ePlex Blood Culture Identification Gram Negative (BCID-GN) Panel

{0} # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM A. 510(k) Number: K182619 B. Purpose for Submission: To establish performance claims and marketing clearance for the ePlex Blood Culture Identification Gram-Negative (BCID-GN) Panel C. Measurand: Nucleic acid targeted sequences from Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii, Enterobacter cloacae complex, Enterobacter (non-cloacae complex), Escherichia coli, Fusobacterium necrophorum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomonas maltophilia, CTX-M, IMP, KPC, NDM, OXA, and VIM, Pan Gram-Positive, Pan Candida D. Type of Test: A multiplexed nucleic acid test intended for use with the GenMark ePlex instrument for the simultaneous qualitative in vitro detection and identification of multiple bacterial nucleic acids from positive blood culture specimens. E. Applicant: GenMark Diagnostics, Incorporated F. Proprietary and Established Names: ePlex Blood Culture Identification Panel Gram Negative (BCID-GN) Common Name: ePlex BCID-GN Panel G. Regulatory Information: 1. Regulation section: 21 CFR 866.3365 2. Classification: Class II 3. Product code(s): PEN, PAM, PEO {1} 4. Panel: Microbiology (83) ### H. Intended Use: #### 1. Intended use(s): The GenMark ePlex Blood Culture Identification Gram-Negative (BCID-GN) 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-negative bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GN Panel is capable of detecting several gram-positive bacteria (Pan Gram-Positive assay) and several Candida species (Pan Candida assay). The ePlex BCID-GN Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-negative organism. The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GN Panel: Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii, Enterobacter cloacae complex, Enterobacter (non-cloacae complex), Escherichia coli, Fusobacterium necrophorum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomonas maltophilia, CTX-M (blaCTX-M), IMP (blaIMP), KPC (blaKPC), NDM (blaNDM), OXA (blaOXA) (OXA-23 and OXA-48 groups only), and VIM (blavIM). The ePlex BCID-GN Panel contains assays for the detection of genetic determinants associated with resistance to antimicrobial agents including CTX-M(blaCTX-M), which is associated with resistance to extended spectrum beta-lactamase (ESBL)-mediated resistance to penicillins, cephalosporins and monobactams, as well as OXA (blaOXA) (OXA-23 and OXA-48 groups only), KPC (blaKPC), and metallo-beta-lactamases IMP (blaIMP), VIM (blaVIM), and NDM (blaNDM), which is associated with carbapenemase-mediated resistance. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance assays do not indicate susceptibility, as there are multiple mechanisms of resistance in gram-negative bacteria. The ePlex BCID-GN 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-Positive assay (which is designed to detect Bacillus cereus group, Bacillus subtilis group, Enterococcus, Staphylococcus, and Streptococcus), as well as a Pan Candida assay, which is designed to detect four Candida species: Candida albicans, Candida glabrata, Candida krusei, and Candida parapsilosis. The detection and identification of specific bacterial and fungal nucleic acids from {2} 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-GN 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-GN 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-GN 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 bloodstream infection. # 2. Indication(s) for use: Same as intended use. # 3. Special conditions for use statement(s): For prescription use only Limitations: - This product should not be used with blood culture media that contains charcoal. - Bacterial and fungal nucleic acids may be present in blood culture, independent of bacterial or fungal viability. Detection of an assay target does not guarantee that the corresponding bacteria or fungi are infectious or are the causative agents for clinical symptoms. - Decreased sensitivity has been observed for some targets in BD BACTEC Lytic Anaerobic blood culture bottles. - In mixed cultures, the ePlex BCID-GN Panel may not identify all organisms in the specimen, depending upon the concentration of each target present. - Antimicrobial resistance can occur via multiple mechanisms. A “Not Detected” result for the BCID-GP antimicrobial resistance gene assays does not indicate antimicrobial susceptibility. Subculturing and standard susceptibility testing of isolates is required to determine antimicrobial susceptibility. - The BCID-GN Pan Candida and Pan Gram-Positive assays are designed to detect Candida and gram-positive microorganisms in mixed blood cultures where these microorganisms might be missed by Gram stain. Lower than expected clinical sensitivity was observed for these analytes, which was likely due to the presence of Candida spp. or gram-positive microorganisms in mixed cultures at concentrations below the respective limits of detection for the Pan Candida and Pan GP targets. {3} - For Enterococcus saccharolyticus, Serratia odorifera, and Streptococcus thoraltensis, 100% detection was not observed at expected bottle positivity concentrations. Decreased sensitivity may be observed for these strains. - False negative results may occur for specimens containing Fusobacterium necrophorum because the assay's limit of detection for F. necrophorum can be near the concentration observed at the time of bottle positivity. # 4. Special instrument requirements: GenMark ePlex Instrument and Software # I. Device Description: The ePlex Blood Culture Identification Gram-Negative (BCID-GN) Panel is based on the principles of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription (RT-PCR) and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization. Nucleic acid extraction from biological specimens occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified. During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal proves (labeled with electrochemically active ferrocenes) bind to specific target sequence/region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets. A summary of microorganisms and resistance markers detected by the ePlex BCID-GN Panel are presented in Table 1: Table 1: ePlex BCID-GN Panel, organism and resistance marker targets | Bacterial Targets | | | --- | --- | | Acinetobacter baumannii | Klebsiella pneumoniae group | | Bacteroides fragilis | Morganella morganii | | Citrobacter | Neisseria meningitidis | | Cronobacter sakazakii | Proteus | {4} | Enterobacter cloacae complex | Proteus mirabilis | | --- | --- | | Enterobacter (non-cloacae complex) | Pseudomonas aeruginosa | | Escherichia coli | Salmonella | | Fusobacterium necrophorum | Serratia | | Fusobacterium nucleatum | Serratia marcescens | | Haemophilus influenzae | Stenotrophomonas maltophilia | | Klebsiella oxytoca | | | Antimicrobial Resistance Markers(See Table 5 For More Detail) | | | CTX-M (\( bla_{CTX-M} \)) | NDM (\( bla_{NDM} \)) | | IMP (\( bla_{IMP} \)) | OXA (\( bla_{OXA} \)) | | KPC (\( bla_{KPC} \)) | VIM (\( bla_{VIM} \)) | | Pan Targets | | | Pan Gram-Positive | Pan Candida | ### Materials provided ePlex Blood Culture Identification Gram-Negative Panel kit containing 12 individual test cartridges. Storage is at 2-8°C. ### Materials needed but not provided ### Equipment • GenMark ePlex instrument and software • Pipettes calibrated to deliver 50 \( \mu \) l - Printer (optional) ### Consumables - Pipette tips, aerosol resistant, RNase/DNase-free • Disposable, powder free gloves • 10% bleach for decontamination of appropriate surfaces • 70% ethanol or isopropyl alcohol • 1.5 mL RNase/DNase-free microcentrifuge tube or equivalent ### Interpretation of Results Results interpretation of the ePlex BCID-GP Panel is performed by the ePlex instrument, and the interpretation of results on the ePlex BCID-GP Panel Detection Report for each targeted analyte is summarized in Table 2. {5} Table 2: Interpretation of results, ePlex BCID-GN Panel Detection Report | Target Result | Explanation | Action | | --- | --- | --- | | Detected | The test was completed successfully and the target has generated signal above its defined threshold and the Internal Control was reported as PASS. | All results are displayed on the ePlex BCID-GN Panel Detection Report. Test is valid, report results. | | Not Detected | The test was completed successfully and the target did not generate signal above its defined threshold and the Internal Control was reported as PASS. | All results are displayed on the ePlex BCID-GN Panel Detection Report. Test is valid, report results. | | Invalid | The test has not successfully completed and results for this test are not valid. This may be due to an instrument or software error. | No results are displayed on the ePlex BCID-GN Panel Detection Report. Test is not valid, repeat test. | Genus and group assay result