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 Description

The Unyvero LRT BAL Application automates and integrates DNA purification and eight parallel multiplex endpoint PCR reactions. It provides qualitative detection of nucleic acids from multiple lower respiratory pathogens using hybridization on PCR chamber arrays in a single use cartridge from a single bronchoalveolar lavage (BAL)-like specimen (BAL or mini-BAL).

The Unyvero LRT BAL Application identifies 20 microorganisms and 10 antibiotic resistance markers as listed in the Intended Use Statement, below.

The Unyvero LRT BAL Application uses a multiplex PCR approach following array hybridization which targets 30 individual analytes (microorganisms (N = 20) and antibiotic resistance markers (N = 10) divided into eight separate PCR reactions that are performed in individual reaction chambers simultaneously on a Unyvero LRT BAL Application cartridge. Multiplex compositions are designed to avoid any expected common occurrence of certain analytes within the same multiplex to largely reduce competitive PCR inhibition. Individual analyte assays of the Unyvero LRT BAL panel are designed to exhibit low or absent cross-reactivity with the relevant bronchoalveolar lavage (BAL)like specimens (BAL or mini-BAL) sample matrix or 'close neighbor' strains. Array oligonucleotides are designed for similar hybridization and melting temperatures (approx. 65 - 80 ℃, varying by amplicon). Hybridization and melting temperatures are used to exclude non-specific hybridization signals for improved signal specificity.

The instrumentation consists of one (or more) Unyvero L4 Lysator, one (or more) Unyvero A50 Analyzer, a Unyvero C8 Cockpit, and four single-use consumables: the Unyvero LRT BAL Cartridge, the Unyvero Sample Tube, Sample Tube Cap and the Unyvero Master Mix. A Unyvero Sample Tube Holder is supplied as accessory to simplify the sample filling step.

Unyvero LRT BAL Cartridge contains DNA isolation and purification reagents, . primers, hybridization and wash buffers, and oligonucleotides for detection.
Unyvero T1 Sample Tube and Transport Cap contains glass beads and buffers to lyse ● microorganisms and liquefy the sample.
. 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. ●

An internal control (a synthetic gene without any homology to known sequences) is processed in every chamber in order to verify the DNA purification, array hybridization, and detection.

Other than the built-in controls, no external materials are supplied with Unyvero LRT BAL Application devices and consumables. Good laboratory practice recommends running external positive and negative controls using samples cultured in the laboratory.

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).

How to perform a Unyvero LRT BAL test:

1. Remove the Unyvero Sample Tube from its packaging and slide it in the Unyvero Sample Tube Holder in the upright position with the barcoded end at the bottom.
1. Remove the Transport Cap from the Sample Tube by pulling it upward.
1. Pipette 180 uL of vortexed patient specimen into the Sample Tube and close it using the Unyvero Cap provided in the LRT BAL kit: align the small nodules on the neck of the Sample Tube with the Tube with the openings on the Cap and press down to lock in place.
1. Scan the Sample Tube and place it into the Lysator. Close the Lysator lid to start the Lysator.
1. Remove Master Mix from freezer and thaw at room temperature (15 ℃ 25 ℃) for approximately 30 minutes.
1. When lysis is complete, remove the Sample Tube from the Lysator and place it into the labeled position on the left-hand side of the Unyvero LRT BAL Cartridge.
1. Place the thawed Master Mix into the labeled position on the right-hand side of the Cartridge.
1. Scan the Cartridge on the Cockpit and insert it into the position indicated on the Analyzer. The software provides on-screen instructions to start the test.
1. View results after completion of the run.

During the automated analysis (Step 8), which is entirely controlled by the A50 Analyzer, the sample is mixed with ethanol and then transferred onto the DNA purification column, where buffers purify and elute the 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, consisting of one labeled and one non-labeled primer for the respective multiplex endpoint PCR. After specific amplification, PCR products are hybridized to the corresponding array probes. Each array has been manufactured with probes corresponding to the amplicons for the targeted microorganism sequences described above. A total of up to 49 spots per array allows for redundant detection with at least four spots per analyte, as well as spots for intensity calibration, and orientation markers for the image processing software. Binding of amplicons to specific probes is detected by analyzing fluorescence images of the arrays. Result data are displayed on the C8 Cockpit for visualization, printout, temporary storage and electronic data export.

A test run is completed after 4 to 5 hrs, and results for panel microorganisms and corresponding antibiotic resistance markers are displayed on the Cockpit screen. Four screens are provided:

. The Result Summary screen provides a quick overview of all detected LRT BAL panel microorganisms, together with all detected corresponding antibiotic resistance markers.
. The Microorganisms screen provides a list of all panel microorganisms grouped in Grampositive bacteria, non-fermenting bacteria, Enterobacteriaceae and other microorganisms together with the analysis result (reported as detected, not detected or invalid).
The Result Details screen provides a list of all analyzed microorganisms and the ● corresponding antibiotic resistance markers together with the analysis result (reported as detected, not detected, invalid or NA).
Test Details screen showing user name, lot numbers of used consumables, expiration dates of ● the consumables and start and stop times and dates of the test.

Results can be reviewed on the cockpit or, optionally, be printed out. All results are saved in a database on the Unyvero Cockpit for later review and printing.

The Unyvero software is designed to:

Manage analysis workflow (Cockpit) ●
Carry out sample lysis (Lysator) ●
Execute the analysis and generate the analytical result (Analyzer) .
Manage communication among units (Cockpit, Lysator, Analyzer) ●
Monitor internal mechanical / electrical actuators (Lysator, Analyzer) ●
Present analysis results (Cockpit) .
Store analysis results (Cockpit) ●

Each device (Cockpit, Lysator. Analyzer) is a subsystem within the overall system, and each consists of hardware and software components. The different devices are interconnected by an Ethernet based communication interface, and system functionality is provided by the interaction of all three device types. Only the Cockpit presents a rich user interface and allows interaction with the operator. The Lysator and Analyzer units include a simple display for showing device status. Optional HIS/LIS connectivity allows transferring results to a hospital or laboratory information system.

AI/ML Overview

This looks like a medical device submission, specifically a 510(k) summary for the "Unyvero LRT BAL Application." Let's break down the acceptance criteria and study data provided.

Acceptance Criteria and Device Performance (Summary derived from the provided text)

The document doesn't explicitly state "acceptance criteria" numerical thresholds in a single, consolidated table. However, regulatory submissions often imply acceptance criteria based on the demonstrated performance that is deemed sufficient for clearance. Based on the performance tables provided, particularly the "Comparison to Composite Comparator Reference" (Table 28), we can infer the device's demonstrated performance for each microorganism. For antibiotic resistance markers, the "Comparison of antibiotic resistance markers to corresponding molecular reference assays" (Table 29) shows reported performance.

Inferred Acceptance Criteria (Implicit from demonstrated performance):

For Microorganism Detection (PPA & NPA values compared to a Composite Comparator): The FDA generally expects high Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) to ensure the device is effective and safe. While no explicit thresholds are given, the achieved percentages, predominantly in the range of 80-100% for PPA and 90-100% for NPA across various microorganisms, suggest these levels were acceptable to the FDA.
For Antibiotic Resistance Marker Detection (PPA & NPA values compared to Molecular Reference): Similar high PPA and NPA values were demonstrated (e.g., 88.9-100% for PPA and 69.5-100% for NPA), indicating these ranges were likely acceptable.
Reproducibility: High agreement rates (over 90% for most cases, 100% for negative samples) for both moderate and low concentrations.
Inclusivity & Exclusivity: Detection of target strains at or near LoD, and no cross-reactivity with non-target or common flora organisms.
Limit of Detection (LoD): Demonstrated specific LoD values for each target (as listed in Tables 2 and 3).

