Unyvero LRT BAL Application
Device Facts
| Record ID | K191967 |
|---|---|
| Device Name | Unyvero LRT BAL Application |
| Applicant | Curetis GmbH |
| Product Code | QBH · Microbiology |
| Decision Date | Dec 20, 2019 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3985 |
| Device Class | Class 2 |
Indications for Use
The Unyvero LRT BAL Application is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms (N = 20) and antibiotic resistance markers (N = 10) in bronchoalveolar lavage (BAL)-like specimens (BAL or mini-BAL) from adult hospitalized patients with suspected lower respiratory tract infections. The Unyvero LRT BAL Application performed on the Unyvero System is indicated as an aid in the diagnosis of lower respiratory tract infection in adult hospitalized patients with signs and symptoms of lower respiratory infection; results should be used in conjunction with other clinical and laboratory findings. As BAL specimens may contain colonizing microorganisms, detection of Unyvero LRT BAL microbial targets does not indicate that the microorganism is the disease. Unyvero positive results do not rule out co-infection with other microorganisms. Negative results do not preclude lower respiratory infection, as the causative agent may be a microorganism not detected by this test. A negative result for any antibiotic resistance marker does not indicate that detected microorganisms are susceptible to applicable antimicrobial agents. Detected antibiotic resistance markers cannot be definitively linked to specific microorganisms, and may be present in organisms that are not detected by the Unyvero LRT BAL Application. Microbiology cultures of BALs should be performed to obtain isolates for species identification and antimicrobial susceptibility testing and to identify potential microorganisms not targeted by the Unyvero LRT BAL Application.
Device Story
The Unyvero LRT BAL Application is a multiplex molecular assay for the qualitative detection of 20 lower respiratory pathogens and 10 antibiotic resistance markers in BAL or mini-BAL specimens. The device uses the Unyvero System (Lysator, Analyzer, Cockpit). The process involves specimen lysis, DNA extraction, and eight parallel multiplex endpoint PCR reactions followed by array hybridization in a single-use cartridge. The system provides automated processing and analysis. Results are displayed on the Unyvero Cockpit for clinician review. The device is intended to aid in the diagnosis of lower respiratory tract infections in hospitalized adults. It provides rapid identification of potential pathogens and resistance markers, which, when used in conjunction with clinical and laboratory findings, can assist in clinical decision-making regarding antimicrobial therapy. It does not replace standard microbiology cultures, which are required for definitive species identification and antimicrobial susceptibility testing.
Clinical Evidence
Clinical performance was established via a prospective study (N=1,016) and an archived study (N=392). Prospective study PPA/NPA for typical microorganisms compared to SoC culture ranged widely (e.g., P. aeruginosa PPA 95.8%, NPA 95.4%; S. aureus PPA 88.7%, NPA 95.7%). A composite comparator (SoC culture + molecular PCR/sequencing) was also used. Antibiotic resistance marker performance was evaluated against molecular reference assays. Contrived samples supplemented data for rare analytes. Bench testing confirmed LoD, inclusivity, and exclusivity.
Technological Characteristics
The system uses multiplex endpoint PCR and array hybridization. Materials include a single-use cartridge containing DNA isolation reagents, primers, and hybridization buffers. The system is networked via Ethernet. Software manages the workflow across the Lysator, Analyzer, and Cockpit. The process is automated, requiring minimal manual intervention after sample loading. No specific ASTM standards for materials are cited; technical performance is defined by analytical sensitivity (LoD) and specificity.
Indications for Use
Indicated for adult hospitalized patients with suspected lower respiratory tract infections. Used as an aid in diagnosis of lower respiratory tract infection in patients with signs and symptoms. Not for definitive linkage of resistance markers to specific organisms; does not rule out co-infection; negative results do not preclude infection.
Regulatory Classification
Identification
A device to detect and identify microorganisms and associated resistance marker nucleic acids directly from respiratory specimens is an in vitro diagnostic device intended for the detection and identification of microorganisms and associated resistance markers in respiratory specimens collected from patients with signs or symptoms of respiratory infection. The device is intended to aid in the diagnosis of respiratory infection in conjunction with clinical signs and symptoms and other laboratory findings. These devices do not provide confirmation of antibiotic susceptibility since mechanisms of resistance may exist other than those detected by the device.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) The intended use for the 21 CFR 809.10 labeling must include a detailed description of what the device detects, the type of results provided to the user, the clinical indications appropriate for test use, and the specific population(s) for which the device is intended. (2) The 21 CFR 809.10(b) labeling must include: (i) A detailed device description, including all device components, control elements incorporated into the test procedure, instrument requirements, ancillary reagents required but not provided, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens. (ii) Performance characteristics from analytical studies, including, but not limited to, limit of detection, inclusivity, reproducibility, cross reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, and linearity, as applicable. (iii) A limiting statement that the device is intended to be used in conjunction with clinical history, signs and symptoms, and results of other diagnostic tests, including culture and antimicrobial susceptibility testing. (iv) A detailed explanation of the interpretation of test results for clinical specimens and acceptance criteria for any quality control testing. (v) A limiting statement that negative results for microorganisms do not preclude the possibility of infection, and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. (vi) If applicable, a limiting statement that detected microorganisms may not be the cause of lower respiratory tract infection and may be indicative of colonizing or normal respiratory flora. (vii) If applicable, a limiting statement that detection of resistance markers cannot be definitively linked to specific microorganisms and that the source of a detected resistance marker may be an organism not detected by the assay, including colonizing flora. (viii) If applicable, a limiting statement that detection of antibiotic resistance markers may not correlate with phenotypic gene expression. (3) The 21 CFR 809.10(b) labeling and any test report generated by the device must include a limiting statement that negative results for resistance markers do not indicate susceptibility of detected microorganisms. (4) Design verification and validation must include: (i) Performance characteristics from clinical studies that include prospective (sequential) samples and, if appropriate, additional characterized samples. The study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained from an FDA accepted reference method and/or FDA accepted comparator method, as appropriate. Results from the clinical studies must include the clinical study protocol (including predefined statistical analysis plan, if applicable), clinical study report, and results of all statistical analyses. (ii) A detailed device description including the following: (A) Thorough description of the assay methodology including, but not limited to, primer/probe sequences, primer/probe design, and rationale for target sequence selection, as applicable. (B) Algorithm used to generate a final result from raw data (e.g., how raw signals are converted into a reported result). (iii) A detailed description of device software, including, but not limited to, validation activities and outcomes. (iv) As part of the risk management activities, an appropriate end user device training program must be offered as an effort to mitigate the risk of failure from user error.
Predicate Devices
- Unyvero LRT Application (DEN170047)
Submission Summary (Full Text)
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Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
# I Background Information:
A 510(k) Number
K191967
B Applicant
Curetis GmbH
C Proprietary and Established Names
Unyvero Lower Respiratory Tract (LRT) BAL Application
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QBH | Class II | 21 CFR 866.3985 - Device To Detect And Identify Microorganisms And Associated Resistance Marker Nucleic Acids Directly In Respiratory Specimens | MI - Microbiology |
# II Submission/Device Overview:
A Purpose for Submission:
Clearance of the Unyvero Lower Respiratory Tract (LRT) BAL Application for use on the Unyvero System.
B Measurand:
Nucleic acid sequences from the following microorganisms and associated resistance markers.
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| Microorganism Targets | Antibiotic Resistance Marker Targets |
| --- | --- |
| Acinetobacter spp. | ctx-M (blaCTX-M, subgroup 1 only) |
| Chlamydia pneumoniae | kpc (blaKPC) |
| Citrobacter freundii | mecA |
| Escherichia coli | ndm (blaNDM) |
| Enterobacter cloacae complex | oxa-23 (blaOXA-23) |
| Haemophilus influenzae | oxa-24 (blaOXA-24) |
| Klebsiella oxytoca | oxa-48 (blaOXA-48) |
| Klebsiella pneumoniae | oxa-58 (blaOXA-58) |
| Klebsiella variicola | tem (blaTEM) |
| Legionella pneumophila | vim (blaVIM) |
| Moraxella catarrhalis | |
| Morganella morganii | |
| Mycoplasma pneumoniae | |
| Proteus spp. | |
| Pseudomonas aeruginosa | |
| Serratia marcescens | |
| Staphylococcus aureus | |
| Stenotrophomonas maltophilia | |
| Streptococcus pneumoniae | |
### C Type of Test:
Multiplex molecular assay for detection of lower respiratory pathogens and associated resistance markers.
### III Intended Use/Indications for Use:
### A Intended Use:
The Unyvero LRT BAL Application is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms (N = 20) and antibiotic resistance markers (N = 10) in bronchoalveolar lavage (BAL)-like specimens (BAL or mini-BAL) from adult hospitalized patients with suspected lower respiratory tract infections.
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| Microorganism | Associated Antibiotic Resistance Marker |
| --- | --- |
| Acinetobacter spp.^{a} | ctx-M^{b}, kpc, ndm, oxa-23, oxa-24, oxa-58, vim |
| Chlamydia pneumoniae | - |
| Citrobacter freundii | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Enterobacter cloacae complex^{c} | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Escherichia coli | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Haemophilus influenzae | tem |
| Klebsiella oxytoca | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Klebsiella pneumoniae^{d} | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Klebsiella variicola | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Legionella pneumophila | - |
| Moraxella catarrhalis | - |
| Morganella morganii | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Mycoplasma pneumoniae | - |
| Pneumocystis jirovecii | - |
| Proteus spp.^{e} | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Pseudomonas aeruginosa | ctx-M^{b}, kpc, ndm, vim |
| Serratia marcescens | ctx-M^{b}, kpc, ndm, oxa-48, vim |
| Staphylococcus aureus | mecA |
| Stenotrophomonas maltophilia | - |
| Streptococcus pneumoniae | - |
$^{a}$ Acinetobacter spp. includes: A. baumannii, A. calcoaceticus, A. haemolyticus, A. junii, A. lwoffii, A. nosocomialis, A. parvus, A. pittii (detected by LRT BAL Application) and A. ursingii (not detected by LRT BAL Application).
$^{b}$ ctx-M1 subgroup.
$^{c}$ Enterobacter cloacae complex includes: E. asburiae, E. chengduensis, E. chuandaensis, E. cloacae, E. hormaechei (incl. ssp. xiangfangensis), E. kobei, E. ludwigii, E. roggenkampii, E. sichuanensis as well as E. bugandensis (not yet recognized as member of the E. cloacae complex).
$^{d}$ Klebsiella pneumoniae includes two variants: K. pneumoniae (variant 1), and K. quasipneumoniae (variant 2).
$^{e}$ Proteus spp. includes P. cibarius, P. hauseri, P. mirabilis, P. penneri and P. vulgaris.
The Unyvero LRT BAL Application performed on the Unyvero System is indicated as an aid in the diagnosis of lower respiratory tract infection in adult hospitalized patients with signs and symptoms of lower respiratory infection; results should be used in conjunction with other clinical and laboratory findings. As BAL specimens may contain colonizing microorganisms, detection of Unyvero LRT BAL microbial targets does not indicate that the microorganism is the cause of the disease. Unyvero positive results do not rule out co-infection with other microorganisms. Negative results do not preclude lower respiratory infection, as the causative agent may be a microorganism not detected by this test.
A negative result for any antibiotic resistance marker does not indicate that detected microorganisms are susceptible to applicable antimicrobial agents. Detected antibiotic
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resistance markers cannot be definitively linked to specific microorganisms, and may be present in organisms that are not detected by the Unyvero LRT BAL Application.