interpretation While many results on the ePlex BCID-GN Panel are based on a single assay, the ePlex BCID-GN Panel uses two assays for each of the Proteus and Serratia results. The ePlex BCID-GN Proteus result is based on two assays: the species-specific Proteus mirabilis and the broad Proteus assay. The broad Proteus assay will detect Proteus mirabilis, however, its primary purpose is to detect non-mirabilis Proteus species. See Table 3 for detailed Proteus target call logic. Table 3: Proteus target results from ePlex BCID-GN Panel Detection Report | Proteus Result | Proteus Assay | Proteus mirabilis Assay | Description | | --- | --- | --- | --- | | Not Detected | Negative | Negative | No Proteus species detected | | Detected | Positive | Positive | Proteus mirabilis detected | | Detected | Positive | Negative | Unspecified Proteus detected | The ePlex BCID-GN Serratia result is based on two assays: the species-specific Serratia marcescens and the broad Serratia assay. The broad Serratia assay will detect Serratia marcescens, however, its primary purpose is to detect Serratia species other than Serratia marcescens. See Table 4 for detailed Serratia target call logic. Table 4 Serratia target results from ePlex BCID-GN Panel Detection Report | Serratia Result | Serratia Assay | Serratia marcescens Assay | Description | | --- | --- | --- | --- | | Not Detected | Negative | Negative | No Serratia species detected | | Detected | Positive | Positive | Serratia marcescens detected | | Detected | Positive | Negative | Unspecified Serratia detected | {6} Test results for antimicrobial resistance genes are only reported when an associated organism is detected in the same specimen. Table 5 specifies organism targets that are reported with each the six resistance markers on the ePlex BCID Panel (indicated by X in the table). Table 5. Resistance marker organism associations | Organism | Resistance Gene | | | | | | | --- | --- | --- | --- | --- | --- | --- | | | CTX-M | IMP | KPC | NDM | OXA | VIM | | Acinetobacter baumannii | X | X | X | X | X | X | | Bacteroides fragilis | | | | | | | | Citrobacter | X | X | X | X | X | X | | Cronobacter sakazakii | | | X | | | | | Enterobacter cloacae complex | X | X | X | X | X | X | | Enterobacter (non-cloacae complex) | X | X | X | X | X | X | | Escherichia coli | X | X | X | X | X | X | | Fusobacterium necrophorum | | | | | | | | Fusobacterium nucleatum | | | | | | | | Haemophilus influenzae | | | | | | | | Klebsiella oxytoca | X | X | X | X | X | X | | Klebsiella pneumoniae group | X | X | X | X | X | X | | Morganella morganii | X | X | X | X | X | X | | Neisseria meningitidis | | | | | | | | Proteus | X | X | X | X | X | X | | Proteus mirabilis | X | X | X | X | X | X | | Pseudomonas aeruginosa | X | X | X | X | X | X | | Salmonella | X | X | X | X | X | X | | Serratia | X | X | X | X | X | X | | Serratia marcescens | X | X | X | X | X | X | | Stenotrophomonas maltophilia | X | | | | | | ### Pan Assay Results The ePlex BCID-GN Panel Pan Gram-Positive assay detects one or more of the following gram-positive organisms: Enterococcus, Bacillus cereus group, Bacillus subtilis group, Staphylococcus, and/or Streptococcus as shown in Table 6. Table 6: Pan gram-positive target results from ePlex BCID-GN Panel Detection Report | Pan Gram-Positive Result | Description | | --- | --- | | Not Detected | No specified gram-positive organism detected. | | Detected | One or more of the following gram-positive organisms has been detected: Enterococcus, Bacillus cereus group, Bacillus subtilis group, Staphylococcus, and/or Streptococcus. Additional testing for speciation is suggested. | The ePlex BCID-GN Panel Pan Candida detects one or more of the following Candida species: Candida albicans, Candida glabrata, Candida krusei, and/or Candida parapsilosis as shown in Table 7. {7} Table 7: Pan Candida Target Results from ePlex BCID-GN Panel Detection Report | Pan Candida Result | Description | | --- | --- | | Not Detected | No specified Candida species detected. | | Detected | One or more of the following Candida organisms has been detected: Candida albicans, Candida glabrata, Candida krusei, and/or Candida parapsilosis. Additional testing for identification is recommended. | ### ePlex BCID-GN Panel reports: Several different reports are available on the ePlex System. Results are provided in a printable format and may be viewed electronically or exported for additional analysis. Reports can be customized with account specific information such as the address, logo and institutional specific footers on each report. For more information on ePlex Reports, refer to the ePlex Operator Manual. #### Detection Report The ePlex BCID-GN Panel Detection Report includes the results for each individual sample run on the ePlex System. The Summary section indicates the overall test result and lists all detected targets in that sample. The Results section includes a list of all targets on the panel with an individual result for each target. Results are reported as Detected, Not Detected, or Invalid (displayed as a red x); results for the Internal Control are reported as PASS, FAIL, INVALID, or N/A. #### External Control Report The ePlex BCID-GN Panel External Control Report is generated for an external control that has been pre-defined in the ePlex BCID-GN Panel Software. For more information on defining external controls on the ePlex System, refer to the ePlex Operator Manual. The Summary section indicates the overall result (PASS or FAIL status) and lists all detected targets for that external control. The Results section includes a list of all panel targets with the result, expected result, and PASS/FAIL status for each. Results are reported as Detected, Not Detected, or Invalid (displayed as a red x). A target is reported as PASS if the actual result matches the expected result (as defined for that control); a target is reported as FAIL if the actual result does not match the expected result. If the actual result for each target matches the expected result (all targets reported as PASS), the overall result for the external control is reported as PASS in the Summary section. If the actual result for any target does not match the expected result, the overall result for the external control is reported as FAIL in the Summary section. #### Summary Report The Summary Report allows the operator to use searchable criteria to create customized reports, using specified targets, dates, range of dates, sample, external control, test bay, or operator. For more information on creating Summary Reports, refer to the ePlex Operator Manual. {8} ## J. Substantial Equivalence Information: 1. Predicate device name(s): FilmArray Blood Culture Identification Panel 2. Predicate 510(k) number(s): K130914 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | New Device ePlex Blood Culture Identification Gram Negative Panel (K182619) | Predicate Device FilmArray Blood Culture Identification Panel (K130914) | | Classification | 21 CFR 866.3365 Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures | 21 CFR 866.3365 Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures | | Device Class | Class II | Class II | | Product Code | PEN, PAM, PEO | P PEN, PAM, PEO | | Intended Use | The ePlex Blood Culture Identification Gram-Negative (BCID-GN) 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 gram negative bacterial organisms and select determinants of antimicrobial resistance in positive blood culture. In addition, the ePlex Panel is capable of detecting several gram-positive bacteria (Pan Gram-Positive assay), and several Candida species (Pan Candida assay). The ePlex BCID-GN Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrates the presence of organisms as confirmed by Gram stain. | The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray systems. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrate the presence of organisms as determined by Gram stain. | | Organisms Detected | Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii, Enterobacter cloacae complex, Enterobacter (non-cloacae | Enterococci, Listeria monocytogenes, Staphylococci (including specific differentiation of Staphylococcus aureus), Streptococci (with specific | {9} | Similarities | | | | --- | --- | --- | | | complex), *Escherichia coli*, *Fusobacterium necrophorum*, *Fusobacterium nucleatum*, *Haemophilus influenzae*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Morganella morganii*, *Neisseria meningitidis*, *Proteus*, *Proteus mirabilis*, *Pseudomonas aeruginosa*, *Salmonella*, *Serratia*, *Serratia marcescens*, *Stenotrophomonas maltophilia*, Pan Gram-Positive (selected organisms), Pan Candida (selected species) | differentiation of *Streptococcus agalactiae*, *Streptococcus pneumoniae*, and *Streptococcus pyogenes*), *Acinetobacter baumannii*, *Enterobacteriaceae* (including specific differentiation of the *Enterobacter cloacae* complex, *Escherichia coli*, *Klebsiella oxytoca*, *Klebsiella pneumoniae*, *Proteus*, and *Serratia marcescens*), *Haemophilus influenzae*, *Neisseria meningitidis* (encapsulated), *Pseudomonas aeruginosa*, *Candida albicans*, *Candida glabrata*, *Candida krusei*, *Candida parapsilosis*, and *Candida tropicalis* | | **Analyte** | DNA | DNA | | **Technological Principles** | Multiplex nucleic acid amplification test (NAAT) | Multiplex nucleic acid amplification test (NAAT) | | **Specimen Type** | Blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrates the presence of organisms (Gram-positive, Gram-negative, yeasts) as confirmed by Gram stain | Blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrates the presence of organisms (Gram-positive, Gram-negative, yeasts) as confirmed by Gram stain | | **Test Interpretation** | Automated test interpretation and report generation. User cannot access raw data. | Automated test interpretation and report generation. User cannot access raw data. | | **Sample Preparation Method** | Sample processing is automated in the ePlex BCID-GN cartridge | Sample processing is automated in the FilmArray BCID pouch | | **Controls** | Each cartridge includes two types of internal controls that together monitor performance of each step of the testing process, including extraction, amplification and detection processes. | Two controls are included in each reagent pouch to control for each sample processing and both stages of PCR and melt analysis. | | Differences | | | | --- | --- | --- | | Item | New Device ePlex Blood Culture Identification Gram Negative Panel (K182619) | Predicate Device FilmArray Blood Culture Identification Panel (K130914) | | **Resistance Genes** | Assay detects *ctx*-M, *imp*, *kpc*, *ndm*, *oxa*, and *vim* | Assay detects *mecA*, *vanA*, *vanB* and *kpc*. | | **Chemistry** | Reagents contained within cartridge to allow: sample lysis and nucleic acid extraction, PCR amplification, and hybridization-based electrochemical detection | Reagents contained with a pouch to allow: sample lysis, nucleic acid extraction, multiplex nested PCR followed by high resolution melting analysis to confirm identity of amplified product | {10} | Differences | | | | --- | --- | --- | | Item | New Device ePlex Blood Culture Identification Gram Negative Panel (K182619) | Predicate Device FilmArray Blood Culture Identification Panel (K130914) | | Instrumentation | ePlex Instrument | FilmArray Instrument | | Reagent Storage | Cartridge containing reagents is stored at refrigerated temperature | Reagents are stored at room temperature. | | Gram Stain | Specimens tested have gram negative organisms based on Gram stain prior to testing. | Specimens tested have organisms present based on Gram stain. | ### K. Standard/Guidance Document Referenced: - Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures (May 2015) - CLSI MM17-A, Vol 28, No. 9, Verification and Validation of Multiplex Nucleic Acid Assays - CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition (June 2012) - CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline – Second Edition (November 2005) - CLSI EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline (May 2013) ### L. Test Principle: The ePlex instrument automates all aspects of sample processing and nucleic acid testing in a single cartridge associated with the ePlex BCID-GN Panel. Multiple bloodstream pathogens can be detected from a single blood culture specimen. Automation of DNA extraction, amplification, and target detection is accomplished within the cartridge using a combination of electrowetting and GenMark's eSensor technology. Electrowetting, or digital microfluidics, uses electrical fields to directly manipulate discrete droplets on the surface of a hydrophobically coated printed circuit board (PCB). Sample and reagents are moved in a programmable fashion in the ePlex cartridge to complete all portions of the sample processing and detection. eSensor technology is based on the principles of competitive DNA hybridization and electrochemical detection, which is highly specific and is not based on fluorescent or optical detection. A sample is loaded onto the ePlex cartridge and nucleic acids are extracted and purified from the specimen via magnetic solid phase extraction. PCR is used to create double-stranded DNA, which is treated with exonuclease to create single-stranded DNA in preparation for eSensor detection. The target DNA is mixed with ferrocene-labeled signal probes that are complementary to the specific targets on the panel. Target DNA hybridizes to its complementary signal probe and target-specific capture probes, which are bound to gold-plated electrodes, forming a hybridization complex as shown below in Figure 1. The presence of each target is determined by voltammetry which generates specific electrical signals from the ferrocene-labeled signal probe. {11}  # M. Performance Characteristics (if/when applicable): # 1. Analytical performance: # a. Precision/Reproducibility: A multisite reproducibility study was conducted at three testing sites (two external sites and one internal site). The study evaluated multiple sources of variability, such as site-to-site, lot-to-lot, day-to-day, and operator-to-operator variability. One ePlex instrument with four towers was used at each study site. Two operators each tested the reproducibility panel at each site on five nonconsecutive days with three unique lots of ePlex BCID-GN Panel cartridges. The reproducibility panel consisted of a seven-member test panel comprised of six panel members representing 17 BCID-GN analytes (including five resistance markers) and one panel member containing an off-panel organism, Cutibacterium granulosum. Panel members were prepared by spiking cultured isolates into negative blood culture matrix (NBM). The NBM was prepared by incubating 10 mL of negative whole blood in BD BACTEC Standard/10 Aerobic/F blood culture bottles per the manufacturer's instructions for a minimum of eight hours on a continuous monitoring blood culture system. After incubation and prior to use in analytical studies, the NBM was pre-screened to ensure the matrix is negative for all BCID-GN targets. Each of the on-panel organisms was evaluated at two concentrations: (1) Bottle positivity {12} and (2) One log higher than bottle positivity (to mimic organism concentrations present after eight hours additional incubation on the blood culture instrument). The organisms and concentrations evaluated in the study are summarized in Table 8. Table 8: Bottle Positivity Concentrations | Organism | Bottle Positivity Concentration | Bottle Positivity +8 Hours Concentration | | --- | --- | --- | | Acinetobacter baumannii (OXA+) | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Enterobacter cloacae (CTX-M+, KPC+) | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Escherichia coli (IMP+) | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Fusobacterium nucleatum | 1 x 10^{7} CFU/mL | 1 x 10^{8} CFU/mL | | Haemophilus influenzae | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Klebsiella oxytoca | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Neisseria meningitidis | 3 x 10^{7} CFU/mL | 3 x 10^{8} CFU/mL | | Pseudomonas aeruginosa (VIM+) | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Serratia marcescens | 1 x 10^{8} CFU/mL | 1 x 10^{9} CFU/mL | | Candida albicans (Pan Candida target) | 1 x 10^{6} CFU/mL | 1 x 10^{7} CFU/mL | | Staphylococcus aureus (Pan Gram-Positive target) | 1 x 10^{7} CFU/mL | 1 x 10^{8} CFU/mL | | Cutibacterium granulosum | Incubated on instrument until bottle positivity plus eight hours | N/A | The seven panel members were tested 108 times each (excluding repeat testing): 3 replicates x 3 sites x 2 operators x 3 lots x 2 days of testing/operator/lot, for a minimum number of 756 tests. A summary of results for the ePlex BCID-GN Panel reproducibility study is provided in Table 9. For the negative panel member, 100% of replicates generated the expected negative results for all ePlex BCID-GN Panel analytes. Table 9. Reproducibility of the ePlex BCID-GN Panel | Analyte | Concentration Tested | Expected Results | Agreement with Expected Result | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | Site 1 | Site 2 | Site 3 | All Sites (95% CI) | | **Organisms** | | | | | | | | Acinetobacter baumannii | BP+8* 1x10^{9} CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108 (96.6-100) | | | BP** 1x10^{8} CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108 (96.6-100) | | | Negative | Not Detected | 179/179 | 178/179 | 180/180 | 537/538 (99.0-100) | | Enterobacter cloacae complex | BP+8 1x10^{9} CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108 (96.6-100) | | | BP 1x10^{8} CFU/mL | Detected | 35/35 | 36/36 | 36/36 | 107/107 (96.5-100) | | | Negative | Not Detected | 180/180 | 179/179 | 180/180 | 539/539 (99.3-100) | {13} | Analyte | Concentration