Reported Device Performance (from Tables 28 & 29 for clinical accuracy):

Microorganism	PPA (%) (95% CI)	NPA (%) (95% CI)	PPV (%) (95% CI)	NPV (%) (95% CI)
Acinetobacter spp.	88.9 (67.2 - 96.9)	99.5 (98.8 - 99.8)	76.2 (54.9 - 89.4)	99.8 (99.3 - 99.9)
Chlamydia pneumoniae	NA	100.0 (99.6 - 100.0)	NA	100.0 (99.6 - 100.0)
Citrobacter freundii	100.0 (20.7 - 100.0)	99.7 (99.1 - 99.9)	25.0 (4.6 - 69.9)	100.0 (99.6 - 100.0)
Enterobacter cloacae complex	81.8 (61.5 - 92.7)	99.8 (99.3 - 99.9)	90.0 (69.9 - 97.2)	99.6 (99.0 - 99.8)
Escherichia coli	82.4 (66.5 - 91.7)	98.1 (97.0 - 98.8)	59.6 (45.3 - 72.4)	99.4 (98.7 - 99.7)
Haemophilus influenzae	90.9 (72.2 - 97.5)	96.4 (95.0 - 97.4)	35.7 (24.5 - 48.8)	99.8 (99.2 - 99.9)
Klebsiella oxytoca	80.0 (49.0 - 94.3)	99.4 (98.7 - 99.7)	57.1 (32.6 - 78.6)	99.8 (99.3 - 99.9)
Klebsiella pneumoniae	84.0 (65.3 - 93.6)	99.1 (98.3 - 99.5)	70.0 (52.1 - 83.3)	99.6 (99.0 - 99.8)
Klebsiella variicola	0.0 (0.0 - 65.8)	99.9 (99.4 - 100.0)	0.0 (0.0 - 79.3)	99.8 (99.3 - 99.9)
Legionella pneumophila	100.0 (20.7 - 100.0)	100.0 (99.6 - 100.0)	100.0 (20.7 - 100.0)	100.0 (99.6 - 100.0)
Moraxella catarrhalis	75.0 (50.5 - 89.8)	99.7 (99.1 - 99.9)	80.0 (54.8 - 93.0)	99.6 (99.0 - 99.8)
Morganella morganii	100.0 (43.9 - 100.0)	100.0 (99.6 - 100.0)	100.0 (43.9 - 100.0)	100.0 (99.6 - 100.0)
Mycoplasma pneumoniae	100.0 (43.9 - 100.0)	99.7 (99.1 - 99.9)	50.0 (18.8 - 81.2)	100.0 (99.6 - 100.0)
Pneumocystis jirovecii	80.0 (60.9 - 91.1)	99.8 (99.3 - 99.9)	90.9 (72.2 - 97.5)	99.5 (98.8 - 99.8)
Proteus spp.	100.0 (72.3 - 100.0)	100.0 (99.6 - 100.0)	100.0 (72.3 - 100.0)	100.0 (99.6 - 100.0)
Pseudomonas aeruginosa	93.5 (87.1 - 96.8)	98.7 (97.7 - 99.2)	89.3 (82.2 - 93.8)	99.2 (98.4 - 99.6)
Serratia marcescens	93.8 (71.7 - 98.9)	99.8 (99.3 - 99.9)	88.2 (65.7 - 96.7)	99.9 (99.4 - 100.0)
Staphylococcus aureus	83.3 (74.6 - 89.5)	97.4 (96.1 - 98.2)	76.9 (68.0 - 84.0)	98.2 (97.2 - 98.9)
Stenotrophomonas maltophilia	82.9 (68.7 - 91.5)	99.3 (98.5 - 99.7)	82.9 (68.7 - 91.5)	99.3 (98.5 - 99.7)
Streptococcus pneumoniae	100.0 (74.1 - 100.0)	99.8 (99.3 - 99.9)	84.6 (57.8 - 95.7)	100.0 (99.6 - 100.0)

Antibiotic Resistance Markers (Table 29):

Antibiotic Resistance Marker	PPA (%) (95% CI)	NPA (%) (95% CI)
ctx-M	88.9 (56.5 - 98.0)	99.5 (97.2 - 99.9)
kpc	100.0 (43.9 - 100.0)	99.5 (97.3 - 99.9)
mecA	100.0 (85.1 - 100.0)	69.5 (58.9 - 78.4)
ndm	100.0 (20.7 - 100.0)	100.0 (98.2 - 100.0)
oxa-23	100.0 (43.9 - 100.0)	100.0 (82.4 - 100.0)
oxa-24	100.0 (43.9 - 100.0)	94.1 (73.0 - 99.0)
oxa-48	100.0 (20.7 - 100.0)	100.0 (96.7 - 100.0)
oxa-58	NA	100.0 (84.5 - 100.0)
tem	100.0 (70.1 - 100.0)	85.1 (72.3 - 92.6)
vim	NA	99.5 (97.3 - 99.9)

Study Details

Here's a breakdown of the information regarding the studies:

Sample size used for the test set and the data provenance:
- Prospective Study: 1,016 bronchoalveolar lavage (BAL) or mini-BAL specimens from hospitalized adult patients with suspected lower respiratory tract infections. Data provenance: Collected at nine US clinical sites between June 2015 and July 2016 for a previous study, stored frozen. Retrospective collection of prospectively collected samples.
- Archived Study: 392 lavage specimens (BAL or mini-BAL) from patients (age 18 or older) with suspected lower respiratory infection and at least one positive LRT BAL panel microorganism by standard-of-care (SoC). Data provenance: Collected at 11 US clinical sites between 2015 and 2019, complemented by a few specimens from other sites. This included 197 specimens from a previous study and 195 new specimens. Retrospective collection.
- Contrived Study: Used to supplement rare analytes. Consisted of 60 contrived specimens per target analyte (total not explicitly stated for all analytes combined, but implies 60 for each of the 13 microorganisms and relevant antibiotic resistance markers used in this component). Prepared by spiking pooled microorganisms into negative lavage specimens.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The document describes "Standard-of-Care (SoC) culture results" as one reference method for microorganisms and "molecular multiplex PCR comparator assay for which all positive PCR results were followed by bi-directional sequencing" as another.
- For "atypical microorganisms," a combination of two different validated PCR comparator assays (each targeting different genetic loci) was used.
- For antibiotic resistance markers, "PCR assays corresponding to each LRT BAL antibiotic resistance marker assay were included into the multiplex PCR comparator assay as a (single) molecular reference. Positive PCRs were followed by bi-directional sequencing."
- "Cultured isolates had been collected for positive prospective specimens whenever possible. Isolates were regrown and evaluated by MALDI-TOF to confirm strain identities and by whole genome sequencing using a next generation sequencing (NGS) approach to screen for presence or absence of LRT BAL panel antibiotic resistance markers."
- "Phenotypic AST results for positive specimens as reported by SoC culture were collected to correlate antibiotic resistance markers detected by the LRT BAL Application to resistance phenotypes."
- The document does NOT explicitly mention the number or specific qualifications of "experts" (e.g., medical doctors, microbiologists, or other specialists) routinely involved in establishing the ground truth measurements (like interpreting SoC culture results, performing PCR/sequencing or AST). It refers to "standard-of-care" clinical laboratory procedures and "molecular reference assays," implying trained laboratory personnel, but no explicit "expert panel" or count is given as typically seen for imaging studies.
Adjudication method (e.g., 2+1, 3+1, none) for the test set:
- For microbiology results: Discrepant results for false positives were analyzed by performing "singleplex PCRs/bi-directional sequencing using primer pairs targeting different genetic loci compared to the corresponding LRT BAL assays on specimen DNA extracts for each discrepant LRT BAL analyte." This indicates a form of molecular adjudication for discrepancies, rather than a consensus by human experts in the traditional sense, but still a specific method for resolving differences.
- For antibiotic resistance markers, "Correlation of Detected Antibiotic Resistance Markers to Strain Genotypes and Phenotypes" involved comparing the device's results to both molecular sequencing of isolates and phenotypic antimicrobial susceptibility testing (AST) results. This also serves as a multi-modal "adjudication" or reference verification process.
- No explicit "2+1" or "3+1" human reader adjudication method, common in imaging studies, is mentioned here, as it's a diagnostic test based on molecular/culture evidence.
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, this is not applicable. The Unyvero LRT BAL Application is a qualitative nucleic acid multiplex test for microorganism and resistance marker detection. It is a standalone diagnostic device, not an AI-assisted interpretation tool for human readers (like radiologists interpreting images). Therefore, an MRMC comparative effectiveness study involving human reader improvement with/without AI is not relevant to this device.
If a standalone (i.e., algorithm only without human-in-the-loop performance) was done:
- Yes, this was a standalone performance study. The device itself performs automated DNA purification, multiplex PCR, hybridization, and interpretation. The clinical performance data presented (PPA, NPA, etc.) represent the performance of the "Unyvero LRT BAL Application" as a full system, comparing its direct output to the established reference methods (SoC culture, molecular comparator assays, sequencing). There is no "human-in-the-loop" aspect to the test's result generation.
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- For "typical" microorganisms: The primary ground truth for the prospective study was Standard-of-Care (SoC) culture results, supplemented by a composite comparator (SoC culture combined with molecular multiplex PCR and bi-directional sequencing) to address limitations of culture.
- For "atypical" microorganisms: The primary ground truth was a composite comparator consisting of two different validated PCR comparator assays (each with bi-directional sequencing).
- For antibiotic resistance markers: Ground truth involved comparison to molecular reference assays (PCR/sequencing) for the specific marker and phenotypic antimicrobial susceptibility testing (AST) results for cultured isolates.
The sample size for the training set:
- The document describes performance studies (prospective, archived, contrived) used for validation. It does NOT specify a distinct "training set" or its size. This is typical for a traditional (non-AI/machine learning) diagnostic device validation, where the focus is on a comprehensive validation dataset rather than a separate training and test split. The LoD, inclusivity, exclusivity, interference, and reproducibility studies use various laboratory-prepared samples.
How the ground truth for the training set was established:
- Not applicable, as a specific "training set" is not mentioned in the context of this device. The validation studies utilized reference methods as described in point 6 above.