Microbiology cultures of BALs should be performed to obtain isolates for species identification and antimicrobial susceptibility testing and to identify potential microorganisms not targeted by the Unyvero LRT BAL Application.
# **B Indication(s) for Use:**
Same as Intended Use
# **C Special Conditions for Use Statement(s):**
Rx - For Prescription Use Only
# **D Special Instrument Requirements:**
Unyvero System, including the Unyvero Lysator, Analyzer and Cockpit
# **IV Device/System Characteristics:**
# **A Device Description:**
The Unyvero LRT BAL Application integrates DNA purification and eight parallel multiplex endpoint PCR reactions, each followed by array hybridization with all reactions performed simultaneously in a single use cartridge. The assay is performed with bronchial alveolar lavage (BAL or mini-BAL) specimens and provides qualitative detection of nucleic acids from 20 lower respiratory pathogens and 10 antibiotic resistance markers. Multiplex reagent compositions are designed to limit the occurrence of certain analytes within the same multiplex assay and to reduce the potential for competitive inhibition. Individual analyte assays of the Unyvero LRT Array oligonucleotides are designed for similar hybridization and melting temperatures (approx. 65 - 80°C, varying by amplicon). Hybridization and melting temperatures are used to exclude non-specific hybridization signals for improved signal specificity.
The Unyvero instrumentation consists of one (or more) Unyvero Lysators, one (or more) Unyvero Analyzers, a Unyvero Cockpit. Four single-use consumables are required for testing: the Unyvero LRT BAL Cartridge, the Unyvero Sample Tube, Sample Tube Cap and the Unyvero Master Mix. A Unyvero Sample Tube Holder is also supplied for use in the specimen inoculation step. The four assay consumables consist of the following:
- Unyvero LRT BAL Cartridge contains DNA isolation and purification reagents, primers, hybridization and wash buffers, and oligonucleotides for detection.
- Unyvero T1 Sample Tube, contains glass beads and buffers to lyse microorganisms and liquefy the specimen.
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- Unyvero T1 Sample Tube Cap seals the Unyvero Sample Tube and contains Proteinase K and a synthetic control gene for process monitoring.
- Unyvero M1 Master Mix Tube contains reagents for DNA amplification.
## Controls
An internal control (a synthetic gene with no known homology to assay target sequences) is processed in each of the eight PCR chambers. Results are used to verify adequate DNA purification, amplification, array hybridization, and detection.
Other than the built-in controls, external control materials are not supplied with Unyvero LRT BAL Application devices and consumables. The assay labeling recommends running external positive and negative controls regularly. Suggested positive controls included characterized specimens inoculated with well-characterized microorganisms.
## Reagents
No additional reagents are required to perform the Unyvero LRT BAL Application; all reagents are supplied within the cartridge or within the other consumables with the exception of the polymerase Master Mix, which is provided separately (frozen).
## B Principle of Operation:
The Unyvero LRT BAL Application uses a multiplex PCR approach followed by array hybridization. The Unyvero LRT Application is a qualitative PCR-based assay that detects DNA sequences of microorganisms and associated resistance markers in BAL specimens. The assay includes specimen processing (lysis), DNA extraction and isolation, multiplex PCR with eight parallel multiplex endpoint PCR reactions, and qualitative detection of amplified DNA products using hybridization arrays.
A BAL specimen is first pipetted into the Unyvero Sample Tube and closed with the Unyvero Sample Tube Cap. Closing the sample tube automatically adds the lysis reagent and the internal control gene template to the specimen. The sample tube which fits into the Unyvero Lysator only if closed with the Unyvero sample tube cap is then placed on the Lysator. After the specimen is lysed in the Lysator, the Sample Tube and Master Mix are loaded into the Unyvero LRT Cartridge which is then inserted into the position assigned by the Unyvero Analyzer for automated processing and analysis.
In the Unyvero LRT Cartridge, the remaining testing steps are automated by the Unyvero Analyzer. The lysed specimen is further processed and then transferred onto a DNA purification column for nucleic acid extraction and elution of genomic DNA. Eluted DNA is transferred to a chamber, where mixing with the Master Mix takes place. This mixture is distributed into eight separate PCR reaction chambers each containing multiple primer pairs. After amplification, PCR products are hybridized to the corresponding array probes. Multiple spots per array allow for redundant detection with at least four spots per analyte, as well as
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spots for intensity calibration, and orientation markers for the imaging software. Binding of amplicons to specific probes is detected by analyzing fluorescence images of each separate array. Result data are transferred to the Unyvero Cockpit for visualization and result printout. A test run is completed after approximately 4.5 hours, and results for panel microorganisms and associated antibiotic resistance markers are displayed on the Unyvero Cockpit screen.
### C Instrument Description Information:
| Modes of Operation | Yes | No |
| --- | --- | --- |
| Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device? | ☑ | ☐ |
| Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission? | ☐ | ☑ |
| **Software** | | |
| FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types. | ☑ | ☐ |
1. Instrument Name:
Unyvero System
2. Specimen Identification:
Specimen Identification information can be manually entered or automatically entered using the integrated barcode reader.
3. Specimen Sampling and Handling:
Before starting the test, the user scans the clinical identification (barcode) from the primary specimen container using the built-in barcode reader of the Unyvero Cockpit or the information may be entered manually on the cockpit on-screen keyboard.
The specimen is initially vortexed and then manually pipetted into the Unyvero Sample Tube. After the specimen is placed in the Sample Tube, the user then places the Unyvero Cap on the Sample Tube, scans the Sample Tube barcode and places it in the Unyvero Lysator. After processing on the Lysator, the user places the Sample Tube and thawed Mastermix into the Unyvero LRT Cartridge, scans the Cartridge barcode and places it into the indicated position in the Unyvero Analyzer. The Unyvero software then instructs the user to start the test which is fully automated until completion.
4. Calibration:
Calibration is not required by the user.
5. Quality Control:
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See section L1(c) for information on internal and external controls.
## V Substantial Equivalence Information:
### A Predicate Device Name(s):
Curetis Unyvero LRT Application
### B Predicate 510(k) Number(s):
DEN170047
### C Comparison with Predicate:
| Device & Predicate Device(s): | K191967 | DEN170047 |
| --- | --- | --- |
| Device Trade Name | **Unyvero Lower Respiratory Tract BAL Application** | **Unyvero LRT Application** |
| **General Device Characteristic Similarities** | | |
| Microorganisms Detected | ‘Typical’ Bacteria: *Acinetobacter* spp. *Citrobacter freundii* *Enterobacter cloacae* complex *Escherichia coli* *Haemophilus influenzae* *Klebsiella oxytoca* *Klebsiella pneumoniae* *Klebsiella variicola* *Moraxella catarrhalis* *Morganella morganii* *Proteus* spp. *Pseudomonas aeruginosa* *Serratia marcescens* *Staphylococcus aureus* *Stenotrophomonas maltophilia* *Streptococcus pneumoniae* ‘Atypical’ Bacteria: *Chlamydia pneumoniae* *Legionella pneumophila* *Mycoplasma* | ‘Typical’ Bacteria: *Acinetobacter* spp. *Citrobacter freundii* *Enterobacter cloacae* complex *Escherichia coli* *Haemophilus influenzae* *Klebsiella oxytoca* *Klebsiella pneumoniae* *Klebsiella variicola* *Moraxella catarrhalis* *Morganella morganii* *Proteus* spp. *Pseudomonas aeruginosa* *Serratia marcescens* *Staphylococcus aureus* *Stenotrophomonas maltophilia* *Streptococcus pneumoniae* ‘Atypical’ Bacteria: *Chlamydia pneumoniae* *Legionella pneumophila* *Mycoplasma* |
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| | pneumoniae Fungus: Pneumocystis jirovecii | pneumoniae |
| --- | --- | --- |
| Antimicrobial Resistance Markers Detected | ctx-M kpc mecA ndm oxa-23 oxa-24 oxa-48 oxa-58 tem vim | Same |
| Technology | Multiplex PCR followed by probe array | Same |
| Result Type | Qualitative | Same |
| Instrumentation | Unyvero System | Same |
| Test Interpretation | Automated test interpretation and report generation | Same |
| General Device Characteristic Differences | | |
| Specimen Type | BAL specimens | Endotracheal aspirate specimens |
### VI Standards/Guidance Documents Referenced:
None
### VII Performance Characteristics (if/when applicable):
#### A Analytical Performance:
Sample matrix: Unless otherwise specified, analytical study samples were prepared using a simulated matrix (ARM, Artificial Respiratory Matrix) which was adapted from a composition published by Dinesh et al. ARM was diluted to an appropriate consistency as a surrogate for BAL matrix (25% (v/v) ARM, 60% (v/v) PBS and 15% (v/v) glycerol). The final 0.25x ARM also includes 5 mg/mL mucin type II from pig stomach and 1 mg/mL fish sperm DNA.
##### 1. Reproducibility:
Assay reproducibility was established with contrived samples that were prepared using viable microorganism strain suspensions inoculated into 0.25 ARM (artificial respiratory
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matrix). Representative target organisms evaluated included Gram-positive, Gram-negative and atypical microorganisms. The following LRT BAL Application analytes were included in the study: Acinetobacter spp., C. pneumoniae, C. freundii, H. influenzae, K. pneumoniae (carrying LRT BAL antibiotic resistance markers ctx-M, ndm, and tem), M. morganii, Proteus spp. and, S. aureus (carrying LRT BAL antibiotic resistance marker mecA). Reproducibility testing included samples prepared with moderate microorganism concentrations (2.5x - 10x LoD), samples prepared at low concentrations (1x - 4x LoD), and negative samples (surrogate matrix only (0.25 ARM)). Three different Unyvero systems were used in the study with each system consisting of one Unyvero Cockpit, three Unyvero Lysators, up to six Unyvero Analyzers, Testing was performed by three operators.
Each operator performed 3 replicates each of moderate, low and negative samples per test shift using one Unyvero system (maximum: 18 replicates per test day). Each operator performed a minimum of 30 replicates per concentration level (90 total replicates per Unyvero system, 270 replicates in total for all three Unyvero systems with 90 replicates of each concentration level). Testing was performed over a minimum of five test days. Failed runs as well as runs with single invalid analyte results were repeated to achieve a minimum number of at least 30 valid results for each of the target analytes and test concentration.
The study demonstrated acceptable test reproducibility by the LRT BAL Application for all analytes and concentrations evaluated. Three cartridge runs performed on different test days and with different operators/test systems generated unexpected positive results with weak signals close to assay thresholds (2x E. coli, for one 'moderate' and one 'low' test sample, 1x oxa-23, for one 'moderate' test sample).
Tables 1 to 3 summarize the study results for the microorganism and resistance marker target evaluated in the study.