Tested | Expected Results | Agreement with Expected Result | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | Site 1 | Site 2 | Site 3 | All Sites (95% CI) | | Escherichia coli | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 35/35 | 36/36 | 107/107(96.5-100) | | | Negative | Not Detected | 179/179 | 180/180 | 180/180 | 539/539(99.3-100) | | Fusobacterium nucleatum | BP+8\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 35/35 | 36/36 | 107/107(96.5-100) | | | BP\( 1 \times 10^{7} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 179/179 | 180/180 | 180/180 | 539/539(99.3-100) | | Haemophilus influenzae | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 179/179 | 179/179 | 180/180 | 538/538(96.6-100) | | Klebsiella oxytoca | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 179/179 | 179/179 | 180/180 | 538/538(96.6-100) | | Neisseria meningitidis | BP+8\( 3 \times 10^{8} \) CFU/mL | Detected | 35/35 | 36/36 | 36/36 | 107/107(96.5-100) | | | BP\( 3 \times 10^{7} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 180/180 | 179/179 | 180/180 | 539/539(99.3-100) | | Pseudomonas aeruginosa | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 35/35 | 36/36 | 107/107(96.5-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 179/179 | 180/180 | 180/180 | 539/539(99.3-100) | | Serratia | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 35/35 | 36/36 | 36/36 | 107/107(96.5-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 180/180 | 179/179 | 180/180 | 539/539(99.3-100) | | Serratia marcescens | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 35/35 | 36/36 | 36/36 | 107/107(96.5-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 180/180 | 179/179 | 180/180 | 539/539(99.3-100) | {14} | Analyte | Concentration Tested | Expected Results | Agreement with Expected Result | | | | | --- | --- | --- | --- | --- | --- | --- | | | | | Site 1 | Site 2 | Site 3 | All Sites (95% CI) | | Pan Candida | BP+8\( 1 \times 10^{7} \) CFU/m) | Detected | 35/35 | 36/36 | 36/36 | 107/107(96.5-100) | | | BP\( 1 \times 10^{6} \) CFU/mL | Detected | 35/36 | 36/36 | 36/36 | 107/108(94.9-99.8) | | | Negative | Not Detected | 180/180 | 179/179 | 180/180 | 539/539(99.3-100) | | Pan Gram-Positive(Staphylococcus aureus) | BP+8\( 1 \times 10^{8} \) CFU/mL | Detected | 34/36 | 35/35 | 36/36 | 105/107(93.4-99.5) | | | BP\( 1 \times 10^{7} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 179/179 | 179/180 | 180/180 | 538/539(99.0-100) | | Resistance Markers | | | | | | | | CTX-M(Enterobacter cloacae) | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 35/35 | 36/36 | 36/36 | 107/107(96.5-100) | | | Negative | Not Detected | 144/144 | 143/143 | 144/144 | 431/431(99.1-100) | | IMP(Escherichia coli) | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 35/35 | 35/36 | 106/107(94.9-99.8) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 35/35 | 35/36 | 106/107(94.9-99.8) | | | Negative | Not Detected | 143/143 | 144/144 | 144/144 | 431/431(99.1-100) | | KPC(Enterobacter cloacae) | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 35/35 | 36/36 | 36/36 | 107/107(96.5-100) | | | Negative | Not Detected | 144/144 | 143/143 | 144/144 | 431/431(99.1-100) | | OXA(Acinetobacter baumannii) | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 143/143 | 143/143 | 144/144 | 430/430(99.1-100) | | VIM(Pseudomonas aeruginosa) | BP+8\( 1 \times 10^{9} \) CFU/mL | Detected | 36/36 | 35/35 | 36/36 | 107/107(96.5-100) | | | BP\( 1 \times 10^{8} \) CFU/mL | Detected | 36/36 | 36/36 | 36/36 | 108/108(96.6-100) | | | Negative | Not Detected | 143/143 | 144/144 | 144/144 | 431/431(99.1-100) | *BP +8 – Bottle Positivity + 8 hours **BP – Bottle Positivity Five false positive results were observed in the BCID-GN Panel reproducibility study. Bacteroides fragilis (2), Acinetobacter baumannii, Fusobacterium necrophorum, and Pan Gram-Positive were detected in five different samples that had other organisms at positive concentrations. Four of the five samples with false positive results were observed at one testing site across two cartridge lots on four different days. On two of the days that {15} false positive results were observed (i.e., Pan Gram-Positive and *Acinetobacter baumannii* targets), external controls containing those targets were also tested. These results suggest a potential site-specific root cause (e.g., site-specific contamination) rather than a systematic performance issue with the BCID-GN Panel. During the study, 812 runs were attempted with six pre-flight check failures (1.2% pre-flight errors) for which a new cartridge had to be tested. The pre-flight check is an ePlex system process that tests the integrity of the cartridge/instrument interface prior to initiating sample processing. An additional 10 runs were aborted by the system (1.2% system abort errors) resulting in 796 completed runs, including repeat runs due to invalid results. Of the 756 samples initially tested, 715 (94.6%) had valid results and 41 (5.4%) had invalid results. Of the 40 samples re-tested, 39 had valid results and 1 had a second invalid result upon re-test. The final validity rate (95% CI) was 99.7% (99.0%-99.9%). # *b. Linearity/assay reportable range:* Not applicable, qualitative assay. # *c. Traceability, Stability, Expected values (controls, calibrators, or methods):* ### Assay Controls #### Internal Controls Each ePlex BCID-GN Panel cartridge includes internal controls that monitor performance of each step of the testing process, including extraction, amplification and detection of DNA targets. Each of the eight amplification reactions has an internal control, and in each reaction, either the internal control or a target must generate signal above the defined threshold for a valid test result. Internal control results are interpreted by the ePlex Software and displayed on the ePlex BCID-GN Panel Reports as Internal Control with a result of PASS, FAIL, N/A, or INVALID. Table 10 includes details on the interpretation of Internal Control results. {16} **Table 10: Internal control results** | Internal Control Result | Explanation | Action | | --- | --- | --- | | **PASS** | Signal above threshold has been detected from each amplification reaction. The test was completed and internal controls were successful, indicating valid results were generated. | All results are displayed on the ePlex BCID-GN Panel Detection Report. Test is valid, report results. | | **FAIL** | Signal above threshold has not been detected from at least one amplification reaction. The test was completed but internal controls were not detected, indicating that results may not be valid. | No results are displayed on the ePlex BCID-GN Panel Detection Report. Test is not valid, repeat the test using a new cartridge. | | **N/A** | The internal control in every amplification reaction does not generate signal above the threshold, but a target in every amplification reaction does generate signal above the threshold. The test was completed and internal controls were not successful, however detection of signal above the threshold for a target in every amplification reaction indicates valid results were generated. | All results are displayed on the ePlex BCID-GN Panel Detection Report. Test is valid, report results. | | **INVALID** | An error has occurred during processing that prevents analysis of signal data. The test has not successfully completed and results for this test are not valid. This may be due to an instrument or software error. | No results are displayed on the ePlex BCID-GN Panel Detection Report. Test is not valid, repeat the test using a new cartridge. | ### External Controls External controls are not provided with the ePlex BCID-GN Panel; however, the package insert includes the following recommendations for external controls: - Positive and negative external controls should be tested with each new lot of reagents or monthly, whichever occurs first. Blood culture medium can be used as the negative control. Previously characterized positive samples or blood culture medium spiked with well characterized organisms can be used as the external positive control. External controls should be run in accordance with laboratory protocols and accrediting organizations, as applicable. During the prospective clinical evaluation of the ePlex BCID-GN Panel, each testing site was provided with positive and negative external control samples. There were four organism mixes that served as positive controls and were prepared at concentrations near or below the concentration expected at bottle positivity. Negative blood culture matrix was used as the negative control. At least one positive and one negative external control were tested each day and cycled throughout the clinical study. The control mixes are described in Table 11. {17} Table 11. External controls used in the BCID-GN Panel Clinical study | External Controls | Expected Calls | | --- | --- | | Mix