Summary

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Image /page/0/Picture/2 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

December 20, 2019

Curetis GmbH % Gail Radcliffe President Radcliffe Consulting, Inc. 231 Fairbanks Street West Boylston, Massachusetts 01583

Re: K191967

Trade/Device Name: Unyvero LRT BAL Application Regulation Number: 21 CFR 866.3985 Regulation Name: Device To Detect And Identify Microorganisms And Associated Resistance Marker Nucleic Acids Directly In Respiratory Specimens Regulatory Class: Class II Product Code: OBH Dated: July 22, 2019 Received: July 23, 2019

Dear Gail Radcliffe:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

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statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Kristian Roth, Ph.D. Branch Chief Bacterial Multiples and Medical Countermeasures Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Ouality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K191967

Device Name Unyvero LRT BAL Application

Indications for Use (Describe)

[continued on page 2]

Type of Use (Select one or both, as applicable)
{ Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

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[continued from Form FDA 3881, page 1]

Microorganism	Associated Antibiotic Resistance Marker
Acinetobacter spp.a	ctx-Mb, kpc, ndm, oxa-23, oxa-24, oxa-58, vim
Chlamydia pneumoniae	-
Citrobacter freundii	ctx-Mb, kpc, ndm, oxa-48, vim
Enterobacter cloacae complexc	ctx-Mb, kpc, ndm, oxa-48, vim
Escherichia coli	ctx-Mb, kpc, ndm, oxa-48, vim
Haemophilus influenzae	tem
Klebsiella oxytoca	ctx-Mb, kpc, ndm, oxa-48, vim
Klebsiella pneumoniaed	ctx-Mb, kpc, ndm, oxa-48, vim
Klebsiella variicola	ctx-Mb, kpc, ndm, oxa-48, vim
Legionella pneumophila	-
Moraxella catarrhalis	-
Morganella morganii	ctx-Mb, kpc, ndm, oxa-48, vim
Mycoplasma pneumoniae	-
Pneumocystis jirovecii	-
Proteus spp.e	ctx-Mb, kpc, ndm, oxa-48, vim
Pseudomonas aeruginosa	ctx-Mb, kpc, ndm, vim
Serratia marcescens	ctx-Mb, kpc, ndm, oxa-48, vim
Staphylococcus aureus	mecA
Stenotrophomonas maltophilia	-
Streptococcus pneumoniae	-

3 Acinetobacter spp. includes: A. baumannii, A. calcoaceticus, A. junii, A. nosocomialis, A. parvus, A. pitiii (detected by LRT BAL Application) and A. ursingii (not detected by LRT BAL Application).

b ctx-M1 subgroup.

° Enterobacter cloacae complex includes: E. chundaensis, E. cloacae, E. hormaechei (incl. ssp. xiang(angensis), E. kobei, E. ludwigii, E. roggenkampii, E. sichuandensis (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.

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510(k) SUMMARY - LRT BAL Application (K191967)

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92. The assigned 510(k) number is K191967.

807.92 (a)(1):	Name:	Curetis GmbH
	Address:	Max-Eyth-Strasse 42Holzgerlingen, Germany 71088
	Phone:	+49 7031 49195-24
	Email:	regulatory@curetis.com
	Contact:	Karsten Mueller, Head of Quality and Regulatory Affairs

807.92 (a)(2): Device name - trade name and common name, and classification

Trade name:

Unyvero LRT BAL Application

Common Name: Detection and identification of microorganisms and associated resistance marker nucleic acids directly in respiratory specimens

Classification / Product Code: 21 CFR Part 866.3985 / QBH

807.92 (a)(3): Identification of the legally marketed predicate devices

The Unyvero LRT BAL Application is substantially equivalent to its predecessor test system, the Unyvero LRT Application (Curetis, GmbH, Germany), granted de novo status under DEN170047 on April 3rd, 2018.

807.92 (a)(4): Device Description

The Unyvero LRT BAL Application identifies 20 microorganisms and 10 antibiotic resistance markers as listed in the Intended Use Statement, below.

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Overview

Instrument. Cartridge, and Other Consumables

Unyvero LRT BAL Cartridge contains DNA isolation and purification reagents, . primers, hybridization and wash buffers, and oligonucleotides for detection.
Unyvero T1 Sample Tube and Transport Cap contains glass beads and buffers to lyse ● microorganisms and liquefy the sample.
. 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 without any homology to known sequences) is processed in every chamber in order to verify the DNA purification, array hybridization, and detection.

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Reagents

Assav Procedures

How to perform a Unyvero LRT BAL test:

1. Remove the Unyvero Sample Tube from its packaging and slide it in the Unyvero Sample Tube Holder in the upright position with the barcoded end at the bottom.
1. Remove the Transport Cap from the Sample Tube by pulling it upward.
1. Pipette 180 uL of vortexed patient specimen into the Sample Tube and close it using the Unyvero Cap provided in the LRT BAL kit: align the small nodules on the neck of the Sample Tube with the openings on the Cap and press down to lock in place.
1. Scan the Sample Tube and place it into the Lysator. Close the Lysator lid to start the Lysator.
1. Remove Master Mix from freezer and thaw at room temperature (15 ℃ 25 ℃) for approximately 30 minutes.
1. When lysis is complete, remove the Sample Tube from the Lysator and place it into the labeled position on the left-hand side of the Unyvero LRT BAL Cartridge.
1. Place the thawed Master Mix into the labeled position on the right-hand side of the Cartridge.
1. Scan the Cartridge on the Cockpit and insert it into the position indicated on the Analyzer. The software provides on-screen instructions to start the test.
1. View results after completion of the run.

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A test run is completed after 4 to 5 hrs, and results for panel microorganisms and corresponding antibiotic resistance markers are displayed on the Cockpit screen. Four screens are provided:

. The Result Summary screen provides a quick overview of all detected LRT BAL panel microorganisms, together with all detected corresponding antibiotic resistance markers.
. The Microorganisms screen provides a list of all panel microorganisms grouped in Grampositive bacteria, non-fermenting bacteria, Enterobacteriaceae and other microorganisms together with the analysis result (reported as detected, not detected or invalid).
The Result Details screen provides a list of all analyzed microorganisms and the ● corresponding antibiotic resistance markers together with the analysis result (reported as detected, not detected, invalid or NA).
Test Details screen showing user name, lot numbers of used consumables, expiration dates of ● the consumables and start and stop times and dates of the test.

