection was obtained for all expected targets (90/90 detected positive hits), and 100% negative calls were observed for the negative samples. Results are summarized in Table 3 below. Table 3. Results of Lot-to-Lot Repeatability Study | Organism | Target Type | Agreement with Expected Results | | | | 95% C.I. | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Lot 1 | Lot 2 | Lot 3 | Overall | Lower | Upper | | *B. cereus* group | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | *Candida* spp. Group 2 | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | *Corynebacterium* | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | *C. neoformans/ gattii* | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | *E. faecalis* | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | *Fusarium* | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (31/31) | 100% (91/91) | 96.0% | 100% | K254194 - Page 14 of 56 {14} | Organism | Target Type | Agreement with Expected Results | | | | 95% C.I. | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Lot 1 | Lot 2 | Lot 3 | Overall | Lower | Upper | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | L. monocytogenes | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | Pan Gram Negative | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (31/31) | 100% (91/91) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | S. aureus | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | S. epidermidis | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (31/31) | 100% (91/91) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | S. anginosus group | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | S. peroris | Off-panel Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | Resistance Markers | | | | | | | | | Aac(6') aph(2') (E. faecalis) | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | Aac(6') aph(2') (S. epidermidis) | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (31/31) | 100% (91/91) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | cfr | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (31/31) | 100% (91/91) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | ermC | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | mecA | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (31/31) | 100% (91/91) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | K254194 - Page 15 of 56 {15} | Organism | Target Type | Agreement with Expected Results | | | | 95% C.I. | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | Lot 1 | Lot 2 | Lot 3 | Overall | Lower | Upper | | mecC | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | tetK | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | VanB | Ring Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | | | Ring Positive +24 Hours | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | 96.0% | 100% | ### 2. Linearity: This study is not applicable as the test device is a qualitative assay. ### 3. Analytical Specificity/Interference: The QIAstat-Dx BCID GPF Plus AMR Panel was evaluated for analytical specificity/cross-reactivity and interference. #### Cross-Reactivity A total of 140 organisms, including those phylogenetically related to the on-panel organisms and organisms likely to be present in clinical blood culture specimens, were tested to verify the absence of cross-reactivity with the candidate device. Samples were prepared by spiking organisms at high concentration into blood culture bottles at a minimum concentration of \(\geq 1.50 \times 10^{8}\) CFU/mL for bacteria and \(\geq 1.50 \times 10^{6}\) CFU/mL for fungi and tested in triplicate. Where cross-reactivity was observed at the initial concentration (D0) tested, samples were diluted and retested until no cross-reactivity was detected. Table 4 lists the organisms subjected to wet testing on the candidate device. Table 5 lists the organisms for which cross-reactivity was observed, the targets detected by the candidate device, and the concentration at which the organism was no longer cross-reactive on the candidate device. Table 4. List of Microorganisms Evaluated for Cross-Reactivity | Organism | Concentration Tested (CFU/mL) | Organism | Concentration Tested (CFU/mL) | | --- | --- | --- | --- | | Acinetobacter junii | 1.5E+08 | Neisseria lactamica | 1.5E+08 | | Actinomyces odontolyticus | 1.5E+08 | Neisseria mucosa | 1.5E+08 | | Aerococcus sanguinicola | 1.5E+08 | Ochrobactrum anthropic | 1.5E+08 | | Aerococcus urinae | 1.5E+08 | Pediococcus acidilactici | 1.5E+08 | | Aerococcus viridans | 1.5E+08 | Pediococcus pentosaceus | 1.5E+08 | | Anaerococcus prevotii | 1.5E+08 | Penicillium/Talaromyces marneffei (gDNA) | 2.35E+07 copies/mL | | Arcanobacterium haemolyticum | 1.5E+08 | Peptostreptococcus anaerobius | 1.5E+08 | | Bacillus amyloliquefaciens | 1.5E+08 | Prevotella oralis | 1.5E+08 | | Bacillus atrophaeus | 1.5E+08 | Propionibacterium propionicum | 1.5E+08 | K254194 - Page 16 of 56 {16} | Organism | Concentration Tested (CFU/mL) | Organism | Concentration Tested (CFU/mL) | | --- | --- | --- | --- | | Bacillus badius | 1.5E+08 | Providencia rettgeri | 1.5E+08 | | Bacillus licheniformis | 1.5E+08 | Pseudomonas putida | 1.5E+08 | | Bacillus pumilus | 1.5E+08 | Raoultella ornithinolytica | 1.5E+08 | | Bacillus subtilis | 1.5E+08 | Rhodococcus equi | 1.5E+08 | | Bacteroides distasonis (Parabacteroides) | 1.5E+08 | Rhodococcus fascians | 1.5E+08 | | Bacteroides eggerthii | 1.5E+08 | Rhodotorula glutinis | 1.5E+08 | | Bacteroides ovatus | 1.5E+08 | Rhodotorula minuta | 1.5E+08 | | Bacteroides ureolyticus (Campylobacter ureolyticus) | 1.5E+08 | Rhodotorula mucilaginosa | 1.5E+08 | | Brochothrix thermosphacta | 1.5E+08 | Rothia dentocariosa | 1.5E+08 | | Burkholderia cepacian | 1.5E+08 | Rothia mucilaginosa | 1.5E+08 | | Candida lambica | 1.5E+08 | Saccharomyces cerevisiae | 1.5E+08 | | Candida lipolytica | 1.5E+08 | Shigella boydii | 1.5E+08 | | Candida metapsilosis | 1.5E+08 | Shigella sonnei | 1.5E+08 | | Candida orthopsilosis | 1.5E+08 | Staphylococcus arlettae | 1.5E+08 | | Candida pelliculosa/Wickerhamomyces | 1.5E+08 | Staphylococcus auricularis | 1.5E+08 | | Candida/Wickerhamiella rugosa | 1.5E+08 | Staphylococcus carnosus | 1.5E+08 | | Candida sake | 1.5E+08 | Staphylococcus chromogenes | 1.5E+08 | | Candida utilis | 1.5E+08 | Staphylococcus cohnii | 1.5E+08 | | Carnobacterium divergens | 1.5E+08 | Staphylococcus gallinarum | 1.5E+08 | | Carnobacterium maltaromaticum | 1.5E+08 | Staphylococcus haemolyticus | 1.5E+08 | | Cellulosimicrobium cellulans | 1.5E+08 | Staphylococcus hycius | 1.5E+08 | | Cellulomonas turbata | 1.5E+08 | Staphylococcus intermedius | 1.5E+08 | | Clostridium clostridioforme | 1.5E+08 | Staphylococcus lentus | 1.5E+08 | | Clostridium perfringens | 1.5E+08 | Staphylococcus muscae | 1.5E+08 | | Propionibacterium/Cutibacterium acnes | 2.13E+08 | Staphylococcus pasteuri | 1.5E+08 | | Priopionibacterium/Cutibacterium granulosum | 1.5E+08 | Staphylococcus pettenkoferi | 1.5E+08 | | Enterobacter cowanii | 1.5E+08 | Staphylococcus pseudintermedius | 1.5E+08 | | Enterococcus avium | 1.5E+08 | Staphylococcus saccharolyticus | 1.5E+08 | | Enterococcus casseliflavus | 1.5E+08 | Staphylococcus saprophyticus | 1.5E+08 | | Enterococcus cecorum | 1.5E+08 | Staphylococcus schleiferi | 1.5E+08 | | Enterococcus dispar | 1.5E+08 | Staphylococcus sciuri | 1.5E+08 | | Enterococcus durans | 1.5E+08 | Staphylococcus simulans | 1.5E+08 | | Enterococcus gallinarum | 1.5E+08 | Staphylococcus vitulinus | 1.5E+08 | | Enterococcus hirae | 1.5E+08 | Staphylococcus warneri | 1.5E+08 | | Enterococcus italicus | 1.5E+08 | Staphylococcus warneri | 1.5E+08 | | Enterococcus lactis | 1.5E+08 | Staphylococcus xylosus | 1.5E+08 | | Enterococcus malodoratus | 1.5E+08 | Streptococcus canis | 1.5E+08 | | Enterococcus pseudoavium | 1.5E+08 | Streptococcus criceti | 1.5E+08 | | Enterococcus raffinosus | 1.5E+08 | Streptococcus dysgalactiae | 1.5E+08 | | Enterococcus saccharolyticus | 1.5E+08 | Streptococcus equi | 1.5E+08 | | Erysipelothrix rhusiopathiae | 1.5E+08 | Streptococcus equinus | 1.5E+08 | | Gemella haemolysans | 1.5E+08 | Streptococcus gallolyticus | 1.5E+08 | | Gemella morbillorum | 1.5E+08 | Streptococcus gordonii | 1.5E+08 | | Geotrichum capitatum (Blastoschizomyces capitatus) | 1.5E+08 | Streptococcus infantarius | 1.5E+08 | | Granulicatella adiacens | 1.5E+08 | Streptococcus infantis | 1.5E+08 | | Granulicatella elegans | 1.5E+08 | Streptococcus mitis | 1.5E+08 | | Kingella kingae | 1.5E+08 | Streptococcus oralis | 1.5E+08 | | Kocuria kristinae | 1.5E+08 | Streptococcus parasanguinis | 1.5E+08 | | Kocuria rhizophila | 1.5E+08 | Streptococcus peroris | 1.5E+08 | | Kytococcus sedentarius | 1.5E+08 | Streptococcus pseudopneumoniae | 1.5E+08 | | Lactobacillus zeae | 1.5E+08 | Streptococcus salivarius | 1.5E+08 | | Lactococcus garvieae | 1.5E+08 | Streptococcus sanguinis | 1.5E+08 | | Lactococcus lactis subsp. | 1.5E+08 | Streptococcus thoraltensis | 1.5E+08 | | Lawsonella clevelandensis | 1.5E+08 | Trichosporon asahii | 8.00E+06 | K254194 - Page 17 of 56 {17} | Organism | Concentration Tested (CFU/mL) | Organism | Concentration Tested (CFU/mL) | | --- | --- | --- | --- | | Leuconostoc carnosum | 1.5E+08 | Trueperella bernardiae | 1.5E+08 | | Leuconostoc citreum | 1.5E+08 | Vagococcus fluvialis | 1.5E+08 | | Leuconostoc mesenteroides | 1.5E+08 | Vibrio parahaemolyticus | 1.5E+08 | | Listeria ivanovii | 1.5E+08 | Weissella paramesenteroides | 1.5E+08 | | Macrococcus caseolyticus (subsp. Hominis) | 1.5E+08 | Corynebacterium simulans | 1.5E+08 | | Mycobacterium tuberculosis (gDNA) | 1.5E+08 copies/mL | Staphylococcus caprae | 1.5E+08 | Table 5. List of Microorganisms Where Cross-Reactivity Observed | Organism | Target Detected | Concentration (CFU/mL) | | --- | --- | --- | | Bacteroides ureolyticus (Campylobacter ureolyticus) | Corynebacterium | 1.64E+06 | | Propionibacterium/Cutibacterium acnes | Corynebacterium | 7.28 E+05 | | Propionibacterium/Cutibacterium granulosum | Corynebacterium | 1.50E+05 | | Mycobacterium tuberculosis | Corynebacterium | 5.20E+06 | | Rhodococcus equi | Corynebacterium | 4.75E+07 | | Rhodococcus fascians | Corynebacterium | 1.50E+07 | | Rothia dentocariosa | Corynebacterium | 5.20E+06 | | Rothia mucilaginosa | Corynebacterium | 1.64E+07 | | Ochrobactrum anthropic | Corynebacterium | 1.64E+07 | | Lawsonella clevelandensis | Corynebacterium | 1.64E+06 | | Enterococcus lactis | Enterococcus faecium | 2.10E+03 | | Staphylococcus caprae | Staphylococcus capitis/hominis | 1.50E+06 | ### In silico Analysis Primers and probes included in the QIAstat-Dx BCID GPF Plus AMR Panel were analyzed to evaluate their specificity using BLAST homology analysis run against the Core nucleotide database from The National Center for Biotechnology Information (NCBI) database. A