Table 1: Reproducibility Study, Moderate Positive Samples
| Microorganism target | x-fold LoD | Unyvero System/Operator | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | # Pos. /# Exp. | % | 95% CI |
| *Acinetobacter baumannii* ATCC 19606 | 5 | operator 1 | 30/31 | 96.8 | 83.8 - 99.4 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **91/92** | **98.9** | **94.1 - 99.8** |
| *Chlamydia pneumoniae* ATCC VR-2282 | 5 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **92/92** | **100.0** | **96.0 - 100.0** |
| *Citrobacter freundii* ATCC 8090 | 5 | operator 1 | 29/30^{a} | 96.7 | 83.3 - 99.4 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **90/91** | **98.9** | **94.0 - 99.8** |
| *Haemophilus influenzae* ATCC 33391 | 5 | operator 1 | 29/31 | 93.5 | 79.3 - 98.2 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **90/92** | **97.8** | **92.4 - 99.4** |
| *Klebsiella pneumoniae* NCTC 13443 | 2.5 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
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| Microorganism target | x-fold LoD | Unyvero System/Operator | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | # Pos. /# Exp. | % | 95% CI |
| | | **total** | **92/92** | **100.0** | **96.0 - 100.0** |
| *ctx*-M NCTC 13443 | 10 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **92/92** | **100.0** | **96.0 - 100.0** |
| *ndm* NCTC 13443 | 5 | operator 1 | 29/30^{a} | 96.7 | 83.3 - 99.4 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **90/91** | **98.9** | **94.0 - 99.8** |
| *tem* NCTC 13443 | 5 | operator 1 | 28/31 | 90.3 | 75.1 - 96.7 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **89/92** | **96.7** | **90.8 - 98.9** |
| *Morganella morganii* ATCC 25830 | 5 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **92/92** | **100.0** | **96.0 - 100.0** |
| *Proteus vulgaris* ATCC 29905 | 5 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 30/30^{a} | 100.0 | 88.7 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **91/91** | **100.0** | **96.0 - 100.0** |
| *Staphylococcus aureus* NCTC 12493 | 8.3 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **92/92** | **100.0** | **96.0 - 100.0** |
| *mecA* NCTC 12493 | 3 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **92/92** | **100.0** | **96.0 - 100.0** |
$^{a}$ Reduced number of available results due to invalid analyte results
Table 2: Reproducibility study results, Low positive samples
| Microorganism Target | x-fold LoD | Unyvero System/Operator | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | # Pos. /# Exp. | % | 95% CI |
| *Acinetobacter baumannii* ATCC 19606 | 2 | operator 1 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | **total** | **89/91** | **97.8** | **92.3 - 99.4** |
| *Chlamydia pneumoniae* ATCC VR-2282 | 2 | operator 1 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | **total** | **91/91** | **100.0** | **96.0 - 100.0** |
| *Citrobacter freundii* ATCC 8090 | 2 | operator 1 | 28/30 | 93.3 | 78.7 - 98.2 |
| | | operator 2 | 26/31 | 83.9 | 67.4 - 92.9 |
| | | operator 3 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | **total** | **83/91** | **91.2** | **83.6 - 95.5** |
| *Haemophilus influenzae* ATCC 33391 | 2 | operator 1 | 27/30 | 90.0 | 74.4 - 96.5 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | **total** | **87/91** | **95.6** | **89.2 - 98.3** |
K191967 - Page 10 of 88
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| Microorganism Target | x-fold LoD | Unyvero System/Operator | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | # Pos. /# Exp. | % | 95% CI |
| Klebsiella pneumoniaeNCTC 13443 | 1 | operator 1 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 28/30 | 93.3 | 78.7 - 98.2 |
| | | total | 89/91 | 97.8 | 92.3 - 99.4 |
| ctx-MNCTC 13443 | 4 | operator 1 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | operator 2 | 30/31 | 96.8 | 83.8 - 99.4 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | total | 89/91 | 97.8 | 92.3 - 99.4 |
| ndmNCTC 13443 | 2 | operator 1 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | operator 2 | 30/31 | 96.8 | 83.8 - 99.4 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | total | 89/91 | 97.8 | 92.3 - 99.4 |
| temNCTC 13443 | 2 | operator 1 | 27/30 | 90.0 | 74.4 - 96.5 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | total | 87/91 | 95.6 | 89.2 - 98.3 |
| Morganella morganiiATCC 25830 | 2 | operator 1 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | operator 2 | 30/31 | 96.8 | 83.8 - 99.4 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | total | 90/91 | 98.9 | 94.0 - 99.8 |
| Proteus vulgaris\( ^a \)ATCC 29905 | 2 | operator 1 | 25/30 | 83.3 | 66.4 - 92.7 |
| | | operator 2 | 24/31 | 77.4 | 60.2 - 88.6 |
| | | operator 3 | 27/30 | 90.0 | 74.4 - 96.5 |
| | | total | 76/91 | 83.5 | 74.6 - 89.7 |
| Staphylococcus aureusNCTC 12493 | 3 | operator 1 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | operator 2 | \( 30/30^b \) | 100.0 | 88.7 - 100.0 |
| | | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | total | 89/90 | 98.9 | 94.0 - 99.8 |
| mecANCTC 12493 | 1.3 | operator 1 | 30/30 | 100.0 | 88.7 - 100.0 |
| | | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | | operator 3 | 29/30 | 96.7 | 83.3 - 99.4 |
| | | total | 90/91 | 98.9 | 94.0 - 99.8 |
\( ^{a} \) Proteus spp. demonstrated a slightly lower than expected positivity rate for the 2x LoD concentration with a positivity rate (for all operators combined) of 83.5% with a 95% CI of 74.6% - 89.7%. However, for an alternative test series performed in parallel for the sample stability study using the same strain and the identical cartridge lot with pooled negative BAL matrix the expected positivity rate at a 2x LoD concentration for Proteus spp. was confirmed (positivity rate: 92/93, 98.9%).
\( ^{b} \) Reduced number of available results due to invalid analyte results.
Table 3: Reproducibility Study Results, Negative Samples
| Microorganism target | Unyvero System/Operator | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- |
| | | # Neg. /# Exp. | % | 95% CI |
| Acinetobacter baumanniiATCC 19606 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| Chlamydia pneumoniaeATCC VR-2282 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| Citrobacter freundii | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
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| Microorganism target | Unyvero System/Operator | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- |
| | | # Neg. /# Exp. | % | 95% CI |
| ATCC 8090 | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| Haemophilus influenzaeATCC 33391 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| Klebsiella pneumoniaeNCTC 13443 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | \( 30/30^a \) | 100.0 | 88.7 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 91/91 | 100.0 | 96.0 - 100.0 |
| ctx-MNCTC 13443 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | \( 30/30^a \) | 100.0 | 88.7 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 91/91 | 100.0 | 96.0 - 100.0 |
| ndmNCTC 13443 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| temNCTC 13443 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| Morganella morganiiATCC 25830 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | \( 30/30^a \) | 100.0 | 88.7 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 91/91 | 100.0 | 96.0 - 100.0 |
| Proteus vulgarisATCC 29905 | operator 1 | \( 30/30^a \) | 100.0 | 88.7 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 91/91 | 100.0 | 96.0 - 100.0 |
| Staphylococcus aureusNCTC 12493 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 92/92 | 100.0 | 96.0 - 100.0 |
| mecANCTC 12493 | operator 1 | 31/31 | 100.0 | 89.0 - 100.0 |
| | operator 2 | \( 30/30^a \) | 100.0 | 88.7 - 100.0 |
| | operator 3 | 30/30 | 100.0 | 88.7 - 100.0 |
| | total | 91/91 | 100.0 | 96.0 - 100.0 |
\( ^{a} \) Reduced number of available results due to invalid analyte results
### 2. Linearity:
Not Applicable
### 3. Analytical Reactivity (Inclusivity):
The analytical reactivity of the Unyvero LRT BAL Application was evaluated with reference strains for each microorganism target. Samples were prepared with microorganism
K191967 - Page 12 of 88
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concentrations near their respective LoDs in pooled negative BAL specimen matrix. Reference strains tested in the LoD study were also included in the study as control strains.
Assay inclusivity was established near LoD (< 5x LoD) for all but one of the tested reference strains. For one strain of M. pneumoniae (ATCC 15531) that was evaluated using genomic DNA extract, inclusivity was demonstrated only at 5x LoD, although in silico analysis demonstrated no primer or probe mismatches to the corresponding LRT BAL assay targets. Results from the study are presented in Table 4.
Table 4: Inclusivity Study, Microorganism Targets
| Reference Strain | Strain ID | Test Conc. [CFU/ml or other as noted by footnote] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- |
| *Acinetobacter baumannii* (pos. control/LoD ref. strain) | ATCC 19606 | 4.0 x 10^{4} | 2x | 6/6 |
| *Acinetobacter baumannii* | NCTC 13305 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter baumannii* | NCTC 13301 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter baumannii* | NCTC 13302 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter baumannii* | Micromyx 6148 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter baumannii* | Micromyx 4410 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter baumannii* | JMI 49755 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter baumannii* | NRZ-00449 | 1.0 x 10^{4} | 0.5x | 2/2 |
| *Acinetobacter baumannii* | UCLA A4 | 1.0 x 10^{4} | 0.5x | 2/2 |
| *Acinetobacter calcoaceticus* | ATCC 23055 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter lwoffii* | ATCC 15309 | 4.0 x 10^{4} | 2x | 2/2 |
| *Acinetobacter haemolyticus* | ATCC 17906 | 4.0 x 10^{4} | 2x | 2/2 |
| ***Chlamydia pneumoniae*** (pos. control/LoD ref. strain) | ATCC VR-2282 | 6.4 x 10^{2 a} | 2x | 4/4 |
| *Chlamydia pneumoniae* | ATCC VR-1310 | 6.4 x 10^{2 a} | 2x | 2/2 |
| *Chlamydia pneumoniae* | ATCC 53592 | 6.4 x 10^{2 a} | 2x | 0/2 |
| | | 1.0 x 10^{3 a} | 3x | 2/2 |
| ***Citrobacter freundii*** (pos. control/LoD ref. strain) | ATCC 8090 | 1.6 x 10^{5} | 2x | 4/4 |
| *Citrobacter freundii* | ATCC 43864 | 1.6 x 10^{5} | 2x | 2/2 |
| *Citrobacter freundii* | NCTC 8581 | 1.6 x 10^{5} | 2x | 2/2 |
| *Citrobacter freundii* | NRZ-00452 | 1.6 x 10^{5} | 2x | 2/2 |
| *Citrobacter freundii* | UCLA C1 | 1.6 x 10^{5} | 2x | 2/2 |
| ***Enterobacter cloacae*** (pos. control/LoD ref. strain) | ATCC 13047 | 4.0 x 10^{5} | 2x | 7/7 |
| *Enterobacter cloacae* | ATCC 23355 | 4.0 x 10^{5} | 2x | 2/2 |
| *Enterobacter cloacae* | ATCC 49141 | 4.0 x 10^{5} | 2x | 2/2 |
| *Enterobacter cloacae* | ATCC BAA-2468 | 4.0 x 10^{5} | 2x | 2/2 |
| *Enterobacter cloacae* | JMI 46239 | 4.0 x 10^{5} | 2x | 2/2 |