Results can be reviewed on the cockpit or, optionally, be printed out. All results are saved in a database on the Unyvero Cockpit for later review and printing.

Software

The Unyvero software is designed to:

Manage analysis workflow (Cockpit) ●
Carry out sample lysis (Lysator) ●
Execute the analysis and generate the analytical result (Analyzer) .
Manage communication among units (Cockpit, Lysator, Analyzer) ●
Monitor internal mechanical / electrical actuators (Lysator, Analyzer) ●
Present analysis results (Cockpit) .
Store analysis results (Cockpit) ●

{8}------------------------------------------------

807.92 (a)(5): 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.

Microorganism	Associated Antibiotic Resistance Marker(s)
Acinetobacter spp.a	ctx-Mb, kpc, ndm, oxa-23, oxa-24, oxa-58, vim
Chlamydia pneumoniae	-
Citrobacter freundii	ctx-Mb, kpc, ndm, oxa-48, vim
Enterobacter cloacae complexc	ctx-Mb, kpc, ndm, oxa-48, vim
Escherichia coli	ctx-Mb, kpc, ndm, oxa-48, vim
Haemophilus influenzae	tem
Klebsiella oxytoca	ctx-Mb, kpc, ndm, oxa-48, vim
Klebsiella pneumoniaed	ctx-Mb, kpc, ndm, oxa-48, vim
Klebsiella variicola	ctx-Mb, kpc, ndm, oxa-48, vim
Legionella pneumophila	-
Moraxella catarrhalis	-
Morganella morganii	ctx-Mb, kpc, ndm, oxa-48, vim
Mycoplasma pneumoniae	-
Pneumocystis jirovecii	-
Proteus spp.e	ctx-Mb, kpc, ndm, oxa-48, vim
Pseudomonas aeruginosa	ctx-Mb, kpc, ndm, vim
Serratia marcescens	ctx-Mb, kpc, ndm, oxa-48, vim
Staphylococcus aureus	mecA
Stenotrophomonas maltophilia	-
Streptococcus pneumoniae	-

® Acinetobacter spp. includes: A. baumannii, A. haemolyticus, A. junii, A. hvoffii, A. nosocomialis, A. purvus, A. pitti (detected by LRT BAL Application), and A. ursingii (not detected by LRT BAL Application).

b ctx-M1 subgroup.

§ Enterobacter cloacae complex includes: E. chengduensis, E. chundaensis, E. cloacae, E. hormaechei (incl. ssp. xiangfangensis), E. kobei, E. ludwigii, E. sichuanensis and E. bugandensis (not yet recognized as member of the E. cloacae complex).

e Proteus spp. includes: P. cibarius, P. hauseri, P. mirabilis, P. penneri, and P. vulgaris.

510(k) Summary - LRT BAL Application K191967 Rev 3.0

d Klebsiella pneumoniae includes two variants: K. pneumoniae (variant 1), and K. quasipneumoniae (variant 2).

{9}------------------------------------------------

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.

807.92 (a)(6): Technological Similarities and Differences to the Predicate

Element	New Device:Curetis Unyvero LRT BAL Application	Predicate:Curetis Unyvero LRT Application(DEN170047)
Specimen Type	Bronchoalveolar lavage (BAL)-likespecimens from adult hospitalized patientswith suspected lower respiratory tractinfections	Endotracheal aspirate specimens from adulthospitalized patients with suspected lowerrespiratory tract infections
OrganismsDetected	Bacteria:Acinetobacter spp.Citrobacter freundiiEnterobacter cloacae complexEscherichia coliHaemophilus influenzaeKlebsiella oxytocaKlebsiella pneumoniaeKlebsiella variicolaMoraxella catarrhalisMorganella morganiiProteus spp.Pseudomonas aeruginosaSerratia marcescensStaphylococcus aureusStenotrophomonas maltophiliaStreptococcus pneumoniaeAtypical Bacteria:Chlamydia pneumoniaeLegionella pneumophilaMycoplasma pneumoniaeFungus: Pneumocystis jiroveciiAntimicrobial Resistance Genes:	Bacteria:Acinetobacter spp.Citrobacter freundiiEnterobacter cloacae complexEscherichia coliHaemophilus influenzaeKlebsiella oxytocaKlebsiella pneumoniaeKlebsiella variicolaMoraxella catarrhalisMorganella morganiiProteus spp.Pseudomonas aeruginosaSerratia marcescensStaphylococcus aureusStenotrophomonas maltophiliaStreptococcus pneumoniaeAtypical Bacteria:Chlamydia pneumoniaeLegionella pneumophilaMycoplasma pneumoniaeAntimicrobial Resistance Genes:

		Table 1: Comparison of the Unyvero LRT Application and the Unyvero LRT BAL Application

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	ctx-M	ctx-M
	kpc	kpc
	mecA	mecA
	ndm	ndm
	oxa-23	oxa-23
	oxa-24	oxa-24
	oxa-48	oxa-48,
	oxa-58	oxa-58,
	tem	tem
	vim	vim
Analyte	DNA	DNA
TechnologicalPrinciples	Multiplex nucleic acid	Multiplex nucleic acid
Result Types	Qualitative for all analytes	Qualitative for all analytes
Instrumentation	Unyvero System	Unyvero System
Time to Result	About 4-5 hrs	About 4-5 hrs
Reagent Storage	Room temperature(below -20 °C, Master Mix Tube only)	Room temperature(below -20 °C, Master Mix Tube only)
TestInterpretation	Automated test interpretation and reportgeneration.	Automated test interpretation and reportgeneration.
Controls	Control included in each test to monitor forthe presence of PCR inhibitors and enablesthe system to detect failures in the testingprocess.	Control included in each test to monitor forthe presence of PCR inhibitors and enablesthe system to detect failures in the testingprocess.
User Complexity	Moderate	Moderate

807.92 (b)(1): Brief Description of Nonclinical Data

Limits-of-Detection (LoDs)

Limits-of-Detection were determined for each LRT BAL panel analyte by serial dilutions of reference strains prepared in pooled negative native lavage specimens as test matrix. The lowest test concentration with a positivity rate of 95% or higher was determined as the LoD for each specific panel analyte (e.g., at least 19 of 20 positive results for at least 20 performed test replicates). Tables 2 and 3 summarize LoDs for all LRT BAL panel microorganisms or antibiotic resistance markers, respectively.

{11}------------------------------------------------

	ReferenceStrain ID	LoD[CFU/mL]
Acinetobacter spp.	ATCC 19606(A. baumannii)	2.0 x 104
Chlamydia pneumoniae	ATCC VR-2282 a	3.2 x 102
Citrobacter freundii	ATCC 8090	8.0 x 104
Enterobacter cloacae complex	ATCC 13047(E. cloacae)	2.0 x 105
Escherichia coli	ATCC 11775	2.0 x 104
Haemophilus influenzae	ATCC 33391	2.0 x 104
Klebsiella oxytoca	ATCC 13182	1.0 x 104
Klebsiella pneumoniae (var. 1)	ATCC 13883	4.0 x 104
Klebsiella quasipneumoniae(K. pneumoniae, var. 2)	ATCC 700603	4.0 x 104
Klebsiella variicola	ATCC BAA-830	2.0 x 104
Legionella pneumophila	ATCC 33152	8.0 x 104
Moraxella catarrhalis	ATCC 25238	1.5 x 105
Morganella morganii	ATCC 25830	2.0 x 104
Mycoplasma pneumoniae	ATCC 29085 b	1.6 x 103
Pneumocystis jirovecii	36_0314 c	5.0 x 105
Proteus spp.	ATCC 29906(P. mirabilis)ATCC 29905(P. vulgaris)	5.0 x 103
Pseudomonas aeruginosa	ATCC 10145	6.3 x 102
Serratia marcescens	ATCC 13880	4.0 x 104
Staphylococcus aureus	ATCC 12600	1.5 x 105
Stenotrophomonas maltophilia	ATCC 13637	5.0 x 103
Streptococcus pneumoniae	ATCC 49619	2.0 x 104

ª Cell supernatant (in IFU/mL).

b Bacterial suspension, quantified by qPCR (in copies/mL).