A | CTX-M-1, Staphylococcus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, E. cloacae complex, Enterobacter aerogenes | | Mix B | Bacillus cereus, IMP, OXA-23, Candida albicans, Citrobacter freundii, Serratia proteamaculans, Neisseria meningitidis, Enterobacter amnigenus, Proteus vulgaris | | Mix C | Bacteroides fragilis, OXA-48, Acinetobacter baumannii, Enterococcus spp., Fusobacterium necrophorum, Morganella morganii, Klebsiella oxytoca, VIM | | Mix D | Cronobacter sakazakii, Stenotrophomonas maltophilia, Streptococcus ssp., Enterobacter asburiae, Haemophilus influenzae, Klebsiella pneumoniae, NDM, Fusobacterium nucleatum, KPC | | Negative control | Negative (Not Detected) | Over 90 days of investigational testing across 6 testing sites, there were 177 external controls tested with valid results: 25 Positive Control A, 22 Positive Control B, 21 Positive Control C, 21 Positive Control D, and 88 Negative Controls. Of these, 167/177 controls generated the expected results. The remaining 10 external control samples generated discordant results as follows: 3 external control samples had 1 or more false negative (FN) results: - 1 Positive Control A sample had 1 FN Pseudomonas aeruginosa result. - 1 Positive Control A sample had 1 FN Enterobacter (non-cloacae complex) - result. - 1 Positive Control A sample had 2 FN results: Proteus mirabilis, Proteus. 7 external control samples had 1 false positive (FP) result: - 1 Negative Control sample had a 1 FP Pan Gram-Positive result. - 2 Positive Control B samples each had 1 FP Salmonella. - 2 Positive Control C samples each had 1 NDM FP result. - 1 Positive Control C sample had 1 Enterobacter cloacae complex FP result. - 1 Positive Control D sample had 1 VIM FP result. ### Specimen stability Clinical specimen stability claims were supported by an analytical study that investigated the effect of different specimen storage conditions/time periods on specimen integrity for specimens tested with the ePlex BCID-GN Panel. Three organism mixes containing representative targets from each of the eight multiplex primer pools on the ePlex BCID-GN Panel were used for this study. Organism mixes whose members were at concentrations consistent with those observed at bottle positivity were stored at \( \leq-80^{\circ}C \) , \( \leq-20^{\circ}C \) , \( 2-8^{\circ}C \) , and at ambient temperature over several time points. Baseline condition ( \( T_{0} \) ) was established with 20 replicates. Ten replicates were tested for each additional temperature and time point. All test conditions that were assessed in this study are summarized in Table 12. {18} Table 12. Specimen stability study design | Storage Condition | Time Points | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Ambient Conditions15°C – 30°C | \( T_0 \) | 1day | 3days | 5days | 7days | 14days | 30days | -- | | Refrigerated Conditions2°C – 8°C | -- | 1day | 3days | 5days | 7days | 14days | 30days | -- | | Frozen Conditions≤-20°C and ≤-80°C | 1 w | 2 w | 1mo | 3mo | 6mo | 12mo | 18mo | 24mo | | Bottle Incubator | Bottle positivity | | 12-hours post positivity | | | | | | The specimen stability criteria were as follows: - Performance at each time point will be considered acceptable if the positivity rate for each analyte (either from the first 10 replicates or from the final 20 replicates in case of additional testing) is equivalent to the positivity rate for the corresponding analyte at time point \( \mathrm{T_0} \) (baseline time point). - If a time point did not meet the positivity acceptance criteria, the next time point was evaluated. If the subsequent time point met the acceptance criteria, the study was continued. If two consecutive time points failed, the specimen stability was determined to be the last time point when the acceptance criteria were met. Summaries of the specimen stability assessment data are in Tables 12-14: Table 12. Ambient storage sample stability (Percent positivity and mean signal) | Target | Day 0 | | Day 1 | | Day 3 | | Day 5 | | Day 7 | | Day 14 | | Day 30 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | | Mix 1 | | | | | | | | | | | | | | | | E. coli | 100 | 391.5 | 100 | 597.8 | 100 | 673.5 | 100 | 72.0 | 100 | 725.8 | 100 | 754.9 | 100 | 758 | | H. influenzae | 100 | 243.9 | 100 | 259.3 | 100 | 281.3 | 100 | 233.0 | 100 | 246.3 | 100 | 251.5 | 100 | 231.3 | | E. cloacae complex | 100 | 230.1 | 100 | 516.3 | 100 | 723.4 | 100 | 818.4 | 100 | 886.6 | 100 | 937.3 | 100 | 919.2 | | Pan-Candida | 100 | 233.1 | 100 | 247.0 | 100 | 217.8 | 100 | 247.0 | 100 | 184.7 | 100 | 233.7 | 100 | 245.3 | | OXA | 100 | 735.7 | 100 | 746.1 | 100 | 726.0 | 100 | 816.5 | 100 | 795.2 | 100 | 823.0 | 100 | 824.0 | | CTX-M | 95 | 292.9 | 100 | 333.5 | 100 | 340.2 | 100 | 310.4 | 100 | 350.4 | 100 | 337.9 | 100 | 387.8 | | Mix 2 | | | | | | | | | | | | | | | | K. oxytoca | 100 | 489.9 | 100 | 513.8 | 100 | 424.9 | 100 | 418.3 | 100# | 492.5 | 100 | 532.7 | 100 | 512.2 | | P. aeruginosa | 100 | 586.8 | 100 | 517.3 | 100 | 739 | 100 | 781.9 | 100# | 783.4 | 100 | 524.7 | 100 | 800.4 | | A. baumannii | 100 | 783.1 | 100 | 788.8 | 100 | 727.8 | 100 | 760.4 | 100# | 759.5 | 100 | 799.9 | 100 | 803.1 | | E. non-cloacae complex | 100 | 566.4 | 100 | 519.7 | 100 | 633.5 | 100 | 639 | 100# | 669.6 | 100 | 472.3 | 100 | 587.8 | | IMP | 100 | 492 | 100 | 491.5 | 100 | 479.2 | 100 | 457.5 | 100# | 447.2 | 100 | 292.8 | 100 | 473.8 | | Mix 3 | | | | | | | | | | | | | | | {19} | Target | Day 0 | | Day 1 | | Day 3 | | Day 5 | | Day 7 | | Day 14 | | Day 30 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | | N. meningitidis | 100 | 306 | 100 | 349.9 | 100 | 307.6 | 100 | 235.6 | 100^ | 277.8 | 100* | 264.7 | 100 | 282.9 | | S. marcescens | 100 | 479 | 100 | 530.7 | 100 | 498.9 | 100 | 545 | 100^ | 397.8 | 100* | 368.1 | 100 | 375.6 | | B. fragilis | 100 | 712.6 | 100 | 730.5 | 100 | 713.2 | 100 | 682.9 | 100^ | 739.8 | 100* | 767.3 | 100 | 903.8 | | Pan Gram-Positive | 100 | 461.3 | 100 | 511.4 | 100 | 625.5 | 100 | 633.4 | 100^ | 590.3 | 100* | 544.7 | 100 | 489.6 | # Pan Gram-Positive false positive observed in 1 of 10 replicates ^ Mix 3; M. morganii and OXA-48 false positive observed in 1 of 20 replicates * Mix 3; P. aeruginosa false positive observed in 1 of 20 replicates Table 13. Refrigerated (2-8°C) storage sample stability (Percent positivity and mean signal) | Target | Day 0 | | Day 1 | | Day 3 | | Day 5 | | Day 7 | | Day 14 | | Day 30 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | | Mix 1 | | | | | | | | | | | | | | | | E. coli | 100 | 391.5 | 100 | 370.8 | 100 | 461.3 | 100 | 418.9 | 100 | 396.8 | 100 | 473 | 100 | 480.6 | | H. influenzae | 100 | 243.9 | 100 | 226.6 | 100 | 308.2 | 100 | 279.2 | 100 | 268.7 | 100 | 285.1 | 100 | 218 | | E. cloacae complex | 100 | 230.1 | 100 | 239.8 | 100 | 244.8 | 100 | 185.2 | 100 | 274.4 | 100 | 359.1 | 100 | 320.1 | | Pan-Candida | 100 | 233.1 | 100 | 220 | 100 | 214.7 | 100 | 227.2 | 100 | 186.7 | 100 | 217.2 | 100 | 259.1 | | OXA | 100 | 735.7 | 100 | 752.4 | 100 | 744.6 | 100 | 756.6 | 100 | 770.5 | 100 | 803.5 | 100 | 852.2 | | CTX-M | 95 | 292.9 | 100 | 308.9 | 100 | 367.9 | 100 | 339.5 | 100 | 312.3 | 100 | 318.2 | 100 | 318.4 | | Mix 2 | | | | | | | | | | | | | | | | K. oxytoca | 100 | 489.9 | 100^ | 413 | 100 | 411.9 | 100 | 360.7 | 100 | 504.6 | 100 | 518 | 100* | 461.2 | | P. aeruginosa | 100 | 586.8 | 100^ | 457.1 | 100 | 755.7 | 100 | 627 | 100 | 734.9 | 100 | 388.6 | 100* | 697.1 | | A. baumannii | 100 | 783.1 | 100^ | 763.4 | 100 | 762.9 | 100 | 655.4 | 100 | 760.7 | 100 | 814.7 | 100* | 691.3 | | E. non-cloacae complex | 100 | 566.4 | 100^ | 519.3 | 100 | 661.8 | 100 | 553.7 | 100 | 660.2 | 100 | 388.2 | 100* | 497.3 | | IMP | 100 | 492 | 100^ | 404.8 | 100 | 490.7 | 100 | 431.8 | 100 | 467.3 | 100 | 244.1 | 95* | 443.4 | | Mix 3 | | | | | | | | | | | | | | | | N. meningitidis | 100 | 306 | 100 | 327.9 | 100 | 305.3 | 100 | 260.1 | 100 | 300.3 | 100 | 274.4 | 100 | 316.5 | | S. marcescens | 100 | 479 | 100 | 526.9 | 100 | 442.1 | 100 | 486 | 100 | 337.1 | 100 | 303 | 100 | 309.5 | | B. fragilis | 100 | 712.6 | 100 | 758.4 | 100 | 719 | 100 | 714.6 | 100 | 747.5 | 10 | 739.6 | 100 | 875 | | Pan Gram-Positive | 100 | 461.3 | 100 | 579.4 | 100 | 551.5 | 100 | 613.4 | 100 | 536.1 | 100* | 543.5 | 100 | 529.7 | ^ Mix 2, Proteus false positive detected in 1 of 20 replicates * Mix 2 had 20 replicates Table 14. Frozen (≤-20°C and ≤-70°C) Storage Sample Stability (Percent Positivity and Mean Signal) | Target | Day 0 | | Week 1 | | Week 2 | | Month 1 | | Month 3 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | | ≤-20°C Condition | | | | | | | | | | | | Mix 1 | | | | | | | | | | | | E. coli | 100 | 391.5 | 100 | 534.7 | 100 | 478.7 | 100 | 547.2 | 100 | 377.1 | | H. influenzae | 100 | 243.9 | 100 | 312.1 | 100 | 256.9 | 100 | 206.8 | 100 | 314.4 | {20} | Target | Day 0 | | Week 1 | | Week 2 | | Month 1 | | Month 3 | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | | E. cloacae complex | 100 | 230.1 | 100 | 382.2 | 100 | 298.5 | 100 | 285.5 | 100 | 278.2 | | Pan-Candida | 100 | 233.1 | 100 | 182.9 | 100 | 235.1 | 100 | 246.7 | 100 | 178.9 | | OXA | 100 | 735.7 | 100 | 804.2 | 100 | 790 | 100 | 801 | 100 | 771.5 | | CTX-M | 95 | 292.9 | 100 | 339.6 | 100 | 293.3 | 100 | 351.5 | 100 | 268.3 | | Mix 2 | | | | | | | | | | | | K. oxytoca | 100 | 489.9 | 100 | 422.3 | 100 | 525.2 | 100 | 445.9 | 100 | 505.4 | | P. aeruginosa | 100 | 586.8 | 100 | 775.7 | 100 | 416.6 | 100 | 532.4 | 100 | 427.1 | | A. baumannii | 100 | 783.1 | 100 | 738.2 | 100 | 826.2 | 100 | 767.6 | 100 | 529.5 | | E. non-cloacae complex | 100 | 566.4 | 100 | 698.3 | 100 | 534.6 | 100 | 541.7 | 100 | 489.1 | | IMP | 100 | 492 | 100 | 492.4 | 100 | 274.4 | 100 | 489.8 | 100 | 289.4 | | Mix 3 | | | | | | | | | | | | N. meningitidis | 100 | 306 | 100 | 336.4 | 100 | 244.4 | 100 | 279.5 | 100 | 436.4 | | S. marcescens | 100 | 479 | 100 | 307.9 | 100 | 300.7 | 100 | 310 | 100 | 499.4 | | B. fragilis | 100 | 712.6 | 100 | 798.5 | 100 | 725 | 100 | 919.6 | 100 | 599.6 | | Pan Gram-Positive | 100 | 461.3 | 100 | 559.5 | 100 | 486.5 | 100 | 541 | 100 | 381.2 | | ≤-70°C Condition | | | | | | | | | | | | Mix 1 | | | | | | | | | | | | E. coli | 100 | 391.5 | 100 | 538.5 | 100 | 502.6 | 100 | 410.2 | 100 | 290.9 | | H. influenzae | 100 | 243.9 | 100 | 304.6 | 100 | 261.5 | 100 | 232.8 | 100 | 324 | | E. cloacae complex | 100 | 230.1 | 100 | 367 | 100 | 296.8 | 100 | 210.6 | 100 | 275.6 | | Pan-Candida | 100 | 233.1 | 100 | 190.7 | 100 | 222.6 | 100 | 271.4 | 100 | 146.5 | | OXA | 100 | 735.7 | 100 | 798.2 | 100 | 784.4 | 100 | 770.7 | 100 | 757.4 | | CTX-M | 100 | 292.9 | 100 | 298.4 | 100 | 286.9 | 100 | 296.8 | 100 | 256.7 | | Mix 2 | | | | | | | | | | | | K. oxytoca | 100 | 489.9 | 100 | 439.4 | 100 | 488.6 | 100 | 435.3 | 100 | 393.2 | | P. aeruginosa | 100 | 586.8 | 100 | 790.9 | 100 | 403.4 | 100 | 604.8 | 100 | 315.2 | | A. baumannii | 100 | 783.1 | 100 | 704.6 | 100 | 820.7 | 100 | 732.4 | 100 | 374.3 | | E. non-cloacae complex | 100 | 566.4 | 100 | 664.6 | 100 | 501 | 100 | 528.2 | 100 | 368.8 | | IMP | 100 | 492 | 100 | 476.1 | 100 | 268.8 | 100 | 482.1 | 100 | 304.2 | | Mix 3 | | | | | | | | | | | | N. meningitidis | 100 | 306 | 100 | 353.9 | 100 | 226.2 | 100 | 271.3 | 100 | 448.5 | | S. marcescens | 100 | 479 | 100 | 324.7 | 100 | 277 | 100 | 359.3 | 100 | 428.8 | | B. fragilis | 100 | 712.6 | 100 | 792.1 | 100 | 681.1 | 100 | 926.2 | 100 | 587.6 | | Pan Gram-Positive | 100 | 461.3 | 100 | 575.5 | 100 | 480.7 | 100 | 562.8 | 100 | 412 | The specimen stability study demonstrated that positive blood culture specimens can be stored up to 30 days at both ambient and refrigerated (2-8°C) temperatures as well as up to three months at both -20°C and -70°C without negatively impacting ePlex BCID-GN Panel performance. Study results also demonstrated that specimens can also be tested after incubation in a blood culture instrument for up to 12 hours post-bottle positivity. {21} ## Freeze-thaw Study An analytical study was conducted to demonstrate the stability of clinical specimens after subjecting samples to one or two freeze-thaw cycles prior to testing on the BCID-GN Panel. One hundred and ten positive clinical samples representing 20 targeted analytes were tested fresh during the BCID-GN clinical study and selected to represent a variety of organisms present in positive blood cultures. All specimens contained gram-negative organisms on Gram stain. Samples were frozen at ≤-70°C and then subjected to one or two thawing cycles, followed by testing with the BCID-GN Panel. Study results are summarized in Table 15. Table 15. Freeze/Thaw study (Positivity and mean signal) | Target | Total Replicates | Fresh (T0) | | 1X Freeze/Thaw | | 2X Freeze/Thaw | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | | Bacteroides fragilis | 5 | 100 | 520.9 | 100 | 606.0 | 100 | 567.4 | | Citrobacter | 3 | 100 | 364.6 | 100 | 643.3 | 100 | 537.8 | | Enterobacter (non-cloacae complex) | 2 | 100 | 553.1 | 100 | 655.1 | 100 | 768.7 | | Enterobacter cloacae complex^{A} | 11 | 100 | 487.0 | 100 | 706.2 | 100 | 654.8 | | Escherichia coli | 20 | 100 | 883.1 | 100 | 684.4 | 100 | 712.6 | | Haemophilus influenzae | 2 | 100 | 378.2 | 100 | 468.0 | 100 | 427.3 | | Klebsiella oxytoca | 3 | 100 | 362.8 | 100 | 514.1 | 100 | 562.0 | | Klebsiella pneumoniae^{A} | 17 | 100 | 439.7 | 100 | 467.7 | 100 | 534.0 | | Morganella morganii | 2 | 100 | 556.6 | 100 | 522.5 | 100 | 567.9 | | Proteus | 5 | 100 | 133.8 | 100 | 185.7 | 100 | 140.1 | | Proteus mirabilis | 5 | 100 | 246.7 | 100 | 290.0 | 100 | 279.8 | | Pseudomonas aeruginosa^{A} | 9 | 100 | 609.9 | 100 | 650.5 | 100 | 645.3 | | Salmonella | 2 | 100 | 93.1 | 100 | 94.3 | 100 | 101.0 | | Serratia | 6 | 100 | 146.1 | 100 | 148.4 | 100 | 143.7 | | Serratia marcescens | 5 | 100 | 206.3 | 100 | 378.4 | 100 | 389.1 | | Stenotrophomonas maltophilia | 2 | 100 | 367.8 | 100 | 495.5 | 100 | 568.4 | | KPC* | 1 | 100 | 713.9 | 100 | 797.7 | 100 | 837.6 | | CTX-M | 4 | 100 | 273.6 | 100 | 348.8 | 100 | 351.8 | | Pane Candida^{A} | 7 | 100 | 162.4 | 100 | 153.4 | 100 | 188.9 | | Pan Gram-Positive | 5 | 100 | 475.5 | 100 | 537.6 | 100 | 614.7 | *There was only one sample tested for the KPC target. Therefore, the nA value is the actual observed value. A. Four samples had false positive results but all expected targets were detected. i. Under the 1X freeze-thaw condition, one positive Enterobacter cloacae complex sample detected Enterobacter cloacae complex and OXA, making OXA a false positive result. ii. Under the 1X freeze-thaw condition, one positive Pan Candida sample detected Pan Candida and Pan Gram-Positive, making Pan Gram-Positive a false positive result. iii. Under the 2X freeze-thaw condition, one positive Pseudomonas aeruginosa sample detected Pseudomonas aeruginosa and Stenotrophomonas maltophilia, making Stenotrophomonas maltophilia a false positive result. iv. Under the 2X freeze-thaw condition, one positive Klebsiella pneumoniae sample detected Klebsiella pneumoniae and OXA, making OXA a false positive result. Data from the freeze-thaw study support that clinical specimens are stable up to two freeze-thaw cycles and frozen specimens and previously frozen specimens are acceptable {22} for testing with the ePlex BCID-GN Panel. ## Open Pouch Stability Study In-Consumable Sample Stability Study An analytical study was conducted to demonstrate that the ePlex BC-GN cartridge (after the pouch has been opened) can be exposed to ambient conditions for up to two hours before loading the sample and testing on the ePlex instrument. A second study was conducted to demonstrate that the cartridge can be stored at room temperature (15–30°C) for two hours after the sample has been loaded but prior to running the cartridge in an ePlex bay. For these two studies, the test panel was comprised of four organisms representing six of the eight multiplex primer pools and covering six targets on the ePlex BCID-GN Panel. Each organism was tested at a concentration approximating bottle positivity. A total of 20 control replicates were also run for each organism. Control samples were loaded within 20 minutes after pouch opening and were run on the ePlex instrument within 20 minutes of sample loading. The open pouch stability study demonstrated that ePlex BCID-GN cartridges are stable for up to 2 hours at room temperature after the pouch has been opened and prior to testing on the ePlex instrument. Results from the study are presented in Table 16. Table 16: Open pouch stability study results | Target | % Positive | | | --- | --- | --- | | | Sample tested within 20min after pouch opening | Sample tested two hours after pouch opening | | Escherichia coli | 100% (20/20) | 100% (20/20) | | Enterobacter cloacae complex | 100% (20/20) | 95% (19/20) | | Haemophilus influenzae | 100% (20/20) | 100% (20/20) | | Pan-Candida | 100% (20/20) | 95% (19/20) | | CTX-M | 95% (19/20) | 100% (20/20) | | OXA | 100% (20/20) | 100% (20/20) | The in-cartridge stability study demonstrated that the BCID-GN cartridge is stable for up to 2 hours at room temperature after the sample has been loaded prior to testing with the ePlex BCID-GN Panel. Results of the study are summarized in Table 17. Table 17: In-Cartridge sample stability study results | Target | % Positive | | | --- | --- | --- | | | Sample loaded in ≤20 minutes | Sample loaded at 120 minutes | | Escherichia coli | 100% (20/20) | 100% (20/20) | | Enterobacter cloacae complex | 100% (20/20) | 100% (20/20) | | Haemophilus influenzae | 100% (20/20) | 100% (20/20) | {23} | Target | % Positive | | | --- | --- | --- | | | Sample loaded in ≤20 minutes | Sample loaded at 120 minutes | | Pan-Candida | 100% (20/20) | 100% (20/20) | | CTX-M | 95% (19/20) | 95% (19/20) | | OXA | 100% (20/20) | 100% (20/20) | ### d. Bottle Positivity: Organism