| *Enterobacter cloacae* | NRZ-00239 | 4.0 x 10^{5} | 2x | 2/2 |
| *Enterobacter cloacae* ssp. *dissolvens* | ATCC 23373 | 4.0 x 10^{5} | 2x | 2/2 |
| *Enterobacter hormaechei* | ATCC 49162 | 4.0 x 10^{5} | 2x | 2/2 |
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| Reference Strain | Strain ID | Test Conc. [CFU/ml or other as noted by footnote] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- |
| Enterobacter asburiae | ATCC 35953 | 4.0 x 10^{5} | 2x | 2/2 |
| Escherichia coli (pos. control/LoD ref. strain) | ATCC 11775 | 4.0 x 10^{4} | 2x | 4/4 |
| Escherichia coli | ATCC 25922 | 4.0 x 10^{4} | 2x | 2/2 |
| Escherichia coli | ATCC 35218 | 4.0 x 10^{4} | 2x | 4/4 |
| Escherichia coli | ATCC BAA-2523 | 4.0 x 10^{4} | 2x | 2/2 |
| Escherichia coli | NCTC 13351 | 4.0 x 10^{4} | 2x | 4/4 |
| Escherichia coli | NCTC 13476 | 4.0 x 10^{4} | 2x | 2/2 |
| Escherichia coli | JMI 50067 | 4.0 x 10^{4} | 2x | 2/2 |
| Haemophilus influenzae (non-typeable/non-capsulated) (pos. control/LoD ref. strain) | ATCC 33391 | 4.0 x 10^{4} | 2x | 3/3 |
| Reference Strain | Strain ID | Test Conc. [CFU/ml] | x-fold LoD | # Pos./ # Exp. |
| Haemophilus influenzae (serotype a) | ATCC 9006 | 4.0 x 10^{4} | 2x | 2/2 |
| Haemophilus influenzae (serotype c) | ATCC 9007 | 4.0 x 10^{4} | 2x | 2/2 |
| Haemophilus influenzae (serotype b) | ATCC 10211 | 4.0 x 10^{4} | 2x | 1/2 |
| | | 6.0 x 10^{4} | 3x | 2/2 |
| Haemophilus influenzae (serotype b) | ATCC 49247 | 4.0 x 10^{4} | 2x | 2/2 |
| Haemophilus influenzae (serotype b) | ATCC 49766 | 4.0 x 10^{4} | 2x | 2/2 |
| Klebsiella oxytoca (pos. control/LoD ref. strain) | ATCC 13182 | 4.0 x 10^{4} | 4x | 3/3 |
| Klebsiella oxytoca | ATCC 43863 | 4.0 x 10^{4} | 4x | 2/2 |
| Klebsiella oxytoca | ATCC 8724 | 4.0 x 10^{4} | 4x | 2/2 |
| Klebsiella oxytoca | ATCC 49131 | 4.0 x 10^{4} | 4x | 2/2 |
| Klebsiella oxytoca | NCIMB 12819 | 4.0 x 10^{4} | 4x | 2/2 |
| Klebsiella oxytoca | NRZ-22060 | 4.0 x 10^{4} | 4x | 2/2 |
| Klebsiella pneumoniae, variant 1 (pos. control/LoD ref. strain) | ATCC 13883 | 8.0 x 10^{4} | 2x | 4/4 |
| Klebsiella quasipneumoniae (K. pneumoniae, variant 2) (pos. control/LoD ref. strain) | ATCC 700603 | 8.0 x 10^{4} | 2x | 3/3 |
| Klebsiella pneumoniae | NCTC 13439 | 8.0 x 10^{4} | 2x | 2/2 |
| Klebsiella pneumoniae | NCTC 13438 | 8.0 x 10^{4} | 2x | 2/2 |
| Klebsiella pneumoniae | NCTC 13442 | 8.0 x 10^{4} | 2x | 2/2 |
| Klebsiella pneumoniae | NCTC 13443 | 2.0 x 10^{4} | 0.5x | 3/3 |
| Klebsiella pneumoniae | Micromyx 4653 | 8.0 x 10^{4} | 2x | 2/2 |
| Klebsiella pneumoniae | Micromyx 4676 | 8.0 x 10^{4} | 2x | 2/2 |
| Klebsiella pneumoniae | JMI 49767 | 2.0 x 10^{4} | 0.5x | 2/2 |
| Klebsiella pneumoniae | NRZ-00002 | 2.0 x 10^{4} | 0.5x | 2/2 |
| Klebsiella pneumoniae | NRZ-00103 | 8.0 x 10^{4} | 2x | 2/2 |
| Klebsiella variicola (pos. control/LoD ref. strain) | ATCC BAA-830 | 4.0 x 10^{4} | 2x | 3/3 |
| Klebsiella variicola | clinical strain 1 | 4.0 x 10^{4} | 2x | 2/2 |
K191967 - Page 14 of 88
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| Reference Strain | Strain ID | Test Conc. [CFU/ml or other as noted by footnote] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- |
| Klebsiella variicola | clinical strain 2 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| Klebsiella variicola | clinical strain 3 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| Klebsiella variicola | clinical strain 4 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| Legionella pneumophila (serotype 1) (pos. control/LoD ref. strain) | ATCC 33152 | \( {1.6} \times {10}^{5} \) | 2x | 7/7 |
| Legionella pneumophila (serotype 2) | ATCC 33154 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila (serotype 3) | ATCC 33155 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila (serotype 6) | ATCC 33215 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila (serotype 8) | ATCC 35096 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila (serotype 10) | ATCC 43283 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila | UCLA L1 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila | UCLA L5 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Legionella pneumophila | UCLA L6 | \( {1.6} \times {10}^{5} \) | 2x | 2/2 |
| Moraxella catarrhalis (pos. control/LoD ref. strain) | ATCC 25238 | \( {3.0} \times {10}^{5} \) | 2x | 4/4 |
| Moraxella catarrhalis | ATCC 43617 | \( {3.0} \times {10}^{5} \) | 2x | 2/2 |
| Moraxella catarrhalis | ATCC 8176 | \( {3.0} \times {10}^{5} \) | 2x | 2/2 |
| Moraxella catarrhalis | ATCC 25240 | \( {3.0} \times {10}^{5} \) | 2x | 2/2 |
| Moraxella catarrhalis | ATCC 23246 | \( {3.0} \times {10}^{5} \) | 2x | 3/3 |
| Moraxella catarrhalis | ATCC 49143 | \( {3.0} \times {10}^{5} \) | 2x | 2/2 |
| Morganella morganii (pos. control/LoD ref. strain) | ATCC 25830 | \( {4.0} \times {10}^{4} \) | 2x | 2/3 |
| Morganella morganii | ATCC 8019 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| Morganella morganii | ATCC 25829 | \( {4.0} \times {10}^{4} \) | 2x | 0/1 |
| | | \( {6.0} \times {10}^{4} \) | 3x | 2/2 |
| Morganella morganii | DSM-46262 | \( {4.0} \times {10}^{4} \) | 2x | 1/2 |
| | | \( {6.0} \times {10}^{4} \) | 3x | 2/2 |
| Morganella morganii ssp. sibonii | ATCC 49948 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| Mycoplasma pneumoniae (pos. control/LoD ref. strain) | ATCC 29085 | \( {3.2} \times {10}^{3}{}^{\mathrm{b}} \) | 2x | 5/7 |
| Mycoplasma pneumoniae | ATCC 29343 | \( {3.2} \times {10}^{3}{}^{\mathrm{b}} \) | 2x | 2/2 |
| Mycoplasma pneumoniae | ATCC 15531c,d | \( {3.2} \times {10}^{3}{}^{\mathrm{b}} \) | 2x | 0/2 |
| | | \( {5.0} \times {10}^{3}{}^{\mathrm{b}} \) | 3x | 1/2 |
| | | \( {6.4} \times {10}^{3}{}^{\mathrm{b}} \) | 4x | 1/2 |
| | | \( {7.0} \times {10}^{3}{}^{\mathrm{b}} \) | 4.4x | 1/2 |
| | | \( {8.0} \times {10}^{3}{}^{\mathrm{b}} \) | 5x | 2/2 |
| Mycoplasma pneumoniae | ATCC 15293 | \( {3.2} \times {10}^{3}{}^{\mathrm{b}} \) | 2x | 2/2 |
| Mycoplasma pneumoniae | ATCC 49894 | \( {3.2} \times {10}^{3}{}^{\mathrm{b}} \) | 2x | 2/2 |
| Pneumocystis jirovecii (pos. control/LoD ref. strain) | pos. specimen 1 | \( {1.0} \times {10}^{6}{}^{\mathrm{b}} \) | 2x | 1/1 |
| Pneumocystis jirovecii | pos. specimen 2 | \( {1.0} \times {10}^{6}{}^{\mathrm{b}} \) | 2x | 2/2 |
| Pneumocystis jirovecii | pos. specimen 3 | \( {1.0} \times {10}^{6}{}^{\mathrm{b}} \) | 2x | 2/2 |
| Pneumocystis jirovecii | pos. specimen 4 | \( {1.0} \times {10}^{6}{}^{\mathrm{b}} \) | 2x | 2/2 |
| Pneumocystis jirovecii | pos. specimen 5 | \( {1.0} \times {10}^{6}{}^{\mathrm{b}} \) | 2x | 2/2 |
K191967 - Page 15 of 88
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| Reference Strain | Strain ID | Test Conc. [CFU/ml or other as noted by footnote] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- |
| Proteus vulgaris (pos. control/LoD ref. strain) | ATCC 29905 | \(1.0 \times 10^{4}\) | 2x | 7/7 |
| Proteus mirabilis (pos. control/LoD ref. strain) | ATCC 29906 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus mirabilis | ATCC 12453 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus mirabilis | ATCC 14153 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus mirabilis | ATCC 25933 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus vulgaris | ATCC 6380 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus vulgaris | ATCC 8427 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus hauseri | ATCC 700826 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Proteus penneri | ATCC 33519 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Pseudomonas aeruginosa (pos. control/LoD ref. strain) | ATCC 10145 | \(1.3 \times 10^{3}\) | 2x | 3/3 |
| Pseudomonas aeruginosa | ATCC 27853 | \(1.3 \times 10^{3}\) | 2x | 2/2 |
| Pseudomonas aeruginosa | NCTC 13437 | \(1.3 \times 10^{3}\) | 2x | 2/2 |
| Pseudomonas aeruginosa | Micromyx 2562 | \(2.0 \times 10^{3}\) | 3x | 2/2 |
| Pseudomonas aeruginosa | NRZ-00196 | \(1.3 \times 10^{3}\) | 2x | 2/2 |
| Pseudomonas aeruginosa | UCLA P20 | \(1.3 \times 10^{3}\) | 2x | 2/2 |
| Serratia marcescens (pos. control/LoD ref. strain) | ATCC 13880 | \(8.0 \times 10^{4}\) | 2x | 3/3 |
| Serratia marcescens | ATCC 14756 | \(8.0 \times 10^{4}\) | 2x | 2/2 |
| Serratia marcescens | ATCC 15365 | \(8.0 \times 10^{4}\) | 2x | 2/2 |
| Serratia marcescens | ATCC 27117 | \(8.0 \times 10^{4}\) | 2x | 2/2 |
| Serratia marcescens | ATCC 43861 | \(8.0 \times 10^{4}\) | 2x | 2/2 |
| Serratia marcescens ssp. sakuensis | DSM-17174 | \(8.0 \times 10^{4}\) | 2x | 2/2 |
| Staphylococcus aureus (pos. control/LoD ref. strain) | ATCC 12600 | \(3.0 \times 10^{5}\) | 2x | 7/7 |
| Staphylococcus aureus | ATCC BAA-2312 | \(3.0 \times 10^{5}\) | 2x | 2/2 |
| Staphylococcus aureus | NCTC 12493 | \(3.0 \times 10^{5}\) | 2x | 2/2 |
| Staphylococcus aureus | ATCC 33591 | \(3.0 \times 10^{5}\) | 2x | 2/2 |
| Staphylococcus aureus | DSM-17091 | \(3.0 \times 10^{5}\) | 2x | 2/2 |
| Staphylococcus aureus | ATCC 29213 | \(3.0 \times 10^{5}\) | 2x | 2/2 |
| Staphylococcus aureus | ATCC 43300 | \(3.0 \times 10^{5}\) | 2x | 2/2 |
| Stenotrophomonas maltophilia (pos. control/LoD ref. strain) | ATCC 13637 | \(1.0 \times 10^{4}\) | 2x | 4/4 |
| Stenotrophomonas maltophilia | ATCC 13636 | \(1.0 \times 10^{4}\) | 2x | 0/2 |
| | | \(1.5 \times 10^{4}\) | 3x | 2/2 |
| Stenotrophomonas maltophilia | ATCC 17666 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Stenotrophomonas maltophilia | ATCC 49130 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Stenotrophomonas maltophilia | DSM-50173 | \(1.0 \times 10^{4}\) | 2x | 2/2 |
| Stenotrophomonas maltophilia | DSM-21874 c [NCIMB 9528] | \(1.0 \times 10^{4}\) | 2x | 0/2 |
| | | \(1.5 \times 10^{4}\) | 3x | 0/2 |
| | | \(2.0 \times 10^{4}\) | 4x | 2/2 |
K191967 - Page 16 of 88
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| Reference Strain | Strain ID | Test Conc. [CFU/ml or other as noted by footnote] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- |
| *Streptococcus pneumoniae* serotype 19F (pos. control/LoD ref. strain) | ATCC 49619 | 4.0 x 10^{4} | 2x | 4/7^{f} |
| *Streptococcus pneumoniae* serotype 3 | ATCC 6303 | 4.0 x 10^{4} | 2x | 2/2 |
| *Streptococcus pneumoniae* serotype 5 | ATCC 6305 | 4.0 x 10^{4} | 2x | 2/2 |
| *Streptococcus pneumoniae* | ATCC 49150^{g} | 4.0 x 10^{4} | 2x | 0/2 |
| | | 8.0 x 10^{4} | 4x | 2/2 |
| *Streptococcus pneumoniae* | ATCC 33400^{g} | 4.0 x 10^{4} | 2x | 1/2 |
| | | 6.0 x 10^{4} | 3x | 1/2 |
| | | 8.0 x 10^{4} | 4x | 2/2 |
| *Streptococcus pneumoniae* serotype 2 | ATCC 27336 | 4.0 x 10^{4} | 2x | 2/2 |
| *Streptococcus pneumoniae* serotype 1 | ATCC 6301 | 4.0 x 10^{4} | 2x | 2/2 |
| *Streptococcus pneumoniae* serotype 9V | DSM-11865 | 4.0 x 10^{4} | 2x | 2/2 |
| *Streptococcus pneumoniae* serotype 23F | DSM-11866 | 4.0 x 10^{4} | 2x | 2/2 |
| | | 6.0 x 10^{4} | 3x | 1/2 |
| | | 8.0 x 10^{4} | 4x | 2/2 |
| *Streptococcus pneumoniae* serotype 6B | DSM-11867 | 4.0 x 10^{4} | 2x | 2/2 |
$^{a}$ In IFU/mL for *C. pneumoniae*.