& Positive clinical specimen, quantified by qPCR (in copies/mL). LoD was determined using DNA extracted from a positive clinical specimen, and then confirmed by testing dilutions of this clinical specimen at the LoD concentration (20/20 positive results).

{12}------------------------------------------------

	ReferenceStrain ID	Host Microorganism	LoD[CFU/mL]
ctx-M	NCTC 13443	Klebsiella pneumoniae	1.0 x 104
kpc	NCTC 13438	Klebsiella pneumoniae	4.0 x 104
mecA	NCTC 12493	Staphylococcus aureus	4.0 x 105
ndm	NCTC 13443	Klebsiella pneumoniae	2.0 x 104
oxa-23	NCTC 13301	Acinetobacter baumannii	1.0 x 107
oxa-24	NCTC 13302	Acinetobacter baumannii	5.0 x 103
oxa-48	NCTC 13442	Klebsiella pneumoniae	3.0 x 105
oxa-58	NCTC 13305	Acinetobacter baumannii	5.0 x 104
tema	NCTC 13443	Klebsiella pneumoniae	2.0 x 104
vim	NCTC 13437	Pseudomonas aeruginosa	2.0 x 104

Table 3: LoDs for LRT BAL panel antibiotic resistance markers.

ª Although the LRT BAL Application reports tem results only for H. influenzae as corresponding host microorganism, LoD was determined with a tem positive E. coli strain. Note that inclusivity testing was also successfully performed with tem positive H. influenzae strains.

Inclusivity

LRT BAL Microorganisms

Inclusivity wet testing was performed with reference strains for each microorganism analyte at concentrations near the LoD with contrived samples using pooled native negative lavage specimens as sample matrix (Table 4). Reference strains used for LoD determination are considered inclusive and were used as positive controls. Inclusivity was established near LoD (< 5x LoD) for all but one of the tested reference strains. For one reference strain of M. pneumoniae, tested as genomic DNA extract, inclusivity was demonstrated with a reduced analytical sensitivity at 5x LoD, although no primer or probe deviations to the corresponding LRT BAL assay were present.