dedicated analysis was performed to determine the specificity of the PCR assays detecting AMR genes. Results of the in silico analysis are shown in Table 6 below. Target organisms for which sequences predicted potential cross-reactivity are listed. Table 6. Summary Results of In silico Cross-Reactivity Analysis | Target | In silico Prediction | | --- | --- | | L. monocytogenes | Cross-reaction with L. innocua | | Corynebacterium | Cross-reaction with M. luteus | | S. anginosus group | Cross-reaction with S. periodonticum | | S. capitis / S. hominis | Cross-reaction with S. caprae, S. debuckii and S. piscifermentans | | Pan Gram-Negative | Additional detection of N. brasiliensis by N. meningitidis. Additional detection of S. acidaminiphila, S. riyadhensis, and S. tuberculopleuritidis strains by S. maltophilia. | ### Competitive Inhibition (Microbial Interference) A competitive inhibition study was conducted to evaluate the capability of the QIAstat-Dx BCID GPF Plus AMR Panel to detect multiple analytes in one sample, when one pathogen is present at a high concentration and another pathogen is present at a lower concentration. K254194 - Page 18 of 56 {18} Twenty-five samples from commercial suppliers or clinical isolates were used to create representative combinations of on-panel and off-panel organisms that are commonly found in polymicrobial positive blood cultures. Samples were prepared by inoculating culture bottles with isolates and whole blood and incubated in a blood culture instrument until “positive ring” plus 24 hours to ensure a high positive concentration. Mixtures were prepared by spiking one organism at high concentration into Negative Blood Culture Matrix (NBCM) (bottle positivity + 24 hours of incubation), and the second organism at a lower concentration (3x LoD). Studies were conducted in triplicate in BD BACTEC Plus Aerobic bottles. Table 7 below lists the sample mixtures and concentrations used. Mixtures 4 (Tet(M) harbored by E. faecium), 9 (S. capitis), 12 (S. aureus), and 18 (A. baumannii) were only detected when the concentration was increased to 30x-300x LoD (~10⁶ CFU/mL). Table 7. List of Tested Organism Mixtures in Competitive Inhibition Study | Mixture | High Positive | | Low Positive | | | --- | --- | --- | --- | --- | | | Organism | Concentration (CFU/mL) | Organism | Concentration (CFU/mL) | | 01 | Candida albicans (Candida Group 1) | 1.83E+07 | Candida glabrata | 6.20E+05 | | | Candida glabrata (Candida Group 2) | 6.21E+07 | Candida albicans | 2.04E+06 | | 02 | Candida albicans (Candida Group 1) | 1.83E+07 | Enterococcus faecalis | 8.25E+05 | | | Enterococcus faecalis | 4.64E+08 | Candida albicans | 2.04E+06 | | 03 | Candida glabrata (Candida Group 2) | 6.21E+07 | Streptococcus pneumoniae | 2.10E+04 | | | Streptococcus pneumoniae | N/A | Candida glabrata | 6.20E+05 | | 04 | Enterococcus faecium | 8.96E+08 | Enterococcus faecalis | 1.50E+06 | | | Enterococcus faecalis | 7.25E+08 | Enterococcus faecium | 4.08E+04 | | 05 | Staphylococcus aureus | 2.09E+08 | Enterococcus faecalis | 1.50E+06 | | | Enterococcus faecalis | 7.25E+08 | Staphylococcus aureus | 1.43E+04 | | 06 | Streptococcus pneumoniae | N/A | Enterococcus faecalis | 1.50E+06 | | | Enterococcus faecalis | 7.25E+08 | Streptococcus pneumoniae | 2.10E+04 | | 07 | Streptococcus agalactiae | 4.16E+08 | Streptococcus anginosus | 1.45E+05 | | | Streptococcus anginosus | 3.80E+07 | Streptococcus agalactiae | 9.00E+05 | | 08 | Staphylococcus aureus | 2.09E+08 | Staphylococcus epidermidis | 1.03E+05 | | | Staphylococcus epidermidis | 7.29E+08 | Staphylococcus aureus | 1.43E+04 | | 09 | Staphylococcus epidermidis | 7.29E+08 | Staphylococcus capitis | 2.31E+05 | | | Staphylococcus capitis | 2.86E+09 | Staphylococcus epidermidis | 1.03E+05 | | 10 | Streptococcus pyogenes | 9.45E+07 | Micrococcus luteus | 8.65E+05 | | | Micrococcus luteus | 2.13E+08 | Streptococcus pyogenes | 3.00E+05 | | 11 | Staphylococcus lugdunensis | 1.96E+08 | Corynebacterium striatum | 2.14E+06 | | | Corynebacterium striatum | 9.54E+08 | Staphylococcus lugdunensis | 4.50E+03 | | 12 | Streptococcus agalactiae | 4.16E+08 | Staphylococcus aureus | 1.43E+04 | | | Staphylococcus aureus | 2.09E+08 | Streptococcus agalactiae | 9.00E+05 | | 13 | Streptococcus pneumoniae | N/A | Escherichia coli (Pan GN) | 1.21E+07 | | | Escherichia coli (Pan GN) | 4.16E+08 | Streptococcus pneumoniae | 2.10E+04 | | 14 | Staphylococcus aureus | 2.09E+08 | Acinetobacter baumannii (Pan GN) | 4.17E+04 | | | Acinetobacter baumannii (Pan GN) | 2.72E+10 | Staphylococcus aureus | 1.43E+04 | | 15 | Pseudomonas aeruginosa (Pan GN) | 1.41E+09 | Micrococcus luteus | 8.65E+05 | | 16 | Cutibacterium acnes | 1.71E+08 | Bacillus cereus | 1.84E+06 | | 17 | Klebsiella pneumoniae (Pan GN) | 1.40E+09 | Staphylococcus aureus | 1.43E+04 | | 18 | Serratia marcescens (Pan GN) | 4.82E+08 | Enterococcus faecalis | 8.25E+05 | | 19 | Proteus mirabilis (Pan GN) | 3.17E+09 | Staphylococcus aureus | 1.43E+04 | | 20 | Bacteroides fragilis (Pan GN) | 2.27E+09 | Streptococcus anginosus | 1.45E+05 | | 21 | Enterobacter cloacae (Pan GN) | 1.08E+09 | Candida albicans | 2.04E+06 | ### Endogenous/Exogenous Interference An interfering substances study was conducted to evaluate whether potentially interfering endogenous or exogenous substances that may be present in blood culture specimens impact the performance of the QIAstat-Dx BCID GPF