$^{b}$ In copies/mL for *M. pneumoniae* and *P. jirovecii*.
$^{c}$ Quantified DNA extract (reference material) from a commercial provider.
$^{d}$ For *M. pneumoniae* ATCC 15531, a positivity rate of 2/2 was obtained at a 5x LoD concentration. Sequencing did not reveal any mismatches to primer and probe binding sites and the observed slightly reduced sensitivity is likely due to the different source material (DNA extract instead of a cell suspension).
$^{e}$ *S. maltophilia* strain DSM-21874 was positive at a 4x LoD concentration. Sequencing did not reveal any mismatches to primer or probe sequences.
$^{f}$ Initial testing *S. pneumoniae* LoD reference strain ATCC 46916 generated inconsistent detection at 2x LoD. Repeat testing using a freshly prepared counted culture stock generated 8/8 correct results at a 2x LoD concentration.
$^{g}$ *S. pneumoniae* strain ATCC 49150 was positive at 4x LoD. Sequencing did not reveal any mismatches to primer or probe sequences.
$^{h}$ *S. pneumoniae* strain ATCC 33400 was consistently positive at 4x LoD. Sequencing did not reveal any mismatches to primer or probe sequences.
To supplement inclusivity testing, *in silico* GenBank BLAST analyses of LRT BAL primer and probe sequences were performed for each LRT BAL microorganism (search performed July 2019) for all applicable strain entries. BLAST analyses identified strain entries for which detection by LRT BAL is predicted at LoD (match of relevant primer and probe sequences), strains predicted to be detected with reduced sensitivity (typically, single relevant mismatches of primer or probe sequences; strains for which detection is likely at higher than LoD concentrations only), or strains for which detection is not predicted (multiple relevant mismatches in primer and probe sequences).
Table 5 lists microorganisms for which inclusivity was demonstrated by wet testing and are supported by *in silico* analysis (predicted at LoD or predicted with reduced sensitivity). Also presented are *in silico* predictions for strains/species for which *in silico* analysis that were not evaluated by wet-testing. These *in silico* results are provided to the user as supplementary data only. Results are not intended to be a surrogate for wet testing and do not assure that specific strains will be detected.
K191967 - Page 17 of 88
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NOTE: The performance of the Unyvero LRT BAL Application has not been established for those microorganism species that were evaluated by in silico analysis only.
Table 5: Inclusivity, Wet Testing and/or Predicted Detection by In silico Analysis
| Analyte | Wet Testing | in silico: at LoD* | in silico: Reduced Sensitivity* | Comment |
| --- | --- | --- | --- | --- |
| Acinetobacter spp. | | | | |
| A. baumannii | x | x | - | |
| A. calcoaceticus | x | x | - | |
| A. lwoffii | x | x | - | |
| A. haemolyticus | x | x | - | |
| A. nosocomialis | - | x | - | |
| A. pittii | - | x | - | |
| A. junii | - | x | - | |
| A. parvus | - | x | - | |
| A. lactucae | - | x | - | |
| A. oleivorans | - | x | - | |
| A. schindleri | - | x | - | |
| A. guillouiae | - | - | x1 entry | |
| A. radioresistens | - | - | x3 entries | |
| A. soli | - | - | x1 entry | |
| A. ursingii | - | - | x1 entry | reference strain DSM-16037 tested negative at 1.5 x 107CFU/mL |
| Chlamydia pneumoniae | x | x | - | |
| Citrobacter freundii | x | x26 entries | x4 entries | |
| Enterobacter cloacae complex | | | | |
| E. cloacae | x | x | - | including ssp. dissolvens |
| E. hormaechei | x | x | - | including ssp. oharae, steigerwaltii, hoffmannii |
| E. hormaechei ssp. xiangfangensis | - | x7 entries | x1 entry | |
| E. kobei | - | x | - | |
| E. ludwigii | - | x | - | |
| E. sichuanensis | - | x | - | |
| E. chuandaensis | - | x | - | |
| E. roggenkampii | - | x | - | |
| E. asburiae | x | x11 entries | x1 entry | |
| Escherichia coli | x | x | - | |
| Haemophilus influenzae | x | x65 entries | x1 entry | |
| Klebsiella oxytoca | x | x16 entries | x6 entries | |
| Klebsiella pneumoniae | | | | |
| variant 1 (K. pneumoniae) | x | x | - | |
| variant 2 (K. quasi-pneumoniae) | x | x11 entries | x10 entries | |
| Klebsiella variicola | x | x | - | |
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| Analyte | Wet Testing | in silico: at LoD* | in silico: Reduced Sensitivity* | Comment |
| --- | --- | --- | --- | --- |
| *Legionella pneumophila* | x | x | - | some entries predict detection at LoD, other entries predict a possible slight performance reduction; strains for both variants were included in inclusivity wet testing and were detected at concentrations near LoD. |
| *Moraxella catarrhalis* | x | x | - | |
| *Morganella morganii* | x | x | - | |
| *Mycoplasma pneumoniae* | x | x | - | |
| *Pneumocystis jirovecii* | x | x | - | |
| **Proteus spp.** | | | | |
| *P. mirabilis* | x | x | - | |
| *P. vulgaris* | x | x | - | |
| *P. hauseri* | x | x | - | |
| *P. penneri* | x | x | - | |
| *P. cibarius* | - | x | - | |
| *Pseudomonas aeruginosa* | x | x | - | |
| *Serratia marcescens* | x | x | - | |
| *Staphylococcus aureus* | x | x > 450 entries | x 1 entry | |
| *Stenotrophomonas maltophilia* | x | x | - | |
| *Streptococcus pneumoniae* | x | x > 240 entries | x 1 entry | including serotype 7F |
* When in silico analyses predicts detection of strains at higher than LoD concentrations for one or more applicable entries, numbers of GenBank entries are listed.
Similar to microorganism testing, inclusivity wet testing was performed with reference strains carrying antibiotic resistance markers targeted by the LRT BAL Application. Contrived samples were prepared in pooled negative clinical BAL matrix with organism concentrations near the LoD for each resistance marker target.
Inclusivity of the LRT BAL Application for detection of targeted resistance markers was demonstrated for all strains evaluated in the study. Results are presented in Table 6 below with resistance marker variants/subgroups designated, if known. If available, results from phenotypic antibiotic susceptibility testing (AST) results are also presented (e.g., Carbapenem$^{R}$ = resistant to carbapenems, Carbapenem$^{S}$ = susceptible to carbapenems).