Reference Strain	Strain ID	Test Conc.[CFU/ml]	x-fold LoD	# Pos./# Exp.
analyte: Acinetobacter spp.
Acinetobacter baumannii(pos. control/LoD ref. strain)	ATCC 19606	4.0 x 104	2x	6/6
Acinetobacter baumannii	NCTC 13305	4.0 x 104	2x	2/2
Acinetobacter baumannii	NCTC 13301	4.0 x 104	2x	2/2
Acinetobacter baumannii	NCTC 13302	4.0 x 104	2x	2/2
Acinetobacter baumannii	Micromyx 6148	4.0 x 104	2x	2/2
Acinetobacter baumannii	Micromyx 4410	4.0 x 104	2x	2/2
Acinetobacter baumannii	JMI 49755	4.0 x 104	2x	2/2
Acinetobacter baumannii	NRZ-00449	1.0 x 104	0.5x	2/2
Acinetobacter baumannii	UCLA A4	1.0 x 104	0.5x	2/2
Acinetobacter calcoaceticus	ATCC 23055	4.0 x 104	2x	2/2
Acinetobacter lwoffii	ATCC 15309	4.0 x 104	2x	2/2
Acinetobacter haemolyticus	ATCC 17906	4.0 x 104	2x	2/2
Chlamydia pneumoniae	ATCC VR-2282	6.4 x 102 a	2x	4/4
Reference Strain	Strain ID	Test Conc. [CFU/ml]	x-fold LoD	# Pos./ # Exp.
(pos. control/LoD ref. strain)
Chlamydia pneumoniae	ATCC VR-1310	$6.4 x 10^2 a$	2x	2/2
Chlamydia pneumoniae	ATCC 53592	$1.0 x 10^3 a$	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
analyte: Enterobacter cloacae complex
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
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
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
Reference Strain	Strain ID	Test Conc.[CFU/ml]	x-fold LoD	# Pos./# Exp.
Klebsiella pneumoniae, variant 1(pos. control/LoD ref. strain)	ATCC 13883	8.0 x 104	2x	4/4
Klebsiella quasipneumoniae(K. pneumoniae, variant 2)(pos. control/LoD ref. strain)	ATCC 700603	8.0 x 104	2x	3/3
Klebsiella pneumoniae	NCTC 13439	8.0 x 104	2x	2/2
Klebsiella pneumoniae	NCTC 13438	8.0 x 104	2x	2/2
Klebsiella pneumoniae	NCTC 13442	8.0 x 104	2x	2/2
Klebsiella pneumoniae	NCTC 13443	2.0 x 104	0.5x	3/3
Klebsiella pneumoniae	Micromyx 4653	8.0 x 104	2x	2/2
Klebsiella pneumoniae	Micromyx 4676	8.0 x 104	2x	2/2
Klebsiella pneumoniae	JMI 49767	2.0 x 104	0.5x	2/2
Klebsiella pneumoniae	NRZ-00002	2.0 x 104	0.5x	2/2
Klebsiella pneumoniae	NRZ-00103	8.0 x 104	2x	2/2
Klebsiella variicola(pos. control/LoD ref. strain)	ATCC BAA-830	4.0 x 104	2x	3/3
Klebsiella variicola	clinical strain 1	4.0 x 104	2x	2/2
Klebsiella variicola	clinical strain 2	4.0 x 104	2x	2/2
Klebsiella variicola	clinical strain 3	4.0 x 104	2x	2/2
Klebsiella variicola	clinical strain 4	4.0 x 104	2x	2/2
Legionella pneumophila (serotype 1)(pos. control/LoD ref. strain)	ATCC 33152	1.6 x 105	2x	7/7
Legionella pneumophila (serotype 2)	ATCC 33154	1.6 x 105	2x	2/2
Legionella pneumophila (serotype 3)	ATCC 33155	1.6 x 105	2x	2/2
Legionella pneumophila (serotype 6)	ATCC 33215	1.6 x 105	2x	2/2
Legionella pneumophila (serotype 8)	ATCC 35096	1.6 x 105	2x	2/2
Legionella pneumophila (serotype 10)	ATCC 43283	1.6 x 105	2x	2/2
Legionella pneumophila	UCLA L1	1.6 x 105	2x	2/2
Legionella pneumophila	UCLA L5	1.6 x 105	2x	2/2
Legionella pneumophila	UCLA L6	1.6 x 105	2x	2/2
Moraxella catarrhalis(pos. control/LoD ref. strain)	ATCC 25238	3.0 x 105	2x	4/4
Moraxella catarrhalis	ATCC 43617	3.0 x 105	2x	2/2
Moraxella catarrhalis	ATCC 8176	3.0 x 105	2x	2/2
Moraxella catarrhalis	ATCC 25240	3.0 x 105	2x	2/2
Moraxella catarrhalis	ATCC 23246	3.0 x 105	2x	3/3
Moraxella catarrhalis	ATCC 49143	3.0 x 105	2x	2/2
Morganella morganii(pos. control/LoD ref. strain)	ATCC 25830	4.0 x 104	2x	2/3
Morganella morganii	ATCC 8019	4.0 x 104	2x	2/2
Morganella morganii	ATCC 25829	4.0 x 104	2x	0/1
		6.0 x 104	3x	2/2
Morganella morganii	DSM-46262	4.0 x 104	2x	1/2
		6.0 x 104	3x	2/2
Morganella morganii ssp. sibonii	ATCC 49948	4.0 x 104	2x	2/2
Mycoplasma pneumoniae (type 1)(pos. control/LoD ref. strain)	ATCC 29085	3.2 x 103 b	2x	5/7
Reference Strain	Strain ID	Test Conc.[CFU/ml]	x-fold LoD	# Pos./# Exp.
Mycoplasma pneumoniae (type 1)	ATCC 29343	3.2 x 103 b	2x	2/2
		3.2 x 103 b	2x	0/2
		5.0 x 103 b	3x	1/2
Mycoplasma pneumoniae (type 2)	ATCC 15531 c, d	6.4 x 103 b	4x	1/2
		7.0 x 103 b	4.4x	1/2
		8.0 x 103 b	5x	2/2
Mycoplasma pneumoniae (type 1)	ATCC 15293	3.2 x 103 b	2x	2/2
Mycoplasma pneumoniae (type 2)	ATCC 49894	3.2 x 103 b	2x	2/2
Pneumocystis jirovecii(pos. control/LoD ref. strain)	pos. specimen 1	1.0 x 106 b	2x	1/1
Pneumocystis jirovecii	pos. specimen 2	1.0 x 106 b	2x	2/2
Pneumocystis jirovecii	pos. specimen 3	1.0 x 106 b	2x	2/2
Pneumocystis jirovecii	pos. specimen 4	1.0 x 106 b	2x	2/2
Pneumocystis jirovecii	pos. specimen 5	1.0 x 106 b	2x	2/2
analyte: Proteus spp.
Proteus vulgaris(pos. control/LoD ref. strain)	ATCC 29905	1.0 x 104	2x	7/7
Proteus mirabilis(pos. control/LoD ref. strain)	ATCC 29906	1.0 x 104	2x	2/2
Proteus mirabilis	ATCC 12453	1.0 x 104	2x	2/2
Proteus mirabilis	ATCC 14153	1.0 x 104	2x	2/2
Proteus mirabilis	ATCC 25933	1.0 x 104	2x	2/2
Proteus vulgaris	ATCC 6380	1.0 x 104	2x	2/2
Proteus vulgaris	ATCC 8427	1.0 x 104	2x	2/2
Proteus hauseri	ATCC 700826	1.0 x 104	2x	2/2
Proteus penneri	ATCC 33519	1.0 x 104	2x	2/2
Pseudomonas aeruginosa(pos. control/LoD ref. strain)	ATCC 10145	1.3 x 103	2x	3/3
Pseudomonas aeruginosa	ATCC 27853	1.3 x 103	2x	2/2
Pseudomonas aeruginosa	NCTC 13437	1.3 x 103	2x	2/2
Pseudomonas aeruginosa	Micromyx 2562	2.0 x 103	3x	2/2
Pseudomonas aeruginosa	NRZ-00196	1.3 x 103	2x	2/2
Pseudomonas aeruginosa	UCLA P20	1.3 x 103	2x	2/2
Serratia marcescens(pos. control/LoD ref. strain)	ATCC 13880	8.0 x 104	2x	3/3
Serratia marcescens	ATCC 14756	8.0 x 104	2x	2/2
Serratia marcescens	ATCC 15365	8.0 x 104	2x	2/2
Serratia marcescens	ATCC 27117	8.0 x 104	2x	2/2
Serratia marcescens	ATCC 43861	8.0 x 104	2x	2/2
Serratia marcescens ssp. sakuensis	DSM-17174	8.0 x 104	2x	2/2
Staphylococcus aureus(pos. control/LoD ref. strain)	ATCC 12600	3.0 x 105	2x	7/7
Staphylococcus aureus	ATCC BAA-2312	3.0 x 105	2x	2/2
Staphylococcus aureus	NCTC 12493	3.0 x 105	2x	2/2
Staphylococcus aureus	ATCC 33591	3.0 x 105	2x	2/2
Staphylococcus aureus	DSM-17091	3.0 x 105	2x	2/2
Reference Strain	Strain ID	Test Conc.[CFU/ml]	x-fold LoD	# Pos./# Exp.
Staphylococcus aureus	ATCC 29213	3.0 x 105	2x	2/2
Staphylococcus aureus	ATCC 43300	3.0 x 105	2x	2/2
Stenotrophomonas maltophilia(pos. control/LoD ref. strain)	ATCC 13637	1.0 x 104	2x	4/4
Stenotrophomonas maltophilia	ATCC 13636	1.0 x 104	2x	0/2
Stenotrophomonas maltophilia		1.5 x 104	3x	2/2
Stenotrophomonas maltophilia	ATCC 17666	1.0 x 104	2x	2/2
Stenotrophomonas maltophilia	ATCC 49130	1.0 x 104	2x	2/2
Stenotrophomonas maltophilia	DSM-50173	1.0 x 104	2x	2/2
Stenotrophomonas maltophilia	DSM-21874 e[NCIMB 9528]	1.0 x 104	2x	0/2
Stenotrophomonas maltophilia		1.5 x 104	3x	0/2
Stenotrophomonas maltophilia		2.0 x 104	4x	2/2
Streptococcus pneumoniaeserotype 19F(pos. control/LoD ref. strain)	ATCC 49619	4.0 x 104	2x	4/7 f
Streptococcus pneumoniae serotype 3	ATCC 6303	4.0 x 104	2x	2/2
Streptococcus pneumoniae serotype 5	ATCC 6305	4.0 x 104	2x	2/2
Streptococcus pneumoniae	ATCC 49150g	4.0 x 104	2x	0/2
		8.0 x 104	4x	2/2
Streptococcus pneumoniae	ATCC 33400 h	4.0 x 104	2x	1/2
		6.0 x 104	3x	1/2
		8.0 x 104	4x	2/2
Streptococcus pneumoniae serotype 2	ATCC 27336	4.0 x 104	2x	2/2
Streptococcus pneumoniae serotype 1	ATCC 6301	4.0 x 104	2x	2/2
Streptococcus pneumoniae serotype 9V	DSM-11865	4.0 x 104	2x	2/2
Streptococcus pneumoniae serotype 23F	DSM-11866	4.0 x 104	2x	2/2
		6.0 x 104	3x	1/2
		8.0 x 104	4x	2/2
Streptococcus pneumoniae serotype 6B	DSM-11867	4.0 x 104	2x	2/2

Table 4: Inclusivity study results for LRT BAL panel microorganisms.

510(k) Summary - LRT BAL Application K191967 Rev 3.0

{13}------------------------------------------------

{14}------------------------------------------------

510(k) Summary – LRT BAL Application K191967 Rev 3.0

{15}------------------------------------------------

{16}------------------------------------------------

ª In IFU/mL for C. pneumoniae.

b In copies/mL for M. pneumoniae and P. jirovecii.

& Quantified DNA extract (reference material) from a commercial provider.

4 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 is likely due to the different source material (DNA extract instead of a cell suspension).

e S. maltophilia strain DSM-21874 was positive only at a 4x LoD concentration. Sequencing did not reveal any mismatches to primer or probe sequences.

T S. pneumoniae LoD reference strain ATCC 46916 did not show consistently positive signals at 2x LoD. ATCC 46916 controls were repeated using a freshly prepared counted culture stock. This time, a positivity rate of 8/8 was obtained at a 2x LoD concentration, as expected.

& S. pneumoniae strain ATCC 49150 was only positive at 4x LoD. Sequencing did not reveal any mismatches to primer or probe sequences.

4 S. pneumoniae strain ATCC 33400 was only consistently positive at 4x LoD. Sequencing did not reveal any mismatches to primer or probe sequences.

{17}------------------------------------------------

To supplement inclusivity testing, in silico GenBank BLAST analyses of LRT BAL panel primer and probe sequences were performed for each LRT BAL panel 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), only with reduced sensitivity (typically, single relevant mismatches of primer or probe sequences; detection likely at higher than LoD concentrations only), or 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). Note that additional in silico analysis results are provided for strains that have not been wet tested. Such in silico results are provided 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.