Plus AMR Panel. Two positive sample mixes K254194 - Page 19 of 56 {19} were prepared by combining five and three representative, on-panel isolates covering the range of target organisms, with different reaction chambers detecting different targets. Stock isolates were inoculated with whole blood into blood culture bottles and incubated in a blood culture monitoring system until “positive ring”. Positive samples were then prepared by combining equal volumes of each culture (at 3x LoD). The list of representative organisms is shown in Table 8. Interfering substances were individually spiked into the positive mix to achieve the concentrations listed in Table 9. All targets were detected, and no interference was observed from any substance evaluated. Table 8. List of Organisms in Test Mixtures | Mix | Organism | Concentration (CFU/mL) | | --- | --- | --- | | 1 | S. aureus | 1.43E+04 | | | C. gattii | 3.00E+04 | | | L. monocytogenes | 6.36E+04 | | | E. faecalis | 6.36E+05 | | | S. anginosus | 7.35E+04 | | 2 | B. cereus | 3.86E+06 | | | C. glabrata | 1.31E+06 | | | A. baumannii | 8.78E+06 | Table 9. List of Potentially Interfering Substances | Substance | Concentration in Sample (units/mL) | Solvent | | --- | --- | --- | | Acetaminophen | 0.156mg/mL | Water | | Amoxicillin Clavulanate | 0.075mg/mL | Water | | Amphotericin B | 0.002mg/mL | DMSO | | Ara-C Triphosphate | 2.64μg/mL | Water | | Biotin (Vitamin B7) | 0.00351mg/mL | Water | | Sodium hypochlorite solution (Bleach) | 5% (v/v)/55.5mg/mL | N/A | | Caspofungin Diacetate | 0.005mg/mL | DMSO | | Ceftriaxone Disodium Hemiheptahydrate | 0.966mg/mL | Water | | Ciprofloxacin | 0.01mg/mL | Water | | Ethanol | 5% (v/v)/39.5mg/mL | N/A | | Fluconazole | 0.075mg/mL | Ethanol | | 5-Fluorocytosine | 0.09mg/mL | Water | | Gentamicin sulfate | 0.03mg/mL | Water | | Heparin Sodium | 3Units/mL | Water | | Ibuprofen | 0.219mg/mL | DMSO | | Imipenem Monohydrate | 0.5mg/mL | Water | | Povidone (iodinated) | 5mg/mL | Water | | Salicylic Acid | 0.0286mg/mL | Water | | Sodium Polyanethol Sulfonate (SPS) | 0.0025mg/mL | Water | | Tetracycline Hydrochloride | 0.015mg/mL | Water | | Vancomycin (hydrochloride) | 0.12mg/mL | Water | | Bilirubin | 0.25mg/mL | Chloroform | | Cholesterol | 0.5mg/mL | Ethanol | | D-Glucose | 5mg/mL | Water | | γ-globulin | 60mg/mL | NaCl | | Hemoglobin | 5mg/mL | Water | | Magnesium Sulphate (MgSO4) | 0.1mg/mL | Water | | Human genomic DNA | 10.55μg/mL | N/A | K254194 - Page 20 of 56 {20} | Human Serum Albumin | 25mg/mL | Water | | --- | --- | --- | | Triglycerides mixtures (Lipid standards) | 15mg/mL | Chloroform | | Fibrinogen | 0.5mg/mL | NaCl | ### 4. Detection Limit and Assay Reportable Range: #### Limit of Detection Studies Limit of detection (LoD) studies were conducted to determine the LoD for the QIAstat-Dx BCID GPF Plus AMR Panel, defined as the lowest concentration of organism detected in positive blood culture samples at least 95% of the time. The LoD was determined in combined isolate mixtures and single spiked samples. Twenty (20) sample mixtures were prepared by combining up to five organisms. Studies were conducted in BACTEC Plus Aerobic bottles. Stocks were inoculated into blood culture bottles and incubated until “positive ring”. Concentrations (in CFU/mL) in culture were determined, and organisms were combined to form sample mixtures. Mixtures were serially diluted, and if at least 19/20 replicates (95%) were positive for all intended targets and a minimum of two (2) dropouts (< 95%) were observed at the higher dilution (0.1x LoD), then the LoD was established. Table 10 shows the results. Table 10. Results for Combined LoD Studies | Target | Organism | LoD (CFU/mL) | Combined LoD (CFU/mL) | | --- | --- | --- | --- | | Aac(6')aph(2') | E. faecalis | 5.00E+05 | 1.28E+05 | | | S. epidermidis | 1.32E+05 | | | | S. epidermidis | 3.41E+04 | | | | S. epidermidis | 1.00E+04 | | | | S. hominis | 3.87E+04 | | | | E. faecalis | 5.03E+04 | | | B. cereus | B. cereus | 6.11E+05 | 3.13E+05 | | | B. thuringiensis | 1.54E+04 | | | C. auris | C. auris | 1.74E+06 | 1.95E+06 | | | C. auris | 2.17E+06 | | | Candida spp. Group 1 | C. tropicalis | 8.50E+04 | 2.10E+06 | | | C. dublinensis | 3.88E+06 | | | | C. famata | 6.25E+06 | | | | C. guillermondi | 4.75E+05 | | | | C. kefyr | 1.25E+06 | | | | C. albicans | 6.74E+05 | | | Candida spp. Group 2 | C. lusitaniae | 4.83E+06 | 1.31E+06 | | | C. glabrata | 2.07E+05 | | | | C. krusei | 4.37E+04 | | | | C. parapsilosis | 1.55E+05 | | | cfr | S. aureus | 4.75E+03 | 6.84E+04 | | | S. epidermidis | 1.32E+05 | | | Corynebacterium | C. striatum | 7.12E+05 | 2.28E+06 | | | C. jeikeium | 3.84E+06 | | | Cryptococcus neoformans/gattii | C. neoformans | 1.00E+03 | 5.50E+03 | | | C. gattii | 1.00E+04 | | | E. faecalis | E. faecalis | 5.00E+05 | 2.75E+05 | | | E. faecalis | 5.03E+04 | | | E. faecium | E. faecium | 4.30E+04 | 1.36E+04 | | | E. faecium | 1.36E+04 | | | Erm(A) | S. epidermis | 3.41E+04 | 1.73E+04 | | | S. aureus | 4.75E+02 | | K254194 - Page 21 of 56 {21} | Target | Organism | LoD (CFU/mL) | Combined LoD (CFU/mL) | | --- | --- | --- | --- | | Erm(C) | S. epidermidis | 1.00E+04 | 6.94E+03 | | | S. hominis | 3.87E+03 | | | Fusarium | F. oxysporum | 4.83E+03 | 7.72E+03 | | | F. proliferatum | 1.06E+04 | | | L. monocytogenes | L. monocytogenes | 6.50E+03 | 2.12E+04 | | | L. monocytogenes | 3.58E+04 | | | MecA | S. epidermidis | 