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Table 6: Inclusivity Study, Antibiotic Resistance Markers
| Analyte | Subgroup | Reference Strain | Strain ID | Test Conc. [CFU/ml] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- | --- | --- |
| ctx-M | NA* | Klebsiella pneumoniae (pos. control/LoD ref. strain) | NCTC 13443 | 2.0 x 104 | 2x | 3/3 |
| | ctx-M3 | Klebsiella pneumoniae | NRZ-00751 | \( 2.0 \times 10^{4} \) | 2x | 2/2 |
| | ctx-M15 | Klebsiella pneumoniae | NRZ-00249 | \( 2.0 \times 10^{4} \) | 2x | 2/2 |
| | NA | Enterobacter cloacae | JMI 46239 | \( 2.0 \times 10^{4} \) | 2x | 1/2 |
| | | | | \( 3.0 \times 10^{4} \) | 3x | 2/2 |
| | NA | Escherichia coli | JMI 50067 | \( 2.0 \times 10^{4} \) | 2x | 2/2 |
| | NA | Klebsiella pneumoniae | NRZ-00002 | \( 2.0 \times 10^{4} \) | 2x | 1/2 |
| | | | | \( 3.0 \times 10^{4} \) | 3x | 2/2 |
| | NA | Enterobacter cloacae | ATCC BAA-2468 | \( 4.0 \times 10^{4} \) | 4x | 2/2 |
| | NA | Klebsiella pneumoniae | JMI 49831 | \( 4.0 \times 10^{4} \) | 4x | 2/2 |
| | NA | Klebsiella pneumoniae | JMI 49767 | \( 2.0 \times 10^{4} \) | 2x | 2/2 |
| kpc | kpc-3 | Klebsiella pneumoniae (pos. control/LoD ref. strain) | NCTC 13438 | \( 8.0 \times 10^{4} \) | 2x | 5/5 |
| | kpc-2 | Escherichia coli | NRZ-00281 | \( 8.0 \times 10^{4} \) | 2x | 2/2 |
| | kpc-2 | Klebsiella pneumoniae | NRZ-00103 | \( 8.0 \times 10^{4} \) | 2x | 2/2 |
| | kpc-3 | Escherichia coli | NRZ-00222 | \( 8.0 \times 10^{4} \) | 2x | 1/2 |
| | | | | \( 1.2 \times 10^{5} \) | 3x | 2/2 |
| | kpc-3 | Klebsiella pneumoniae (CarbapenemR) | Micromyx 4653 | \( 8.0 \times 10^{4} \) | 2x | 4/4 |
| | kpc-3 | Klebsiella pneumoniae (CarbapenemR) | Micromyx 4676 | \( 8.0 \times 10^{4} \) | 2x | 4/4 |
| mecA | NA | Staphylococcus aureus (MethicillinR, CefoxitinR) (pos. control/LoD ref. strain) | NCTC 12493 | \( 8.0 \times 10^{5} \) | 2x | 3/3 |
| | SCCmecI | Staphylococcus aureus | RKI 07-03165 | \( 8.0 \times 10^{5} \) | 2x | 2/2 |
| | SCCmecII | Staphylococcus aureus | RKI 01-00694 | \( 8.0 \times 10^{5} \) | 2x | 2/2 |
| | SCCmecIII | Staphylococcus aureus (MethicillinR) | ATCC 33591 | \( 8.0 \times 10^{5} \) | 2x | 2/2 |
| | SCCmecIV | Staphylococcus aureus | RKI 09-00187 | \( 8.0 \times 10^{5} \) | 2x | 2/2 |
| | SCCmecV | Staphylococcus aureus | RKI 08-02492 | \( 8.0 \times 10^{5} \) | 2x | 2/2 |
| | NA | Staphylococcus aureus (MethicillinR) | DSM-17091 | \( 8.0 \times 10^{5} \) | 2x | 2/2 |
| | SCCmecII | Staphylococcus aureus (MethicillinR) | ATCC 43300 | \( 3.0 \times 10^{5} \) | 0.8x | 2/2 |
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| Analyte | Subgroup | Reference Strain | Strain ID | Test Conc. [CFU/ml] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- | --- | --- |
| ndm | ndm-1 | Klebsiella pneumoniae (pos. control/LoD ref. strain) | NCTC 13443 | \( {4.0} \times {10}^{4} \) | 2x | 5/5 |
| | ndm-1 | Acinetobacter baumannii | JMI 49755 | \( {4.0} \times {10}^{4} \) | 2x | 4/4 |
| | ndm-1 | Enterobacter cloacae (Imipenem \( {}^{\mathrm{R}} \) , Ertapenem \( {}^{\mathrm{R}} \) ) | ATCC BAA-2468 | \( {4.0} \times {10}^{4} \) | 2x | 1/2 |
| | | | | \( {6.0} \times {10}^{4} \) | 3x | 2/2 |
| | ndm-1 | Enterobacter cloacae | JMI 46239 | \( {4.0} \times {10}^{4} \) | 2x | 4/4 |
| | ndm-1 | Escherichia coli | JMI 50067 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| | ndm-1 | Klebsiella pneumoniae | JMI 49767 | \( {2.0} \times {10}^{4} \) | 1x | 2/2 |
| | ndm-1 | Klebsiella pneumoniae | JMI 49831 | \( {4.0} \times {10}^{4} \) | 2x | 2/2 |
| oxa-23 | oxa-23 | Acinetobacter baumannii (pos. control/LoD ref. strain) | NCTC 13301 | \( {2.0} \times {10}^{7} \) | 2x | 3/3 |
| | NA | Acinetobacter baumannii (Carbapenem \( {}^{\mathrm{R}} \) ) | Micromyx 4410 | \( {2.0} \times {10}^{7} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Carbapenem \( {}^{\mathrm{R}} \) ) | Micromyx 6148 | \( {2.0} \times {10}^{7} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Carbapenem \( {}^{\mathrm{R}} \) ) | Micromyx 6149 | \( {2.0} \times {10}^{7} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Carbapenem \( {}^{\mathrm{R}} \) ) | Micromyx 6153 | \( {2.0} \times {10}^{7} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Imipenem \( {}^{\mathrm{R}} \) , Meropenem \( {}^{\mathrm{R}} \) ) | UCLA A5 | \( {2.0} \times {10}^{7} \) | 2x | 2/2 |
| oxa-24 | oxa-25 | Acinetobacter baumannii (pos. control/LoD ref. strain) | NCTC 13302 | \( {1.0} \times {10}^{4} \) | 2x | 3/3 |
| | oxa-72 | Acinetobacter baumannii | NRZ-00449 | \( {1.0} \times {10}^{4} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Imipenem \( {}^{\mathrm{R}} \) , Meropenem \( {}^{\mathrm{R}} \) ) | UCLA A4 | \( {1.0} \times {10}^{4} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Imipenem \( {}^{\mathrm{R}} \) , Meropenem \( {}^{\mathrm{R}} \) ) | clinical strain 1 | \( {1.0} \times {10}^{4} \) | 2x | 2/2 |
| | NA | Acinetobacter baumannii (Imipenem \( {}^{\mathrm{R}} \) , Meropenem \( {}^{\mathrm{R}} \) ) | clinical strain 2 | \( {1.0} \times {10}^{4} \) | 2x | 2/2 |
| oxa-48 | oxa-48 | Klebsiella pneumoniae (pos. control/LoD ref. strain) | NCTC 13442 | \( {6.0} \times {10}^{5} \) | 2x | 3/3 |
| | oxa-48 | Escherichia coli (Ertapenem \( {}^{\mathrm{R}} \) ) | ATCC BAA-2523 | \( {6.0} \times {10}^{5} \) | 2x | 2/2 |
| | oxa-48 | Escherichia coli | NRZ-00176 | \( {6.0} \times {10}^{5} \) | 2x | 1/2 |
| | | | | \( {9.0} \times {10}^{5} \) | 3x | 2/2 |
| | oxa-162 | Escherichia coli | NRZ-00361 | \( {6.0} \times {10}^{5} \) | 2x | 2/2 |
| | oxa-162 | Klebsiella pneumoniae | NRZ-00472 | \( {6.0} \times {10}^{5} \) | 2x | 2/2 |
| | oxa-232 | Klebsiella oxytoca | NRZ-22060 | \( {6.0} \times {10}^{5} \) | 2x | 2/2 |
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| Analyte | Subgroup | Reference Strain | Strain ID | Test Conc. [CFU/ml] | x-fold LoD | # Pos./ # Exp. |
| --- | --- | --- | --- | --- | --- | --- |
| oxa-58 | oxa-58 | *Acinetobacter baumannii* (pos. control/LoD ref. strain) | NCTC 13305 | 1.0 x 10^{5} | 2x | 2/3 |
| | oxa-58 | *Acinetobacter baumannii* | NRZ-00518 | 1.0 x 10^{5} | 2x | 3/3 |
| tem | NA | *Klebsiella pneumoniae* (pos. control/LoD ref. strain) | NCTC 13443 | 4.0 x 10^{4} | 2x | 2/2 |
| | tem-1 | *Escherichia coli* | ATCC 35218 | 4.0 x 10^{4} | 2x | 2/2 |
| | tem-3 | *Escherichia coli* (ESBL) | NCTC 13351 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Citrobacter freundii* | ATCC 43864 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Enterobacter cloacae* | JMI 46239 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Escherichia coli* | JMI 50067 | 2.0 x 10^{4} | 1x | 2/2 |
| | NA | *Klebsiella pneumoniae* | NRZ-00751 | 2.0 x 10^{4} | 1x | 2/2 |
| | NA | *Escherichia coli* | ATCC BAA-2523 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Acinetobacter baumannii* | JMI 49755 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Haemophilus influenzae* (Ampicillin^{R}, Cefinase^{R}) | clinical strain 1 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Haemophilus influenzae* (Cefinase^{R}) | clinical strain 2 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Klebsiella pneumoniae* | Micromyx 4676 | 8.0 x 10^{4} | 4x | 2/2 |
| vim | vim-10 | *Pseudomonas aeruginosa* (pos. control/LoD ref. strain) | NCTC 13437 | 4.0 x 10^{4} | 2x | 3/3 |
| | vim-1 | *Citrobacter freundii* | NRZ-00452 | 4.0 x 10^{4} | 2x | 2/2 |
| | vim-1 | *Pseudomonas aeruginosa* (Ceftazidime^{R}, Imipenem^{R}) | DSM-24600 | 4.0 x 10^{4} | 2x | 2/2 |
| | vim-1 | *Enterobacter cloacae* | NRZ-00239 | 4.0 x 10^{4} | 2x | 2/2 |
| | vim-1 | *Klebsiella pneumoniae* | NCTC 13439 | 4.0 x 10^{4} | 2x | 2/2 |
| | vim-1 | *Klebsiella pneumoniae* | NCTC 13440 | 4.0 x 10^{4} | 2x | 2/2 |
| | NA | *Pseudomonas aeruginosa* | UCLA P20 | 1.3 x 10^{3} | 0.1x | 2/2 |
| | NA | *Pseudomonas aeruginosa* (Carbapenem^{R}) | Micromyx 2562 | 4.0 x 10^{4} | 2x | 2/2 |
*N/A = variant status unknown
To supplement inclusivity testing specifically for antibiotic resistance marker variants, reference sequences for all available variants belonging to individual antibiotic resistance markers or marker subgroups were evaluated using in silico analysis.
Table 7 below includes a summary of subgroups or variants for applicable LRT BAL antibiotic resistance markers for which detection is predicted at LoD (match of relevant primer and probe sequences), detection is predicted with reduced sensitivity (typically, single relevant mismatches of primer or probe sequences; detection likely at higher than LoD concentrations only), or detection is not predicted (multiple relevant mismatches in primer and probe sequences). In silico data are provide in the test labeling as supplementary
K191967 - Page 22 of 88
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data; however results are not intended to be a surrogate for wet testing and do not assure that specific variant will be detected.
NOTE: The performance of the Unyvero LRT BAL Application has not been established for antibiotic resistance marker variants other than those listed in Table 6 above.
Table 7: In silico Analysis: Predicted Detection of Resistance Marker Subgroups and Variants.