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: Inclusive LRT BAL panel microorganisms as demonstrated by inclusivity wet testing and/or as predicted by in silico analysis (at LoD concentration or with reduced sensitivity). For strains for which in silico analysis predicts higher than LoD concentrations for one or more applicable entries, numbers of GenBank entries are additionally listed.

	WetTesting	In Silico:at LoD	In Silico:ReducedSensitivity	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 testednegative at 1.5 x 107 CFU/mL
Chlamydia pneumoniae	X	X	-
Citrobacter freundii	X	X26 entries	X4 entries
Enterobacter cloacae complex
E. cloacae	X	X	-	including ssp. dissolvens
	WetTesting	In Silico:at LoD	In Silico:ReducedSensitivity	Comment
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. chengduensis	-	X	-
E. chuandaensis	-	X	-
E. roggenkampii	-	X	-
E. asburiae	X	X11 entries	X1 entry
E. bugandensis	-	X	-	not yet recognized as member of theE. cloacae complex
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. quasipneumoniae)	X	X11 entries	X10 entries
Klebsiella variicola	X	X	-
Legionella pneumophila	X	X	-	some entries predict detection atLoD, other entries predict a possibleslight performance reduction; strainsfor both variants were included ininclusivity wet testing and weredetected 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	X	-
Pseudomonas aeruginosa	X	X	-
Serratia marcescens	X	X	-
Staphylococcus aureus	X	X> 450entries	X1 entry
Stenotrophomonas maltophilia	X	X	-
Streptococcus pneumoniae	X	X> 240entries	X1 entry	including serotype 7F

510(k) Summary - LRT BAL Application K191967 Rev 3.0

{18}------------------------------------------------

{19}------------------------------------------------

LRT BAL Antibiotic Resistance Markers

Similar to microorganisms, inclusivity wet testing was performed with reference strains carrying antibiotic resistance markers of the LRT BAL panel at concentrations near LoD with contrived samples using pooled negative native lavage specimens as sample matrix (Table 6). Subgroups of antibiotic resistance markers are added, if known (NA: unknown). Where phenotypic antimicrobial susceptibility testing (AST) results are available, this information is also shown (e.g., carbapenems = resistant to carbapenems, carbapenems = susceptible to carbapenems).

Inclusivity was established near LoD for all tested reference strains.

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 x 104	2x	2/2
	ctx-M15	Klebsiella pneumoniae	NRZ-00249	2.0 x 104	2x	2/2
	NA	Enterobacter cloacae	JMI 46239	3.0 x 104	3x	2/2
	NA	Escherichia coli	JMI 50067	2.0 x 104	2x	2/2
	NA	Klebsiella pneumoniae	NRZ-00002	2.0 x 104	2x	1/2
	NA	Enterobacter cloacae	ATCC BAA-2468	4.0 x 104	4x	2/2
	NA	Klebsiella pneumoniae	JMI 49831	4.0 x 104	4x	2/2
	NA	Klebsiella pneumoniae	JMI 49767	2.0 x 104	2x	2/2
kpc	kpc-3	Klebsiella pneumoniae(pos. control/LoD ref. strain)	NCTC 13438	8.0 x 104	2x	5/5
	kpc-2	Escherichia coli	NRZ-00281	8.0 x 104	2x	2/2
	kpc-2	Klebsiella pneumoniae	NRZ-00103	8.0 x 104	2x	2/2
	kpc-3	Escherichia coli	NRZ-00222	8.0 x 104	2x	1/2
	kpc-3	Escherichia coli	NRZ-00222	1.2 x 105	3x	2/2
	kpc-3	Klebsiella pneumoniae(carbapenemR)	Micromyx 4653	8.0 x 104	2x	4/4
	kpc-3	Klebsiella pneumoniae(carbapenemR)	Micromyx 4676	8.0 x 104	2x	4/4
mecA	NA	Staphylococcus aureus(methicillinR, cefoxitinR)(pos. control/LoD ref. strain)	NCTC 12493	8.0 x 105	2x	3/3
	SCCmecI	Staphylococcus aureus	RKI 07-03165	8.0 x 105	2x	2/2
	SCCmecII	Staphylococcus aureus	RKI 01-00694	8.0 x 105	2x	2/2
	SCCmecIII	Staphylococcus aureus(methicillinR)	ATCC 33591	8.0 x 105	2x	2/2
	SCCmecIV	Staphylococcus aureus	RKI 09-00187	8.0 x 105	2x	2/2
	SCCmecV	Staphylococcus aureus	RKI 08-02492	8.0 x 105	2x	2/2
	NA	Staphylococcus aureus(methicillinR)	DSM-17091	8.0 x 105	2x	2/2
	SCCmecII	Staphylococcus aureus(methicillinR)	ATCC 43300	3.0 x 105	0.8x	2/2
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	$4.0 x 10^4$	2x	5/5
	ndm-1	Acinetobacter baumannii	JMI 49755	$4.0 x 10^4$	2x	4/4
	ndm-1	Enterobacter cloacae (imipenemR,ertapenemR)	ATCC BAA-2468	$4.0 x 10^4$	2x	1/2
	ndm-1	Enterobacter cloacae	JMI 46239	$6.0 x 10^4$	3x	2/2
	ndm-1	Escherichia coli	JMI 50067	$4.0 x 10^4$	2x	4/4
	ndm-1	Klebsiella pneumoniae	JMI 49767	$4.0 x 10^4$	2x	2/2
	ndm-1	Klebsiella pneumoniae	JMI 49831	$2.0 x 10^4$	1x	2/2
oxa-23	oxa-23	Acinetobacter baumannii(pos. control/LoD ref. strain)	NCTC 13301	$4.0 x 10^4$	2x	2/2
	NA	Acinetobacter baumannii(carbapenemR)	Micromyx 4410	$2.0 x 10^7$	2x	3/3
	NA	Acinetobacter baumannii(carbapenemR)	Micromyx 6148	$2.0 x 10^7$	2x	2/2
	NA	Acinetobacter baumannii(carbapenemR)	Micromyx 6149	$2.0 x 10^7$	2x	2/2
	NA	Acinetobacter baumannii(carbapenemR)	Micromyx 6153	$2.0 x 10^7$	2x	2/2
	NA	Acinetobacter baumannii(imipenemR, meropenemR)	UCLA A5	$2.0 x 10^7$	2x	2/2
oxa-24	oxa-25	Acinetobacter baumannii(pos. control/LoD ref. strain)	NCTC 13302	$2.0 x 10^7$	2x	2/2
	oxa-72	Acinetobacter baumannii	NRZ-00449	$1.0 x 10^4$	2x	3/3
	NA	Acinetobacter baumannii(imipenemR, meropenemR)	UCLA A4	$1.0 x 10^4$	2x	2/2
	NA	Acinetobacter baumannii(imipenemR, meropenemR)	clinical strain 1	$1.0 x 10^4$	2x	2/2
	NA	Acinetobacter baumannii(imipenemR, meropenemR)	clinical strain 2	$1.0 x 10^4$	2x	2/2
oxa-48	oxa-48	Klebsiella pneumoniae(pos. control/LoD ref. strain)	NCTC 13442	$1.0 x 10^4$	2x	2/2
	oxa-48	Escherichia coli(ertapenemR)	ATCC BAA-2523	$6.0 x 10^5$	2x	3/3
	oxa-48	Escherichia coli	NRZ-00176	$6.0 x 10^5$	2x	2/2
	oxa-48	Escherichia coli	NRZ-00176	$6.0 x 10^5$	2x	1/2
	oxa-162	Escherichia coli	NRZ-00361	$9.0 x 10^5$	3x	2/2
	oxa-162	Klebsiella pneumoniae	NRZ-00472	$6.0 x 10^5$	2x	2/2
	oxa-232	Klebsiella oxytoca	NRZ-22060	$6.0 x 10^5$	2x	2/2
oxa-58	oxa-58	Acinetobacter baumannii(pos. control/LoD ref. strain)	NCTC 13305	$6.0 x 10^5$	2x	2/2
	oxa-58	Acinetobacter baumannii	NRZ-00518	$1.0 x 10^5$	2x	2/3
tem	NA	Klebsiella pneumoniae(pos. control/LoD ref. strain)	NCTC 13443	$1.0 x 10^5$	2x	3/3
	tem-1	Escherichia coli	ATCC 35218	$4.0 x 10^4$	2x	2/3
	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	$4.0 x 10^4$	2x	2/2
	NA	Klebsiella pneumoniae	NRZ-00751	$2.0 x 10^4$	1x	2/2
	NA	Escherichia coli	ATCC BAA-2523	$2.0 x 10^4$	1x	2/2
Analyte	Subgroup	Reference Strain	Strain ID	Test Conc.[CFU/ml]	x-fold LoD	# Pos./# Exp.
	NA	Acinetobacter baumannii	JMI 49755	4.0 x 104	2x	2/2
	NA	Haemophilus influenzae(ampicillinR, cefinaseR)	clinical strain 1	4.0 x 104	2x	2/2
	NA	Haemophilus influenzae(cefinaseR)	clinical strain 2	4.0 x 104	2x	2/2
	NA	Klebsiella pneumoniae	Micromyx 4676	8.0 x 104	4x	2/2
	vim-10	Pseudomonas aeruginosa(pos. control/LoD ref. strain)	NCTC 13437	4.0 x 104	2x	3/3
	vim-1	Citrobacter freundii	NRZ-00452	4.0 x 104	2x	2/2
vim	vim-1	Pseudomonas aeruginosa(ceftazidimeR, imipenemR)	DSM-24600	4.0 x 104	2x	2/2
	vim-1	Enterobacter cloacae	NRZ-00239	4.0 x 104	2x	2/2
	vim-1	Klebsiella pneumoniae	NCTC 13439	4.0 x 104	2x	2/2
	vim-1	Klebsiella pneumoniae	NCTC 13440	4.0 x 104	2x	2/2
	NA	Pseudomonas aeruginosa	UCLA P20	1.3 x 103	0.1x	2/2
	NA	Pseudomonas aeruginosa(carbapenemR)	Micromyx 2562	4.0 x 104	2x	2/2