1.00E+04 | 3.74E+04 | | | S. aureus | 4.75E+03 | | | | S. epidermidis | 3.41E+04 | | | | S. aureus | 4.75E+03 | | | | S. epidermidis | 1.32E+05 | | | | S. hominis | 3.87E+04 | | | MecC | S. aureus | 4.75E+03 | 2.61E+03 | | | S. aureus | 4.75E+02 | | | Micrococcus spp. | M. yunnanensis | 5.50E+05 | 2.89E+05 | | | M. luteus | 2.89E+04 | | | Pan Gram Negative | A. baumannii | 1.39E+06 | 2.72E+06 | | | E. coli | 4.04E+06 | | | S. agalactiae | S. agalactiae | 3.00E+05 | 2.25E+05 | | | S. agalactiae | 1.50E+05 | | | S. anginosus group | S. anginosus | 4.81E+04 | 2.45E+04 | | | S. anginosus | 8.76E+02 | | | S. capitis/hominis | S. capitis | 7.70E+04 | 4.04E+04 | | | S. hominis | 3.87E+03 | | | S. epidermidis | S. epidermidis | 1.00E+04 | 5.01E+04 | | | S. epidermidis | 2.41E+04 | | | | S. epidermidis | 3.41E+04 | | | | S. epidermidis | 1.32E+05 | | | S. lugdunensis | S. lugdunensis | 1.50E+03 | 5.08E+04 | | | S. lugdunensis | 1.00E+05 | | | S. pneumoniae | S. pneumoniae | 7.00E+02 | 3.85E+03 | | | S. pneumoniae | 7.00E+03 | | | S. pyogenes | S. pyogenes | 1.00E+05 | 5.50E+04 | | | S. pyogenes | 1.00E+04 | | | S. aureus | S. aureus | 4.75E+03 | 2.61E+03 | | | S. aureus | 4.75E+03 | | | | S. aureus | 4.75E+02 | | | | S. aureus | 4.75E+02 | | | Tet(K) | S. aureus | 4.75E+03 | 1.12E+04 | | | S. epidermidis | 2.41E+04 | | | | S. aureus | 4.75E+03 | | | Tet(M) | S. aureus | 1.50E+04 | 1.71E+05 | | | E. faecalis | 5.03E+05 | | | | S. aureus | 4.75E+03 | | | VanA | E. faecium | 4.30E+04 | 3.20E+04 | | | E. faecalis | 5.03E+04 | | | VanB | E. faecalis | 5.00E+05 | 3.18E+05 | | | E. faecium | 1.36E+05 | | Twenty-two single spiked samples detecting 29 targets (organism and resistance markers) were prepared in a second study to verify combined LoDs by inoculating into blood culture bottles and incubated until “positive ring”. Concentrations (in CFU/mL) in culture were determined, and samples were serially diluted. If at least 19/20 replicates (95%) were positive for all intended targets and a minimum of two dropouts (< 95%) were observed at the higher dilution (0.1x LoD), then the LoD was established. For 28 out of 29 targets, the K254194 - Page 22 of 56 {22} LoD was successfully confirmed. For the remaining S. pneumoniae target, the LoD was re-established as 2.21 × 10² CFU/mL (the combined LoD was determined as 7.00 × 10³ CFU/mL). Results were compared to the Growth and Detection Study to ensure that LoDs were at or below the concentration in positive blood culture specimens. Table 11. Results for Growth and Detection Study | Target | Organism | Established LoD (CFU/mL) | Bottle Positive Concentration (CFU/mL) | | --- | --- | --- | --- | | B. cereus | B. cereus | 6.11E+05 | 2.78E+08 | | | B. thuringiensis | 1.54E+04 | | | C. auris | C. auris | 1.74E+06 | 8.08E+06 | | | C. auris | 2.17E+06 | | | Candida spp. Group 1 | C. tropicalis | 8.50E+04 | 3.04E+06 | | | C. dublinensis | 3.88E+06 | 9.90E+06 | | | C. famata | 6.25E+06 | N/A* | | | C. guillermondi | 4.75E+05 | 3.80E+07 | | | C. kefyr | 1.25E+06 | 1.29E+09 | | | C. albicans | 6.74E+05 | 1.61E+06 | | Candida spp. Group 2 | C. lusitaniae | 1.31E+06 | 2.90E+07 | | | C. glabrata | 2.07E+05 | 1.55E+07 | | | C. krusei | 4.37E+04 | 7.86E+06 | | | C. parapsilosis | 1.55E+05 | 3.58E+06 | | Corynebacterium | C. striatum | 7.12E+05 | 1.20E+08 | | | C. jeikeium | 3.84E+06 | | | C. neoformans/gattii | C. neoformans | 1.00E+03 | 1.78E+06 | | | C. gattii | 1.00E+04 | | | E. faecalis | E. faecalis | 5.00E+05 | 6.78E+08 | | | E. faecalis | 5.03E+04 | | | E. faecium | E. faecium | 1.36E+04 | 1.06E+09 | | | E. faecium | 1.36E+04 | | | Fusarium | F. oxysporum | 4.83E+03 | 8.10E+04 | | | F. proliferatum | 1.06E+04 | | | L. monocytogenes | L. monocytogenes | 6.50E+03 | 9.63E+07 | | | L. monocytogenes | 3.58E+04 | | | Micrococcus spp. | M. yunnanensis | 5.50E+05 | 2.17E+08 | | | M. luteus | 2.89E+04 | | | Pan Gram Negative | A. baumannii | 1.39E+06 | 2.17E+08 | | | E. coli | 4.04E+06 | | | S. agalactiae | S. agalactiae | 3.00E+05 | 3.02E+08 | | | S. agalactiae | 1.50E+05 | | | S. anginosus group | S. anginosus | 4.81E+04 | 4.38E+07 | | | S. anginosus | 8.76E+02 | | | S. capitis/hominis | S. capitis | 7.70E+04 | 3.60E+06 | | | S. hominis | 3.87E+03 | | | S. epidermidis | S. epidermidis | 1.00E+04 | 4.64E+07 | | | S. epidermidis | 2.41E+04 | | | | S. epidermidis | 3.41E+04 | | | | S. epidermidis | 1.32E+05 | | | S. lugdunensis | S. lugdunensis | 1.50E+03 | 1.28E+07 | | | S. lugdunensis | 1.00E+05 | | | S. pneumoniae | S. pneumoniae | 7.00E+02 | 9.59E+07 | | | S. pneumoniae | 7.00E+03 | | | S. pyogenes | S. pyogenes | 1.00E+05 | 7.37E+08 | | | S. pyogenes | 1.00E+04 | | | S. aureus | S. aureus | 4.75E+03 | 4.12E+06 | | | S. aureus | 4.75E+03 | | | | S. aureus | 4.75E+02 | | K254194 - Page 23 of 56 {23} | | S. aureus | 4.75E+02 | | --- | --- | --- | * Concentration not available for C. famata (Debaryomyces hansenii) due to culture requirements. ### Analytical Reactivity/Inclusivity An analytical reactivity study was performed to evaluate the inclusivity of the QIAstat-Dx BCID GPF Plus AMR Panel through testing of clinically relevant subtypes, strains, genotypes, serotypes, species and AMR gene variants representing the genetic, temporal and geographic diversity for each on-panel reportable target. Strain mixtures containing up to five pathogens were prepared in negative blood culture matrix (NBCM) targeting \( \sim3x \) LoD. All on-panel organisms (n = 209) were tested in triplicate and retested at higher concentrations if they were not initially detected. If 3/3 replicates were detected at a higher concentration, the strain was determined as “detected with reduced sensitivity”. If a strain was not detected at any concentration, it was reported as “not detected”. A list of tested organisms is shown in Table 12. Strains that did not achieve 100% detection and their corresponding test results are shown in Table 13. One false positive, E. faecium (ATCC BAA-2316) for the \( Aac(6')aph(2'') \) target was observed. Table 12. List of Wet Tested Organisms for Inclusivity | Organism | Strain | Organism | Strain | Organism | Strain | | --- | --- | --- | --- | --- | --- | | Acinetobacter baumannii | NCTC 13302 | Bacillus cereus | ATCC 2769 | Bacillus thuringiensis | ATCC 35646 | | Escherichia coli | ATCC BAA 196 | Bacillus cereus | CCUG 36593 | Bacillus cereus | NCTC 10320 | | Bacillus cereus | NCTC 2599 | Bacillus mycoides | NCTC 926 | Bacillus thuringiensis | CCUG 7429T | | Bacteroides fragilis | NCTC 8560 | Brenneria alni | CCUG 48887T | Proteus penneri | CCUG 60543 | | Candida albicans | ATCC 10231 | Candida auris | CBS 12372 | Candida auris | CBS 14144 | | Candida auris | NCPF 8971 | Candida auris | NCPF 8977 | Candida auris | NCPF 8984 | | Candida dublinensis | NCPF 8516 | Candida famata | CBS 767 | Candida glabrata | ATCC 15126 | | Candida glabrata | ATCC 2001 | Candida guilliermondii | NCPF 3896 | Candida krusei | ATCC 32196 | | Candida krusei | NCPF 3930 | Candida lusitaniae | CCUG 69483 | Candida lusitaniae | NCPF 3968 | | Candida parapsilosis | ATCC 58895 | Candida parapsilosis | NCPF 3207 | Candida tropicalis | ATCC 750 | | Cedecea davisae | NCTC 13724 | Chania multitudinisentens | LMG 28304 | Citrobacter koseri | ATCC 27156 | | Corynebacterium amycolatum | CCUG 57527 | Corynebacterium afermentans subsp. Afermentans | CCUG 32103 | Corynebacterium diphtheriae | CCUG 33629 | | Corynebacterium falseni | ATCC BAA-596 | Corynebacterium imitans | CCUG 36877 | Candida kefyr | NCPF 8343 | | Corynebacterium jeikeium | ATCC BAA-949 | Corynebacterium jeikeium | NCTC 11913 | Corynebacterium pseudotuberculosis | ATCC 43924 | | Corynebacterium ulcerans | ATCC 51799 | Corynebacterium urealyticum | ATCC 43044 | Cronobacter sakazakii | ATCC 29004 | | Cryptococcus gattii | ATCC MYA-4071 | Cryptococcus gattii | CCUG 43335 | Cryptococcus neoformans | ATCC 208821 | | Cryptococcus neoformans | ATCC 90112 | Cryptococcus neoformans | CCUG 23479 | Dickeya dadantii | LMG 25991 | | Edwardsiella tarda | NCTC 10396 | Enterobacter asburiae | ATCC 35955 | Enterococcus faecalis | ATCC 51299 | | Enterococcus faecalis | ATCC 51575 | Enterococcus faecalis | ATCC BAA-2365 | Enterococcus faecalis | CCUG 19916 | | Enterococcus faecalis | NCTC 12203 | Enterococcus faecium | ATCC 51559 | Enterococcus faecium | ATCC BAA-2316 | K254194 - Page 24 of 56 {24} | Enterococcus faecium | CCUG 68933 | Enterococcus faecium | JMI 1031982 | Erwinia aphidicola | ATCC 29919 | | --- | --- | --- | --- | --- | --- | | Enterococcus faecalis | NCTC 13780 | Enterococcus faecium | ATCC 51858 | Enterococcus faecium | NCTC 12204 | | Erwinia billingiae | CCUG 50417 | Escherichia coli | CCUG 67180 | Fusarium oxysporum | CBS 267.50 | | Fusarium proliferatum | NCPF 7484 | Fusarium sacchari | CBS 134.73 | Fusarium solani | NCPF 7483 | | Fusarium verticillioides | CBS 115135 | Haemophilus influenzae | CCUG 33775 | Hafnia alvei | CCUG 59411 | | Izhakiella capsodis | LMG 28431 | Klebsiella pneumoniae | CCUG 68728 | Kluyvera cryocrescens | NCTC 10483 | | Kosakonia quasisacchari | NCTC 14272 | Leclercia adecarboxylata | NCTC 13032 | Lelliota nimipressuralis | CCUG 69901 | | Listeria monocytogenes | ATCC 19111 | Listeria monocytogenes | ATCC 19115 | Listeria monocytogenes | CCUG 67296 | | Listeria monocytogenes | NCTC 5105 | Listeria monocytogenes | NCTC 5348 | Micrococcus luteus | NCTC 196 | | Micrococcus luteus | NCTC 2665 | Micrococcus luteus | NCTC 7011 | Micrococcus luteus | NCTC 7743 | | Micrococcus lylae | CCUG 44721 | Micrococcus yunnanensis | CCUG 59864 | Streptococcus pyogenes | CCUG 74645 | | Neisseria meningitidis | CCUG 27650 | Obesubacterium proteusstrain | CCUG 26658 | Phytobacter diazotrophicus | CCUG 74074 | | Streptococcus agalactiae | NCTC 8181 | Phytophthora/Pectobacterium/Dickeya chrysanthemi | CCUG 47021 | Pluralibacter gergoviae | CCUG 33719 | | Pragia fontium | CCUG 23265 | Proteus mirabilis | ATCC BAA-2792 | Providencia stuartii | CCUG 74678 | | Pseudomonas aeruginosa | ATCC BAA-2794 | Rahnella aquatilis | CCUG 52909 | Raoultella terrigena | NCTC 13038 | | Salmonella enterica | ATCC 6962 | Scandinavium goeteborgensis | NCTC 14286 | Serratia liquefaciens | ATCC 27592 | | Shigella sonnei | CCUG 68726 | Shimwellia blattae | NCTC 12127 | Staphylococcus aureus | ATCC 700698 | | Staphylococcus aureus | ATCC BAA-977 | Staphylococcus aureus | ATCC BAA-33591 | Staphylococcus aureus | NCTC 13552 | | Staphylococcus aureus | ATCC BAA-1556 | Staphylococcus aureus | ATCC BAA-1707 | Staphylococcus aureus | ATCC BAA-2312 | | Staphylococcus aureus | CC398 / t011 | Staphylococcus aureus | CCUG 74989 | Staphylococcus aureus | NCTC 13140 | | Staphylococcus aureus | NCTC 14457 | Staphylococcus aureus | NR-10187/HFH-29994 | Staphylococcus aureus | NR-10189/HFH-30364 | | Staphylococcus aureus | NR-46069 | Staphylococcus aureus | NR-46070/USA300-0114 | Staphylococcus aureus | NR-46069 | | Staphylococcus capitis | ATCC 27840 | Staphylococcus capitis | CCUG 35142T | Staphylococcus capitis | CCUG 42761 | | Staphylococcus epidermidis | JMI 1090853 | Staphylococcus epidermidis | JMI 1151922 | Staphylococcus epidermidis | JMI 1171320 | | Staphylococcus epidermidis | NCTC 13360 | Staphylococcus epidermidis | NCTC 13924 | Staphylococcus epidermidis | PA212221 | | Staphylococcus epidermidis | ATCC 35983 | Staphylococcus epidermidis | ATCC 35984 | Staphylococcus lugdunensis | CCUG 71098 | | Staphylococcus hominis | CCUG 35516 | Staphylococcus hominis subsp. hominis | ATCC 25615 | Staphylococcus hominis subsp. novobiosepticus | CCUG 42399T | | Staphylococcus aureus | ATCC BAA-2313 | Staphylococcus lugdunensis | ATCC 49576 | Staphylococcus lugdunensis | CCUG 66510 | | Staphylococcus lugdunensis | CCUG 74993 | Staphylococcus lugdunensis | NCTC 7990 | Stenotrophomonas maltophilia | NCTC 10498 | K254194 - Page 25 of 56 {25} | Streptococcus agalactiae | ATCC 12401 | Streptococcus agalactiae | CCUG 62211 | Streptococcus agalactiae | NCTC 9410 | | --- | --- | --- | --- | --- | --- | | Streptococcus agalactiae | NCTC 9412 | Streptococcus anginosus | ATCC 9895 | Streptococcus constellatus | CCUG 67510 | | Streptococcus constellatus | NCTC 11325 | Streptococcus intermedius | ATCC 27335 | Streptococcus pneumoniae | ATCC 10357 | | Streptococcus pneumoniae | ATCC BAA-334 | Streptococcus pneumoniae | CCUG 70149 | Streptococcus pneumoniae | CCUG 71170 | | Streptococcus pyogenes | ATCC 12384 | Streptococcus pyogenes | CCUG 58107 | Streptococcus pyogenes | NCTC 12696 | | Streptococcus pyogenes | NCTC 5163 | Tatumella ptyseos | NCTC 11468 | Xenorhabdus nematophila | CCUG 14189T | | Yersinia enterocolitica subsp. enterocolitica | NCTC 12982 | Yokenella regensburgei | NCTC 12131 | Proteus vulgaris | CCUG 55607 | | Acinetobacter calcoaceticus | CCUG 57816 | Haemophilus influenzae type A | ATCC 9006 | Providencia alcalifaciens | CCUG 6325T | | Acinetobacter haemolyticus | CCUG 67703 | Haemophilus influenzae Non-typeable | ATCC 51907 | Proteus rettgeri | CCUG 23362 | | Acinetobacter johnsonii | CCUG 61200 | Haemophilus influenzae type c | ATCC 49699 | Pseudescherichia vulneris | CCUG 23001 | | Acinetobacter lwoffii | CCUG 65000 | Haemophilus influenzae type d | ATCC 9008 | Pseudomonas fluorescens | CCUG 67000 | | Acinetobacter nosocomialis | CCUG 74064T | Haemophilus influenzae type e | ATCC 8142 | Pseudomonas putida | CCUG 57833 | | Acinetobacter pittii | CCUG 72793 | Haemophilus influenzae type f | ATCC 700223 | Salmonella bongori | CCUG 63587 | | Citrobacter amalonaticus | CCUG 4860T | Klebsiella aerogenes | NRZ 101370 | Salmonella enterica subsp. enterica serovar Paratyphi | ATCC 51962 | | Citrobacter braakii | CCUG 43795 | Klebsiella michiganensis | CCUG 66352 | Salmonella enterica subsp. enterica serovar Typhi | CECT 409 | | Citrobacter farmeri | CCUG 57349 | Klebsiella oxytoca | NRZ 104905 | Serratia marcescens | NRZ 79544 | | Citrobacter freundii | CCUG 418T | Klebsiella variicola | CCUG 73561 | Serratia odorifera | CCUG 14508T | | Citrobacter werkmanii | CCUG 30793T | Klebsiella/Raoultella planticola | CCUG 63333 | Serratia plymuthica | CCUG 44368 | | Citrobacter youngae | CCUG 30791 | Neisseria meningitidis Serotype A | ATCC 13077 | Serratia proteamaculans | CCUG 51551 | | Enterobacter cloacae | AR Bank 841 | Neisseria meningitidis Serotype B | ATCC 13092 | Shigella boydii | CCUG 49022 | | Enterobacter hormaechei | CCUG 63317 | Neisseria meningitidis Serotype C | ATCC 13102 | Shigella dysenteriae | CECT 584 | | Enterobacter ludwigii | CCUG 64644 | Neisseria meningitidis Serotype Y | ATCC 35561 | Shigella flexneri | CCUG 56027 | | Enterobacter roggenkampii | CCUG 75192 | | | | | Table 13. Summary Results of Strains Tested at Higher Concentrations | Target | Organism | Detection Results | Concentration (CFU/mL) | | --- | --- | --- | --- | | Aac(6') aph(2') | E. faecium | Detected with reduced sensitivity | 8.16E+06 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | S. epidermidis | Detected with reduced sensitivity | 4.66E+06 | K254194 - Page 26 of 56 {26} | B. cereus | B. cereus | Not detected | NA | | --- | --- | --- | --- | | | B. mycoides | Not detected | NA | | C. auris | C. auris | Detected with reduced sensitivity | 5.22E+07 | | Candida spp. | | | | | Group 1 | C. famata (Debaryomyces hansenii) | Detected with reduced sensitivity | 6.25E+06 | | cfr | S. epidermidis | Detected with reduced sensitivity | 3.81E+06 | | | S. epidermidis | Detected with reduced sensitivity | 3.81E+06 | | Corynebacterium | C. afermentans subsp. Afermentans | Detected with reduced sensitivity | 1.52E+08 | | Erm(A) | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | Erm(C) | S. aureus | Detected with reduced sensitivity | 3.93E+05 | | | S. epidermidis | Detected with reduced sensitivity | 3.81E+06 | | | S. epidermidis | Detected with reduced sensitivity | 3.81E+06 | | | S. epidermidis | Not detected | NA | | Fusarium | F. sacchari | Detected with reduced sensitivity | 1.44E+05 | | | F. solani | Detected with reduced sensitivity | 2.32E+05 | | MecA | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | MecC | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | Micrococcus spp. | M. luteus | Detected with reduced sensitivity | 1.26E+07 | | | M. lylae | Not