| Antibiotic Resistance Marker: Subgroup | Detection Predicted at LoD: Variant No. | Detection Predicted with Reduced Sensitivity: Variant No. | Detection Not Predicted: Variant No. |
| --- | --- | --- | --- |
| ctx-M: ctx-M1 subgroup | 1, 3, 10, 11, 12, 15, 22, 23, 28 - 30, 32 - 34, 36, 37, 42, 52 - 55, 57, 58, 60 - 62, 66, 68, 69, 71, 72, 79, 80, 82, 83, 88, 96, 101, 103, 107, 108, 109, 114, 116, 117, 132, 133, 136, 138, 139, 142, 144, 150, 155 - 158, 162 - 164, 166, 167, 169, 170, 172, 173, 175 - 177, 179 - 184, 186, 188 - 190, 193, 194, 197, 202 - 204, 206 - 212, 216, 218, 222, 224 | 64 | - |
| ctx-M: ctxM2 ctx-M8 ctx-M9 ctx-M25 ctx-M45 subgroups | - | - | 2, 4 - 9, 13, 14, 16 - 21, 24 - 27, 31, 35, 38 - 41, 43 - 51, 56, 59, 63, 65, 67, 73 - 78, 81, 84 - 87, 89 - 95, 97 100, 102, 104 - 106, 110 - 113, 115, 121 - 126, 129 - 131, 134, 137, 141, 147, 148, 152, 159-161, 165, 168, 171, 174, 185, 191, 192, 195, 196, 198, 199, 201, 214, 215, 217, 219, 221, 223 |
| kpc | 1 - 39 | - | - |
| ndm | 1 - 24, 27 | - | - |
| oxa: oxa-23 | 23, 27, 49, 73, 134, 146, 165 - 171, 225, 239, 366, 398, 422, 423, 435, 440, 469, 481 - 483, 565, 657, 806 - 808, 810, 813 - 815, 816, 818 | 103, 133, 809, 811, 812, 816 | - |
| oxa: oxa-24 | 24 - 26, 33, 40, 72, 139, 160, 207, 437, 653 | - | - |
| oxa: oxa-48 | 48, 48b, 162, 163, 181, 199, 232, 244, 245, 247, 252, 370, 405, 416, 438, 439, 484, 505, 514, 515, 517, 519, 538, 546, 547, 566, 567, 731, 788, 793 | 204 | 54, 436, 535 |
| oxa: oxa-58 | 58, 96, 97, 164, 397, 467, 512 | - | 420 |
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| Antibiotic Resistance Marker: Subgroup | Detection Predicted at LoD: Variant No. | Detection Predicted with Reduced Sensitivity: Variant No. | Detection Not Predicted: Variant No. |
| --- | --- | --- | --- |
| tem^{1} | 1 - 4, 6, 8 - 12, 15 - 17, 19 - 22, 24, 26, 28 - 30, 32 - 36, 40, 43, 45, 47 - 49, 52 - 55, 57, 60, 63, 67, 68, 70 - 72, 76 - 88, 90 - 99, 101, 102, 104 - 116, 120 - 139, 141 - 150, 152 - 160, 162 - 164, 166 - 169, 171, 176, 177, 181 - 199, 201, 204 - 217, 219, 220, 224 - 228, 229 - 235, 237 | 151 | 178 |
| vim | 1 - 6, 8 - 12, 14 - 20, 23 - 46, 48 - 54, 56, 58, 60, 62 | 57, 59 | 7, 13, 47, 61 |
$^{1}$ NOTE: H. influenzae commonly hosts tem-1. tem will only be reported if H. influenzae is concurrently detected.
#### 4. Analytical Specificity/Cross-reactivity:
An analytical specificity study was conducted to evaluate LRT BAL Application targets for potential cross-reactivity with common respiratory flora microorganisms and species closely related to LRT BAL analytes. Testing was performed in duplicate using samples prepared at a worst case concentration (typically: 1.5 x 10$^{7}$ CFU/mL for bacteria and 1.0 x 10$^{7}$ copies/mL for viruses) by spiking microorganisms into 0.25x ARM (artificial respiratory matrix). Study results are presented in Table 8.
Cross-reactivity was observed with the following microorganisms with the Haemophilus influenzae assay of the LRT BAL Application:
- Haemophilus haemolyticus (ATCC 33390) – at LoD
- Aggregatibacter aphrophilus (ATCC 19415) - at 10x LoD
- Haemophilus parainfluenzae (ATCC 33392) - at > 100x LoD (> 10$^{7}$ CFU/mL).
Table 8: Analytical Specificity
| Strain | Test Concentration | Cross-Reactivity |
| --- | --- | --- |
| Respiratory Flora Microorganisms | [in CFU/mL or other, as specified in footnotes] | |
| Actinomyces odontolyticus | 1.5 x 10^{7} | no |
| Aspergillus fumigatus | 1.5 x 10^{7} | no |
| Candida albicans | 1.5 x 10^{7} | no |
| Candida dubliniensis | 1.5 x 10^{7} | no |
| Candida glabrata | 1.5 x 10^{7} | no |
| Candida krusei | 1.5 x 10^{7} | no |
| Candida parapsilosis | 1.5 x 10^{7} | no |
| Candida tropicalis | 1.5 x 10^{7} | no |
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| Strain | Test Concentration | Cross-Reactivity |
| --- | --- | --- |
| Cardiobacterium hominis | 1.5 x 10^{7} | no |
| Eikenella corrodens | 1.5 x 10^{7} | no |
| Enterococcus faecalis | 1.5 x 10^{7} | no |
| Enterococcus faecium | 1.5 x 10^{7} | no |
| Fusobacterium nucleatum | 5.0 x 10^{6 d} | no |
| Granulicatella adiacens | 1.5 x 10^{7} | no |
| Kingella kingae | 1.5 x 10^{7} | no |
| Lactobacillus acidophilus | 1.5 x 10^{7} | no |
| Micrococcus luteus | 1.5 x 10^{7} | no |
| Mycobacterium bovis | 1.5 x 10^{7 d} | no |
| Mycoplasma orale | 1.5 x 10^{7} | no |
| Neisseria lactamica | 1.5 x 10^{7 d} | no |
| Neisseria sicca | 1.5 x 10^{7} | no |
| Pantoae agglomerans | 1.5 x 10^{7} | no |
| Peptostreptococcus stomatis | 5.0 x 10^{6 d} | no |
| Porphyromonas gingivalis | 1.5 x 10^{7 d} | no |
| Prevotella buccalis | 1.5 x 10^{7} | no |
| Raoultella planticola | 1.5 x 10^{7} | no |
| **Close Neighbor Microorganisms** | [in CFU/mL] | |
| Acinetobacter ursingii | 1.5 x 10^{7} | no |
| Aggregatibacter actinomycetemcomitans | 1.5 x 10^{7 d} | no |
| Aggregatibacter aphrophilus | 1.5 x 10^{7} | yes (H. influenzae)^{a} |
| Citrobacter koseri | 1.5 x 10^{7} | no |
| Haemophilus haemolyticus | 1.5 x 10^{7} | yes (H. influenzae)^{b} |
| Haemophilus parahaemolyticus | 1.5 x 10^{7} | no |
| Haemophilus parainfluenzae | 1.5 x 10^{7} | yes (H. influenzae)^{c} |
| Legionella longbeachae | 1.5 x 10^{7} | no |
| Legionella/Tatlockia micdadei | 1.5 x 10^{7} | no |
| Staphylococcus capitis | 1.5 x 10^{7} | no |
| Staphylococcus epidermidis | 1.5 x 10^{7} | no |
| Staphylococcus haemolyticus | 1.5 x 10^{7} | no |
| Staphylococcus lugdunensis | 1.5 x 10^{7} | no |
| Staphylococcus saprophyticus | 1.5 x 10^{7} | no |
| Streptococcus agalactiae | 1.5 x 10^{7} | no |
| Streptococcus anginosus | 1.5 x 10^{7} | no |
| Streptococcus dysgalactiae | 1.5 x 10^{7} | no |
| Streptococcus gordonii | 1.5 x 10^{7} | no |
| Streptococcus intermedius | 1.5 x 10^{7} | no |
| Streptococcus mitis | 1.5 x 10^{7} | no |
| Streptococcus mutans | 1.5 x 10^{7} | no |
| Streptococcus oralis | 1.5 x 10^{7} | no |
| Streptococcus parasanguinis | 1.5 x 10^{7} | no |
K191967 - Page 25 of 88
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| Strain | Test Concentration | Cross-Reactivity |
| --- | --- | --- |
| *Streptococcus pseudopneumoniae* | 1.5 x 10^{7} | no |
| *Streptococcus pyogenes* | 1.5 x 10^{7} | no |
| *Streptococcus salivarius* | 1.5 x 10^{7} | no |
| *Streptococcus sanguinis* | 1.5 x 10^{7} | no |
| *Streptococcus vestibularis* | 1.5 x 10^{7} | no |
| **Respiratory Viruses^{e}** | [in copies/mL] | |
| Adenovirus 41 | 1.0 x 10^{5} | no |
| Enterovirus 68 | 1.0 x 10^{5} | no |
| Enterovirus 71 | 1.0 x 10^{5} | no |
| Parainfluenzae Virus 1 | 1.0 x 10^{5} | no |
| Parainfluenzae Virus 2 | 1.0 x 10^{5} | no |
| Parainfluenzae Virus 3 | 1.0 x 10^{5} | no |
| Parainfluenzae Virus 4 | 1.0 x 10^{5} | no |
| Rhinovirus | 1.0 x 10^{5} | no |
| RSV A | 1.0 x 10^{5} | no |
| RSV B | 1.0 x 10^{5} | no |
$^{a}$ For *A. aphrophilus*, cross-reactivity to *H. influenzae* was observed down to a concentration of 2.0 x 10$^{5}$ CFU/mL (1/2 tests positive). For a concentration of 1.0 x 10$^{5}$ CFU/mL (equivalent to 5x LoD of *H. influenzae*), cross-reactivity was no longer observed. According to BLAST analysis, cross-reactivity of *A. aphrophilus* to *H. influenzae* is only predicted for some strains (including the tested reference strain ATCC 19415), but not for other strains.
$^{b}$ For *H. haemolyticus*, cross-reactivity to *H. influenzae* was observed down to a concentration of 4.0 x 10$^{4}$ CFU/mL (2/2 tests positive, equivalent to 2x LoD of *H. influenzae*). Sequence comparison by BLAST for *H. haemolyticus* to *H. influenzae* shows a 3' mismatch to one of the assay primers, while the second primer and all internal array probes show a full match. Therefore, the observed cross-reactivity is supported by BLAST analysis.
$^{c}$ For *H. parainfluenzae*, cross-reactivity to *H. influenzae* was observed only at the tested worst-case concentration of 1.5 x 10$^{7}$ CFU/mL (2/4 tests positive, equivalent to 750x LoD of *H. influenzae*). Additional tests at 2.0 x 10$^{6}$ CFU/mL (equivalent to 100x LoD of *H. influenzae*), cross-reactivity was no longer observed.
$^{d}$ Tested as DNA extract (in copies/mL).
$^{e}$ Tested as DNA or RNA extract (in copies/mL).
Additionally, *in silico* analysis (GenBank BLAST) was performed to assess for potential cross-reactivity of non-target microorganisms that may be present in BAL specimens. Table 9 includes predicted cross-reactivity of applicable LRT BAL microorganisms based on *in silico* analysis, exclusivity wet testing and false positive results observed during the prospective and archived studies.