Concentration Study To approximate organism concentrations that may be present in positive blood culture bottles in a clinical setting, a study was conducted to establish the range of expected organism concentrations present in incubated blood cultures at bottle positivity (i.e., bottle ring) and eight hours after bottle positivity/bottle “ring.” Representative organisms were evaluated, consisting of 14 genera that are detected by the ePlex BCID-GN Panel. The volume of negative human whole blood, determined by the manufacturer’s instructions, was inoculated into each organism-appropriate bottle and incubated to bottle positivity and bottle positivity plus eight hours on a continuously monitoring blood culture system. At the time of positivity (and eight hours after positivity), the blood culture was removed from the instrument for determination of organism concentration (CFU/mL using a standard plate counting method) and ePlex BCID Panel testing. All estimated bottle positivity concentrations are equivalent or greater than the established Limit of Detection (LOD) for each of the assays of the ePlex BCID-GP Panel. The following bottle types were used for the study: BD BACTEC Plus Aerobic/F blood culture bottle (E. faecium, S. aureus, S. anginosus, A. baumannii, E. cloacae, E. coli, H. influenzae, K. oxytoca, N. meningitidis, P. aeruginosa, and S. marcescens), and BD BACTEC Lytic/10 Anaerobic/F (B. fragilis and F. nucleatum). Table 18 summarizes the concentration present at bottle positivity and bottle positivity plus eight hours for each organism evaluated. Table 18: Bottle positivity study, organism concentrations | Organism | Average BP Concentration (CFU/mL) | Average BP+8hrs Concentration (CFU/mL) | | --- | --- | --- | | Serratia marcescens | \( 1.2 \times 10^{9} \) | \( 2.2 \times 10^{9} \) | | Escherichia coli | \( 2.3 \times 10^{8} \) | \( 1.5 \times 10^{9} \) | | Enterobacter cloacae | \( 2.8 \times 10^{8} \) | \( 7.7 \times 10^{8} \) | | Neisseria meningitidis | \( 3.2 \times 10^{7} \) | \( 2.1 \times 10^{8} \) | | Haemophilus influenzae | \( 6.9 \times 10^{8} \) | \( 1.15 \times 10^{9} \) | | Pseudomonas aeruginosa | \( 1.6 \times 10^{8} \) | \( 8.4 \times 10^{8} \) | | Fusobacterium nucleatum | \( 6.5 \times 10^{7} \) | \( 4.9 \times 10^{8} \) | | Acinetobacter baumannii | \( 4.4 \times 10^{8} \) | \( 3.8 \times 10^{8} \) | | Klebsiella oxytoca | \( 9.3 \times 10^{8} \) | \( 1.5 \times 10^{9} \) | | Bacteroides fragilis | \( 4.7 \times 10^{8} \) | \( 6.7 \times 10^{9} \) | | Staphylococcus aureus | \( 2.8 \times 10^{7} \) | \( 2.1 \times 10^{7} \) | | Streptococcus anginosus | \( 4.1 \times 10^{7} \) | \( 4.0 \times 10^{8} \) | {24} | Organism | Average BP Concentration (CFU/mL) | Average BP+8hrs Concentration (CFU/mL) | | --- | --- | --- | | *Enterococcus faecium* | 4.9 x10^{7} | 3.6 x10^{7} | | *Candida albicans* | 1.6 x10^{6} | 1.4 x10^{6} | # e. Detection limit: The limit of detection (LoD) was determined for each analyte/group on the ePlex BCID-GN Panel using samples prepared in negative blood culture matrix consisting of EDTA human whole blood (amount consistent with the bottle manufacturer recommendations) spiked into a blood culture bottle and incubated for a minimum of eight hours in a continuously monitoring blood culture instrument. Quantitation of organism preparations was determined in CFU/mL using plating and colony count methods. Analytes were pooled together and serially diluted to create organism mixes containing no more than five organisms in negative blood/blood culture matrix. A minimum of 20 replicates per organism mix were tested to confirm the final LoD for each assay target. The limit of detection was defined as the lowest concentration at which each target is detected at least 95% of the time. Results from the study demonstrated that all targets were detected in ≥95% of tested replicates at ≤1 x 10$^{8}$ CFU/mL for bacterial and resistance marker targets and ≤1 x 10$^{6}$ CFU/mL for fungal targets. It is noted that the LoD for *Fusobacterium necrophorum* approximates the concentration for this organism present at bottle positivity. The confirmed LoD for ePlex BCID-GN Panel organism and resistance marker targets are shown in Table 19. Table 19: LoD results summary | Target | Organism | Strain | LoD Concentration (CFU/mL) | | --- | --- | --- | --- | | *Acinetobacter baumannii* | *Acinetobacter baumannii* | NCTC 13421 | 1 x 10^{6} | | | *Acinetobacter baumannii* | NCTC 13304 | 1 x 10^{6} | | *Bacteroides fragilis* | *Bacteroides fragilis* | ATCC 25285 | 1 x 10^{5} | | | *Bacteroides fragilis* | ATCC 43860 | 1 x 10^{4} | | *Citrobacter* | *Citrobacter freundii* | NCTC 9750 | 1 x 10^{6} | | | *Citrobacter koseri* | ATCC 27156 | 1 x 10^{6} | | *Cronobacter sakazakii* | *Cronobacter sakazakii* | ATCC 29544 | 1 x 10^{5} | | | *Cronobacter sakazakii* | ATCC 29004 | 1 x 10^{6} | | *Enterobacter* (non-*cloacae* complex) | *Enterobacter aerogenes* | CDC#0074 | 1 x 10^{6} | | | *Enterobacter aerogenes* | CDC#0161 | 1 x 10^{5} | | | *Enterobacter amnigenus* | ATCC 33072 | 1 x 10^{6} | | *Enterobacter cloacae* Complex | *Enterobacter cloacae* | CDC#0154 | 1 x 10^{6} | | | *Enterobacter asburiae* | ATCC 35957 | 1 x 10^{6} | {25} | Target | Organism | Strain | LoD Concentration (CFU/mL) | | --- | --- | --- | --- | | | Enterobacter hormaechei | ATCC BAA-2082 | \( 1 \times 10^{6} \) | | Escherichia coli | Escherichia coli | CDC#0118 | \( 1 \times 10^{7} \) | | | Escherichia coli | NCTC 13441 | \( 1 \times 10^{6} \) | | Fusobacterium necrophorum | Fusobacterium necrophorum | ATCC 51357 | \( 1 \times 10^{8} \) | | | Fusobacterium necrophorum | ATCC 27852 | \( 1 \times 10^{7} \) | | Fusobacterium nucleatum | Fusobacterium nucleatum | ATCC 25586 | \( 1 \times 10^{7} \) | | | Fusobacterium nucleatum | ATCC 23726 | \( 1 \times 10^{5} \) | | Haemophilus influenzae | Haemophilus influenzae | ATCC 19418 | \( 1 \times 10^{5} \) | | | Haemophilus influenzae | ATCC 9006 | \( 1 \times 10^{7} \) | | Klebsiella oxytoca | Klebsiella oxytoca | ATCC 43165 | \( 1 \times 10^{7} \) | | | Klebsiella oxytoca | ATCC 8724 | \( 1 \times 10^{7} \) | | Klebsiella pneumoniae group | Klebsiella pneumoniae | CDC#0160 | \( 1 \times 10^{6} \) | | | Klebsiella pneumoniae | CDC#0107 | \( 1 \times 10^{6} \) | | Morganella morganii | Morganella morganii | ATCC 25829 | \( 1 \times 10^{7} \) | | | Morganella morganii | CDC#0133 | \( 1 \times 10^{7} \) | | Neisseria meningitidis | Neisseria meningitidis | ATCC 13090 | \( 1 \times 10^{5} \) | | | Neisseria meningitidis | ATCC 13102 | \( 1 \times 10^{4} \) | | Proteus | Proteus vulgaris | ATCC 6896 | \( 1 \times 10^{7} \) | | | Proteus vulgaris | ATCC 6380 | \( 1 \times 10^{7} \) | | Proteus mirabilis | Proteus mirabilis | CDC#0159 | \( 1 \times 10^{6} \) | | | Proteus mirabilis | ATCC 43071 | \( 1 \times 10^{6} \) | | Pseudomonas aeruginosa | Pseudomonas aeruginosa | CDC#0103 | \( 1 \times 10^{6} \) | | | Pseudomonas aeruginosa | NCTC 13437 | \( 1 \times 10^{6} \) | | Salmonella | Salmonella bongori | ATCC 43975 | \( 1 \times 10^{5} \) | | | Salmonella enterica | ATCC 6962 | \( 1 \times 10^{5} \) | | Serratia | Serratia liquefaciens | ATCC 27592 | \( 1 \times 10^{6} \) | | | Serratia plymuthica | ATCC 53858 | \( 1 \times 10^{7} \) | | Serratia marcescens | Serratia marcescens | ATCC 14041 | \( 1 \times 10^{7} \) | | | Serratia marcescens | ATCC 14756 | \( 1 \times 10^{5} \) | | Stenotrophomonas maltophilia | Stenotrophomonas maltophilia | ATCC 13637 | \( 1 \times 10^{6} \) | | | Stenotrophomonas maltophilia | ATCC 17666 | \( 1 \times 10^{7} \) | | Pan Candida | Candia albicans | ATCC 10231 | \( 1 \times 10^{6} \) | | | Candia glabrata | ATCC 15126 | \( 1 \times 10^{5} \) | | Pan Gram-Positive | Enterococcus faecalis | ATCC 51575 | \( 1 \times 10^{5} \) | | | Enterococcus faecium | ATCC 31282 | \( 1 \times 10^{7} \) | | | Bacillus subtilis | ATCC 21008 | \( 1 \times 10^{6} \) | | | Staphylococcus aureus | ATCC BAA-2313 | \( 1 \times 10^{5} \) | | | Streptococcus agalactiae | ATCC 13813 | \( 1 \times 10^{6} \) | | | Streptococcus anginosus | ATCC 33397 | \( 1 \times 10^{6} \) | {26} | Target | Organism | Strain | LoD Concentration (CFU/mL) | | --- | --- | --- | --- | | CTX-M | Escherichia coli | NCTC 13441 | 1 x 10^{4} | | | Klebsiella pneumoniae | CDC#0107 | 1 x 10^{5} | | IMP | Enterobacter aerogenes | CDC#0161 | 1 x 10^{6} | | | Pseudomonas aeruginosa | CDC#0103 | 1 x 10^{5} | | KPC | Enterobacter hormaechei | ATCC BAA-2082 | 1 x 10^{6} | | | Morganella morganii | CDC#0133 | 1 x 10^{6} | | NDM | Escherichia coli | CDC#0118 | 1 x 10^{5} | | | Proteus mirabilis | CDC#0159 | 1 x 10^{5} | | OXA | Acinetobacter baumannii (OXA-23) | NCTC 13421 | 1 x 10^{5} | | | Acinetobacter baumannii (OXA-27) | NCTC 13304 | 1 x 10^{5} | | | Enterobacter aerogenes (OXA-48) | CDC#0074 | 1 x 10^{6} | | | Klebsiella pneumoniae (OXA-48) | CDC#0160 | 1 x 10^{6} | | VIM | Enterobacter cloacae | CDC#0154 | 1 x 10^{6} | | | Pseudomonas aeruginosa | NCTC 13437 | 1 x 10^{5} | ### f. Analytical Reactivity (Inclusivity) The analytical reactivity of the BCID-GN Panel was evaluated using a collection of 336 bacterial and fungal strains/isolates. Organisms were tested at concentrations expected at bottle positivity with bacteria tested at 1 x 10$^{9}$ CFU/mL or lower and fungal strains tested at 1 x 10$^{6}$ CFU/mL. For organisms that were not evaluated in the bottle positivity study, test samples were prepared at 4.5x10$^{8}$ CFU/mL (i.e., average concentrations for all gram-negative organisms evaluated in the bottle positivity study). Each strain evaluated for inclusivity was tested in triplicate and if not detected in initial testing, the strain was retested at a higher concentration. If detected at the high concentration(s), the species/isolate is indicated as detected with reduced sensitivity and the concentration is indicated in footnotes under Table 20 below. All strains evaluated generated 'detected' results by the ePlex BCID-GN Panel with the following exceptions: - Strains of Citrobacter amalonaticus, Citrobacter farmer, Citrobacter gillenii, Citrobacter murliniae and Citrobacter sedlakii were not detected by the BCID-GN Panel at any concentration evaluated (up to 1x 10$^{9}$ CFU/mL). - Enterobacter kobei and Enterobacter cancerogenous were not detected by the ePlex BCID-GN Panel. - Enterobacter cowanii was not detected by the ePlex BCID-GN Enterobacter non-cloacae complex assay. Instead, this species generated detected results with the Enterobacter cloacae complex. It is noted that this species is not included in the Enterobacter cloacae complex. - The Pan Gram-positive assay target may have reduced sensitivity for {27} Streptococcus thoraltensis and Enterococcus saccharolyticus (1/6 replicates detected for both organisms at \( 1 \times 10^{7} \) CFU/mL). Additional testing at \( 4 \times 10^{8} \) (Streptococcus thoraltensis) and \( 1 \times 10^{8} \) (Enterococcus saccharolyticus) generated 3/3 replicates detected for the Pan Gram-positive target. - For one strain each of Enterobacter amnigenus and Morganella morganii, two of three initially tested replicates were detected. Additional testing for each strain generated positive results for three additional replicates for a total of five of six replicates tested. - One strain of Serratia odorifera was not detected at \( 1 \times 10^{8} \) CFU/mL and was detected in only one of three replicates at \( 1 \times 10^{9} \) CFU/mL. In silico analysis suggest that this species will not be detected by the BCID-GN Panel. - In initial testing of samples prepared at \( 1 \times 10^{6} \) CFU/mL, the Pan Candida target was detected for one of six replicate samples containing Candida krusei (ATCC 34135) and two of six replicates of Candida parapsilosis (ATCC 90018). Additional testing was performed with blood culture samples from culture bottles grown on a blood culture instrument until bottle positivity. For this additional testing, the Pan Candida target was detected for all six replicates of each Candida strain. Organisms and associated resistance markers evaluated in the analytical reactivity study and detected by the ePlex BCID-GN Panel are shown in Table 20. Additional strains were evaluated as part of the LoD study (See Table 19). Table 20: Analytical Reactivity (Inclusivity) | Organism | Strain | | --- | --- | | Acinetobacter baumannii | | | Acinetobacter baumannii | CDC#0052 | | | NCTC 13302 | | | NCTC 13303 | | | NCTC 13305 | | | NCTC 13420 | | | NCTC 13422 | | | NCTC 13423 | | Acinetobacter baumannii (NDM-1) | CDC#0033 | | Acinetobacter baumannii (OXA-23) | ATCC BAA-1605 | | | CDC#0045 | | | CDC#0056 | | | NCTC 13301 | | | NCTC 13424 | | Acinetobacter spp. (IMP only) | JMI4084\( ^{\mathrm{A}} \) | | Bacteroides fragilis | | | Bacteroides fragilis | ATCC 23745 | | | ATCC 700786 | | | NCTC 9343 | | Organism | Strain | | --- | --- | | Citrobacter | | | Citrobacter braakii | ATCC 43162 | | | ATCC 51113 | | Citrobacter freundii | ATCC 6879 | | | ATCC 8090 | | Citrobacter freundii (CTX) | JMI2047 | | Citrobacter freundii (KPC-2) | CDC#0116 | | Citrobacter koseri | ATCC 25409 | | | ATCC 27028 | | | ATCC 29225 | | | ATCC 29936 | | Citrobacter species (CTX-15, NDM-1) | CDC #0157 | | Citrobacter werkmanii | ATCC 51114 | | Citrobacter youngae | ATCC 29935 | | Cronobacter sakazakii | | | Cronobacter sakazakii | ATCC 12868 | | | ATCC BAA-894 | | | FSL F6-0023 | {28} | Organism | Strain | | --- | --- | | Enterobacter (non-cloacae complex) | | | Enterobacter aerogenes | ATCC 13048 | | | ATCC 29010 | | | ATCC 51697 | | Enterobacter amnigenus | ATCC 33731 | | | ATCC 51816\( ^{B} \) | | Enterobacter gergoviae | ATCC 33028 | | | ATCC 33426 | | Enterobacter cloacae complex | | | Enterobacter asburiae | ATCC 35954 | | | ATCC 35955 | | | ATCC 35956 | | Enterobacter cloacae (CTX-15) | CDC#0038 | | Enterobacter cloacae (CTX-9) | NCTC 13464 | | Enterobacter cloacae (CTX-15, KPC-2) | CDC#0163 | | Enterobacter cloacae (CTX, NDM) | JMI53571 | | Enterobacter cloacae subsp. cloacae | ATCC 23355 | | | ATCC 35030 | | Enterobacter cloacae subsp. dissolvens | ATCC 23373 | | Enterobacter hormaechei | ATCC 700323 | | Enterobacter hormaechei subsp. hormaechei | ATCC 49162 | | Enterobacter hormaechei subsp. oharae | ATCC 49163 | | Enterobacter hormaechei subsp. steigerwaltii | CIP108489T | | Enterobacter ludwigii | DSM-16688 | | Escherichia coli | | | Escherichia coli | ATCC 14948 | | | ATCC 25922 | | | ATCC 33605 | | | ATCC 33876 | | | ATCC 35150 | | | ATCC 4157 | | | ATCC 43888 | | | ATCC 51446 | | | ATCC 51755 | | | ATCC 53498 | | | ATCC 700728 | | | NCIMB 8545 | | | NCTC 8620 | | | ATCC 9637 | | | ATCC BAA-196 | | | ATCC BAA-197 | | | ATCC BAA-198 | | | ATCC BAA-199 | | | ATCC BAA-200 | | | ATCC BAA-201 | | ATCC BAA-202 | | | Organism | Strain | | --- | --- | | | ATCC BAA-203 | | ATCC BAA-204 | | LMC_243094647 | | LMC_243098776 | | LMC_243098947 | | LMC_243108047 | | LMC_243109799 | | LMC_243112411 | | LMC_244006281 | | LMC_244006433 | | LMC_244008038 | | LMC_244012579 | | NCTC 13351 | | NCTC 10279 | | ATCC 10536 | | ATCC 10538 | | ATCC 10799 | | ATCC 11229 | | ATCC 13762 | | ATCC 14169 | | Escherichia coli (CTX-14) | CDC#0086 | | Escherichia coli (CTX-15) | ATCC BAA-2326 | | NCTC 13353 | | NCTC 13400 | | NCTC 13450 | | NCTC 13451 | | Escherichia coli (CTX-3) | NCTC 13452 | | Escherichia coli (CTX-1) | NCTC 13461 | | Escherichia coli (CTX-2) | NCTC 13462 | | Escherichia coli (CTX-8) | NCTC 13463 | | Escherichia coli (CTX-15, NDM-6) | CDC#0137 | | Escherichia coli (CTX-15, NDM-7) | CDC#0162 | | Escherichia coli (IMP) | NCTC 13476 | | Escherichia coli (KPC) | ATCC BAA-2340 | | Escherichia coli (NDM-5) | CDC#0150 | | Escherichia coli (OXA) | LMC_DR00012 | | Escherichia coli (VIM) | JMI32465 | | Fusobacterium necrophorum | | Fusobacterium necrophorum subsp. necrophorum | ATCC 25286 | | NCTC 10575 | | NCTC 10577 | | Fusobacterium nucleatum | | Fusobacterium nucleatum subsp. nucleatum | ATCC 31647 | | Fusobacterium nucleatum subsp. fusiforme | ATCC 51190 | | Fusobacterium nucleatum subsp. vincentii | ATCC 49256 | | Haemophilus influenzae | {29} | Organism | Strain | | --- | --- | | Haemophilus influenzae | ATCC 33930 | | | ATCC 43065 | | | ATCC 43163 | | | NCTC 11931 | | | NCTC 12699 | | | NCTC 8143 | | Haemophilus influenzae Type b | ATCC 10211 | | Haemophilus influenzae Type c | ATCC 9007 | | Haemophilus influenzae Type d | ATCC 9332 | | Haemophilus influenzae Type e | NCTC 8472 | | Haemophilus influenzae Type f | ATCC 9833 | | Klebsiella oxytoca | | | Klebsiella oxytoca | ATCC 43086 | | | ATCC 43863 | | | ATCC 49131 | | | ATCC 700324 | | | ATCC 51817 | | Klebsiella oxytoca (KPC-3) | CDC#0147 | | Klebsiella pneumoniae group | | | Klebsiella pneumoniae (CTX-15) | CDC#0109 | | Klebsiella pneumoniae (CTX-25) | NCTC 13465 | | Klebsiella pneumoniae (CTX, KPC) | IMH-C2261309 | | Klebsiella pneumoniae (CTX, NDM-1) | NCTC 13443 | | Klebsiella pneumoniae (CTX-15; NDM-1; OXA-232) | CDC#0153 | | Klebsiella pneumoniae (CTX-15, OXA-232) | CDC#0075 | | | CDC#0066 | | Klebsiella pneumoniae (CTX-15, OXA-181) | CDC#0039 | | | CDC#0140 | | | CDC#0141 | | | CDC#0142 | | Klebsiella pneumoniae (IMP-4) | CDC#0034 | | | CDC#0080 | | Klebsiella pneumoniae (KPC-3) | CDC#0125 | | | CDC#0112 | | | CDC#0113 | | Klebsiella pneumoniae (KPC) | ATCC BAA-1705 | | | IMH-C2260742 | | | IMH-C3151729 | | | IMH-C4151728 | | | IMH-C4171868 | | Klebsiella pneumoniae (OXA-48) | NCTC 13442 | | Organism | Strain | | --- | --- | | Klebsiella pneumoniae (CTX-15; VIM-27) | CDC#0040 | | Klebsiella pneumoniae (VIM-1) | CDC#0135 | | | NCTC 13439 | | | NCTC 13440 | | Klebsiella pneumoniae subsp. Ozaenae | ATCC 11296 | | Klebsiella pneumoniae subsp. pneumoniae | ATCC 13883 | | | ATCC 27736 | | | ATCC 51503 | | | ATCC 51504 | | Klebsiella quasipneumoniae | ATCC 700603 | | Klebsiella pneumoniae subsp. rhinoscleromatis | ATCC 94…