detected | NA | | Pan Gram Negative | Pseudomonas fluorescens | Detected with reduced sensitivity | 1.58E+08 | | | P. putida | Detected with reduced sensitivity | 2.25E+08 | | | S. dysenteriae | Detected with reduced sensitivity | 8.16E+07 | | S. aureus | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | S. lugdunensis | S. lugdunensis | Detected with reduced sensitivity | 2.63E+06 | | S. anginosus group | S. anginosus | Detected with reduced sensitivity | 8.52E+07 | | S. pneumoniae | S. pneumoniae | Detected with reduced sensitivity | 2.78E+08 | | Tet(K) | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | Tet(M) | S. aureus | Detected with reduced sensitivity | 1.87E+05 | | | S. aureus | Detected with reduced sensitivity | 1.87E+06 | | | E. faecium | Detected with reduced sensitivity | 4.08E+06 | | | E. faecium | Detected with reduced sensitivity | 5.00E+06 | An in silico inclusivity assessment was also performed of all complete genomes available for every target organism/group in the Taxonomy Browser Tool of the NCBI database. Sequences were individually examined to predict detection by their corresponding assay primers and probes. A second assessment using a larger set of genomes was performed to predict reactivity using the BLAST homology analysis tool. Finally, a dedicated assessment of the AMR targets was performed using all available sequences in the Comprehensive Antibiotic Resistance Database (CARD). Results are shown in Table 14. K254194 - Page 27 of 56 {27} Table 14. Predicted (In silico) Inclusivity Results | Target | Sequences Analyzed (% predicted to detect) | Detection Prediction | | --- | --- | --- | | L. monocytogenes | 595 (100.00%) | Inclusivity determined for L. monocytogenes (including serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, 7) | | S. pyogenes | 407 (99.75%) | Only a single sequence (LS483520) not detected. | | B. cereus group | 646 (93.34%) | Inclusivity determined for B. cereus, B. thuringiensis, B. albus, B. anthracis, B. bombysepticus, B. mobilis, B. nitratireducens, B. pacificus, B. paramobilis, B. paranthracis, B. shihchuchen, B. tropicus, B. wiedmannii.Partial inclusivity determined for B. mycoides, B. weihenstephanensis.Notes:5/216 and 4/134 not detected sequences for B. cereus and B. thuringiensis, respectively.B. mycoides only 18.33% detected.1/27 not detected sequence of B. paranthracis.1/2 not detected sequences of B. weihenstephanensis. | | Corynebacterium | 702 (100.00%) | Inclusivity determined for all 131 Corynebacterium species described (C. diphtheriae, C. ulcerans, C. pseudotuberculosis, C. urealyticum, C. argentoratense, C. flavum, C. accolens, C. afermentans, C. ammoniagenes, C. amycolatum, C. anserum, C. appendicis, C. aquatimens, C. aquilae, C. atrinae, C. atypicum, C. aurimucosum, C. auris, C. auriscanis, C. belfantii, C. bovis, C. breve, C. callunae, C. camporealensis, C. canis, C. capitovis, C. casei, C. caspium, C. choanae, C. ciconiae, C. comes, C. confusum, C. coyleae, C. crudilactis, C. cystitidis, C. dentalis, C. deserti, C. doosanense, C. durum, C. efficiens, C. endometrii, C. epidermidicanis, C. faecale, C. falsenii, C. felinum, C. flavescens, C. fournieri, C. frankenforstense, C. freiburgense, C. freneyi, C. genitalium, C. gerontici, C. glaucum, C. glucuronolyticum, C. glutamicum, C. glyciniphilum, C. gottingense, C. guangdongense, C. hadale, C. haemomassiliense, C. halotolerans, C. hansenii, C. heidelbergense, C. hesseae, C. hinderae, C. humireducens, C. ihumii, C. imitans, C. incognita, C. jeddahense, C. jeikeium, C. kalinowskii, C. kefirresidentii, C. kroppenstedtii, C. kutscheri, C. lactis, C. liangguodongii, C. lizhenjunii, C. lujinsingii, C. macclintockiae, C. macginleyi, C. marinum, C. maris, C. marquesiae, C. massiliense, C. matruchotii, C. mayonis, C. minutissimum, C. mustelae, C. mycetoides, C. nuruki, C. occultum, C. pelargi, C. phocae, C. poyangense, C. propinquum, C. provencense, C. pseudodiphtheriticum, C. pseudogenitalium, C. pseudokroppenstedtii, C. pseudopelargi, C. pyruviciproducens, C. qintianiae, C. renale, C. resistens, C. rhinophilum, C. riegelii, C. rouxii, C. sanguinis, C. segmentosum, C. silvaticum, C. simulans, C. singulare, C. sphenisci, C. stationis, C. striatum, C. suedekumii, C. terpenotabidum, C. testudinoris, C. timonense, C. tuberculostearicum, C. uberis, C. ureicelerivorans, C. urogenitale, C. uterequi, C. variabile, C. vitaeruminis, C. wankanglinii, C. xerosis, C. yudongzhengii, C. zhongnanshanii). | | S. epidermidis | 328 (98.00%) | Inclusivity determined for S. epidermidis. | | S. pneumoniae | 355 (100.00%) | Inclusivity determined for S. pneumoniae. | | S. lugdunensis | 42 (100.00%) | Inclusivity determined for S. lugdunensis. | | C. neoformans/gatti | 66 (100.00%) | Inclusivity determined for C. neoformans/gatti (including serotype A (C. neoformans var neoformans), serotype D (C. neoformans var grubii), serotypes B and C (C. gattii including all VGI,VGII, VGIII, VGIV molecular types)) | | E. faecalis | 792 (99.87%) | Inclusivity determined for E. faecalis.Note: A single sequence (CP022312) was not detected. | | E. faecium | 951 (99.89%) | Inclusivity determined for E. faecium.Note: A single sequence (CP175041) was not detected. | | S. anginosus group | 56 (96.43%) | Inclusivity determined for the three major species in the group, including S. anginosus, S. constellatus and S. intermedius.Note: 2/32 sequen…