Table 9: *In silico* Prediction of Cross-reactivity, Wet Testing, Cross-reactivity Observed in Clinical Study
| Close Neighbor Strain^{a} | Cross-Reactivity Prediction (*in silico* analysis) | Wet Testing Result | Cross-Reactions Observed in Clinical Study (N = 1,408 prosp. or arch. specimens) |
| --- | --- | --- | --- |
| *Citrobacter freundii* | | | |
| *Citrobacter braakii* | Detection predicted at higher than LoD concentrations (certain strains) / | - | - |
K191967 - Page 26 of 88
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| Close Neighbor Straina | Cross-Reactivity Prediction (in silico analysis) | Wet Testing Result | Cross-Reactions Observed in Clinical Study (N = 1,408 prosp. or arch. specimens) |
| --- | --- | --- | --- |
| | Detection not predicted (other strains)c | | |
| Citrobacter pasteurii | Detection predicted at higher than LoD concentrations | - | - |
| Citrobacter werkmannii | Detection predicted at higher than LoD concentrations (certain strains) / Detection not predicted (other strains)c | - | - |
| Citrobacter youngae | Detection predicted at LoD (certain strains) / Detection predicted at higher than LoD concentrations (other strains)c | - | 1 |
| Kluyvera georgiana | Detection predicted at higher than LoD concentrations | - | - |
| Citrobacter koseri | Detection not predicted | negative ATCC 27156 | - |
| Escherichia coli | | | |
| Shigella dysenteriaeb | Detection predicted at LoD | - | - |
| Shigella boydii b | Detection predicted at LoD | - | - |
| Shigella flexneri b | Detection predicted at LoD | - | - |
| Shigella sonnei b | Detection predicted at LoD | - | - |
| Escherichia albertii | Detection predicted at LoD | - | - |
| Escherichia fergusonii | Detection predicted at LoD | - | - |
| Haemophilus influenzae | | | |
| Haemophilus haemolyticus | Detection predicted at higher than LoD concentrations | positive ATCC 33390 | 5 |
| Haemophilus parahaemolyticus | Detection not predicted | negative ATCC 10014 | - |
| Haemophilus parainfluenzae | Detection not predicted | positive at worst-case concentration only ATCC 33392 | 1 |
| Haemophilus aegyptius b | Detection predicted at LoD | - | - |
| Aggregatibacter actino-mycetemcomitans | Detection not predicted | negative ATCC 33384 | - |
| Aggregatibacter aphrophilus | Detection predicted at higher than LoD concentrations (certain strains) / Detection not predicted (other strains)c | positive ATCC 19415 | 3 |
| Aggregatibacter segnis | Detection predicted at higher than LoD concentrations | - | - |
| Klebsiella oxytoca | | | |
| Klebsiella michiganensis | Detection predicted at LoD (certain strains) / | - | - |
K191967 - Page 27 of 88
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| Close Neighbor Strain^{a} | Cross-Reactivity Prediction (in silico analysis) | Wet Testing Result | Cross-Reactions Observed in Clinical Study (N = 1,408 prosp. or arch. specimens) |
| --- | --- | --- | --- |
| | Detection predicted at higher than LoD concentrations (other strains)^{c} | | |
| **Klebsiella pneumoniae** | | | |
| *Klebsiella quasivariicola* | Detection predicted at higher than LoD concentrations | - | - |
| **Staphylococcus aureus** | | | |
| *Staphylococcus argenteus* | Detection predicted at LoD | - | - |
| CNS: *S. epidermidis* *S. capitis* *S. lugdunensis* *S. haemolyticus* *S. saprophyticus* | Detection not predicted | negative ATCC 51625 ATCC 27840 ATCC 43809 ATCC 29970 ATCC 15305 | - |
| **Streptococcus pneumoniae** | | | |
| Other *Streptococcus* spp.: *S. agalactiae* *S. anginosus* *S. dysgalactiae* *S. gordonii* *S. intermedius* *S. mitis* *S. mutans* *S. oralis* *S. parasanguinis* *S. pseudopneumoniae* *S. pyogenes* *S. salivarius* *S. sanguinis* *S. vestibularis* | Detection not predicted | negative ATCC 13813 ATCC 33397 ATCC 43078 ATCC 10558 ATCC 27335 ATCC 49456 ATCC 25175 ATCC 35037 ATCC 15912 ATCC BAA-960 ATCC 12344 ATCC 7073 ATCC 10556 ATCC 49124 | - |
$^{a}$ For several analyte assays, soil, environmental, plant or animal derived close neighbor strains are also predicted at LoD or at higher than LoD concentrations (Citrobacter freundii:, C. portucalensis, Enterobacter cloacae complex: E. soli, E. mori,
E. nickellidurans; Escherichia coli: E. marmotae; Staphylococcus aureus: S. schweitzeri, S. simiae; Stenotrophomonas maltophilia:
S. nitritireducens, S. daejeonensis, S. acidaminiphila, S. koreensis, S. rhizophila, Xanthomonas spp., Pseudoxanthomonas spp.).
$^{b}$ Clinical relevance unlikely for respiratory infections
$^{c}$ Predictions for available strains distribute into different categories.
### 5. Interference Testing:
Interference testing was performed previously with the Unyvero LRT Application which relies on the same DNA extraction procedures and chemistry and targets the same microorganism and resistance marker sequences as the LRT BAL Application. Refer to DEN170047 for study results.
### 6. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
Sample Stability:
K191967 - Page 28 of 88
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A sample stability study was conducted to evaluated assay performance differences between fresh samples and samples stored at 2-8°C for at least 24 hours. Samples for the study were prepared at low and moderate organism concentrations inoculated into pooled natural negative BAL matrix. Representative targeted microorganisms were evaluated in the study. Four targeted resistance markers carried by selected strains were also evaluated.
Pair-wise tests with the LRT BAL Application were performed for samples tested within 2 hours after test sample preparation (Time 0) and samples tested after a total storage of at least 24 hours including at least 2 hours at a worst-case ambient temperature of 30°C and a minimum of 22 hour storage at 2-8°C.
Tables 10 to 12 summarize the sample stability test results for each concentration level (moderate, low, negative) for both test conditions (tested freshly, tested after storage), together with the agreement rates based on comparison to expected results.
Results of the study support the claimed specimen storage and transport conditions of 2-8°C for up to 24 hours prior to testing with the LRT BAL Application.
Table 10: Sample Stability, Moderate Positive (~5x LoD), 24 hour storage at 2-8°C
| Moderate | x-fold LoD | Test Condition | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | # Pos. /# Exp. | % | 95% CI |
| *Acinetobacter baumannii* ATCC 19606 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Chlamydia pneumoniae* ATCC VR-2282 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Citrobacter freundii* ATCC 8090 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Haemophilus influenzae* ATCC 33391 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Klebsiella pneumoniae* NCTC 13443 | 2.5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *ctx-M* NCTC 13443 | 10 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *ndm* NCTC 13443 | 5 | fresh 24 hrs | 9/10 11/11 | 90.0 100.0 | 59.6 - 98.2 74.1 - 100.0 |
| *tem* NCTC 13443 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Morganella morganii* ATCC 25830 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Proteus vulgaris* ATCC 29905 | 5 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| *Staphylococcus aureus* NCTC 12493 | 8.3 | fresh 24 hrs | 9/9 * 11/11 | 100.0 100.0 | 70.1 - 100.0 74.1 - 100.0 |
| *mecA* NCTC 12493 | 3 | fresh 24 hrs | 10/10 11/11 | 100.0 100.0 | 72.3 - 100.0 74.1 - 100.0 |
| **total (all analytes)** | | **fresh** **24 hrs** | **118/119** **132/132** | **99.2** **100.0** | **95.4 - 99.9** **97.2 - 100.0** |
* = reduced number of available results due to invalid analyte results.
Table 11: Sample Stability, Low Positive (~2x LoD), 24 hr storage at 2-8°C
K191967 - Page 29 of 88
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| Low | x-fold LoD | Test Condition | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | # Pos. /# Exp. | % | 95% CI |
| Acinetobacter baumanniiATCC 19606 | 2 | fresh | 31/32 | 96.9 | 84.3 - 99.4 |
| | | 24 hrs | 31/32 | 96.9 | 84.3 - 99.4 |
| Chlamydia pneumoniaeATCC VR-2282 | 2 | fresh | 32/32 | 100.0 | 89.3 - 100.0 |
| | | 24 hrs | 32/32 | 100.0 | 89.3 - 100.0 |
| Citrobacter freundiiATCC 8090 | 2 | fresh | 31/32 | 96.9 | 84.3 - 99.4 |
| | | 24 hrs | 29/32 | 90.6 | 75.8 - 96.8 |
| Haemophilus influenzaeATCC 33391 | 2 | fresh | 29/31 | 93.5 | 79.3 - 98.2 |
| | | 24 hrs | 31/31 * | 100.0 | 89.0 - 100.0 |
| Klebsiella pneumoniaeNCTC 13443 | 1 | fresh | 31/32 | 96.9 | 84.3 - 99.4 |
| | | 24 hrs | 32/32 | 100.0 | 89.3 - 100.0 |
| ctx-MNCTC 13443 | 4 | fresh | 32/32 | 100.0 | 89.3 - 100.0 |
| | | 24 hrs | 32/32 | 100.0 | 89.3 - 100.0 |
| ndmNCTC 13443 | 2 | fresh | 31/32 | 96.9 | 84.3 - 99.4 |
| | | 24 hrs | 31/32 | 96.9 | 84.3 - 99.4 |
| temNCTC 13443 | 2 | fresh | 30/31 | 96.8 | 83.8 - 99.4 |
| | | 24 hrs | 30/31 * | 96.8 | 83.8 - 99.4 |
| Morganella morganiiATCC 25830 | 2 | fresh | 32/32 | 100.0 | 89.3 - 100.0 |
| | | 24 hrs | 31/32 | 96.9 | 84.3 - 99.4 |
| Proteus vulgarisATCC 29905 | 2 | fresh | 32/32 | 100.0 | 89.3 - 100.0 |
| | | 24 hrs | 30/30 | 100.0 | 88.7 - 100.0 |
| Staphylococcus aureusNCTC 12493 | 3 | fresh | 32/32 | 100.0 | 89.3 - 100.0 |
| | | 24 hrs | 32/32 | 100.0 | 89.3 - 100.0 |
| mecANCTC 12493 | 1.3 | fresh | 32/32 | 100.0 | 89.3 - 100.0 |
| | | 24 hrs | 32/32 | 100.0 | 89.3 - 100.0 |
| total (all analytes) | fresh | 375/382 | 98.2 | 96.3 - 99.1 | |
| | | 24 hrs | 373/380 | 98.2 | 96.2 - 99.1 |
* = reduced number of available results due to invalid analyte results.
Table 12: Sample Stability, Negative Samples, 24 hr storage at 2-8°C
| Negative | Test Condition | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- |
| | | # Neg. /# Exp. | % | 95% CI |
| Acinetobacter baumanniiATCC 19606 | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| Chlamydia pneumoniaeATCC VR-2282 | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| Citrobacter freundiiATCC 8090 | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| Haemophilus influenzaeATCC 33391 | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| Klebsiella pneumoniaeNCTC 13443 | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| ctx-MNCTC 13443 | fresh | 10/10 * | 100.0 | 72.3 - 100.0 |
| | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
K191967 - Page 30 of 88
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| Negative | Test Condition | Agreement with Expected Result | | |
| --- | --- | --- | --- | --- |
| | | # Neg. /# Exp. | % | 95% CI |
| *ndm* | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| NCTC 13443 | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| *tem* | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| NCTC 13443 | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| *Morganella morganii* | fresh | 10/10 * | 100.0 | 72.3 - 100.0 |
| ATCC 25830 | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| *Proteus vulgaris* | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| ATCC 29905 | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| *Staphylococcus aureus* | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| NCTC 12493 | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| *mecA* | fresh | 11/11 | 100.0 | 74.1 - 100.0 |
| NCTC 12493 | 24 hrs | 11/11 | 100.0 | 74.1 - 100.0 |
| **total (all analytes)** | **fresh** | **130/130** | **100.0** | **97.1 - 100.0** |
| | **24 hrs** | **132/132** | **100.0** | **97.2 - 100.0** |
* = reduced number of available results due to invalid analyte results.
### Sample Stability/Post-lysis
Package insert instructions indicate the user to load the Unyvero LRT BAL Cartridge immediately after lysis on the Unyvero Lysator or if needed, within one hour post-lysis. An analytical study was conducted to assess any differences in performance between immediate testing and after a one hour delay. Study samples were prepared with representative analytes close to their respective LoD concentrations. Results were compared for samples tested immediately after lysis and samples tested after a 1 hour or 4 hours delay between lysis completion and Analyzer start. A total of six replicates were evaluated per analyte and storage condition. The study demonstrated equivalent results for all test conditions.
### External Controls
Quantified stored frozen microorganism stocks comprising LoD reference strains for all LRT BAL analytes were used for preparation of positive controls that were regularly tested throughout clinical and analytical studies. A minimum of one strain for each microorganism target was included in the positive control scheme. All targeted resistance marker targets were also represented. Positive controls were prepared as six different microorganism pools with each analyte present at ~3X LoD.…