Table 6: Inclusivity study results for LRT BAL panel antibiotic resistance markers.

{20}------------------------------------------------

{21}------------------------------------------------

To supplement inclusivity testing for further specific antibiotic resistance marker variants, reference sequences for all available variants belonging to individual antibiotic resistance markers or marker subgroups were compiled and analyzed in silico.

Subgroups or variants for applicable LRT BAL panel antibiotic resistance markers for which detection is predicted at LoD (match of relevant primer and probe sequences), with reduced sensitivity (typically, single relevant mismatches of primer or probe sequences; detection likely at higher than LoD concentrations only), or is not predicted (multiple relevant mismatches in primer and probe sequences) are listed in Table 7. Such predictions are provided as supplementary data only. 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 the inclusivity test results table above.

{22}------------------------------------------------

Antibiotic ResistanceMarker:Subgroup	Detection Predicted at LoD:Variant No.	Detection Predicted withReduced 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:ctx-M2ctx-M8ctx-M9ctx-M25ctx-M45subgroups			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
tem	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

Table 7: In silico prediction for LRT BAL panel antibiotic resistance markers for applicable subgroups or variants.

NOTE: H. influenzae commonly hosts tem-1. tem will only be reported by the LRT BAL Application if H. influenzae is concurrently detected.

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Exclusivity and Cross-Reactivity

For each of the LRT BAL panel analytes, cross-reactive species (clinically relevant close neighbor strains) were predicted by GenBank BLAST search. Predictions were complemented with exclusivity wet testing for close-neighbor strains or common respiratory flora microorganisms performed in duplicate at a worst-case concentration (typically: 1.5 x 107 CFU/mL for bacterial microorganisms, or as indicated) by spiking into 0.25x ARM (artificial respiratory matrix) surrogate as sample matrix.

Exclusivity study results are listed in

Table 8. For Haemophilus haemolyticus (ATCC 33390) and Aggregatibacter aphrophilus (ATCC 19415), cross-reactivity to Haemophilus influenzae close to LoD concentrations (for ATCC 33390) or with reduced sensitivity (for ATCC 19415), respectively, was observed. Another strain, Haemophilus parainfluenzae (ATCC 33392) cross-reacted to Haemophilus influenzae only at very high concentrations in some of the tests (> 100x LoD, > 107 CFU/mL).

Strain	Test Concentration	Cross-Reactivity
respiratory flora microorganisms	[in CFU/mL]
Actinomyces odontolyticus	1.5 x 107	no
Aspergillus fumigatus	1.5 x 107	no
Candida albicans	1.5 x 107	no
Candida dubliniensis	1.5 x 107	no
Candida glabrata	1.5 x 107	no
Candida krusei	1.5 x 107	no
Candida parapsilosis	1.5 x 107	no
Candida tropicalis	1.5 x 107	no
Cardiobacterium hominis	1.5 x 107	no
Eikenella corrodens	1.5 x 107	no
Enterococcus faecalis	1.5 x 107	no
Enterococcus faecium	1.5 x 107	no
Fusobacterium nucleatum	5.0 x 106 d	no
Granulicatella adiacens	1.5 x 107	no
Kingella kingae	1.5 x 107	no
Lactobacillus acidophilus	1.5 x 107	no
Micrococcus luteus	1.5 x 107	no
Mycobacterium bovis	1.5 x 107 d	no
Mycoplasma orale	1.5 x 107	no
Neisseria lactamica	1.5 x 107 d	no
Neisseria sicca	1.5 x 107	no
Pantoae agglomerans	1.5 x 107	no
Peptostreptococcus stomatis	5.0 x 106 d	no
Porphyromonas gingivalis	1.5 x 107 d	no
Prevotella buccalis	1.5 x 107	no
Raoultella planticola	1.5 x 107	no
close neighbor microorganisms	[in CFU/mL]
Acinetobacter ursingii	1.5 x 107	no
Aggregatibacter actinomycetemcomitans	1.5 x 107 d	no
Aggregatibacter aphrophilus	1.5 x 107	yes (H. influenzae) a
Citrobacter koseri	1.5 x 107	no
Haemophilus haemolyticus	1.5 x 107	yes (H. influenzae) b
Haemophilus parahaemolyticus	1.5 x 107	no
Haemophilus parainfluenzae	1.5 x 107	yes (H. influenzae) c
Legionella longbeachae	1.5 x 107	no
Legionella/Tatlockia micdadei	1.5 x 107	no
Staphylococcus capitis	1.5 x 107	no
Staphylococcus epidermidis	1.5 x 107	no
Staphylococcus haemolyticus	1.5 x 107	no
Staphylococcus lugdunensis	1.5 x 107	no
Staphylococcus saprophyticus	1.5 x 107	no
Streptococcus agalactiae	1.5 x 107	no
Streptococcus anginosus	1.5 x 107	no
Streptococcus dysgalactiae	1.5 x 107	no
Streptococcus gordonii	1.5 x 107	no
Streptococcus intermedius	1.5 x 107	no
Streptococcus mitis	1.5 x 107	no
Streptococcus mutans	1.5 x 107	no
Streptococcus oralis	1.5 x 107	no
Streptococcus parasanguinis	1.5 x 107	no
Streptococcus pseudopneumoniae	1.5 x 107	no
Streptococcus pyogenes	1.5 x 107	no
Streptococcus salivarius	1.5 x 107	no
Streptococcus sanguinis	1.5 x 107	no
Streptococcus vestibularis	1.5 x 107	no
respiratory viruses e	[in copies/mL]
Adenovirus 41	1.0 x 105	no
Enterovirus 68	1.0 x 105	no
Enterovirus 71	1.0 x 105	no
Parainfluenzae Virus 1	1.0 x 105	no
Parainfluenzae Virus 2	1.0 x 105	no
Parainfluenzae Virus 3	1.0 x 105	no
Parainfluenzae Virus 4	1.0 x 105	no
Rhinovirus	1.0 x 105	no
RSV A	1.0 x 105	no
RSV B	1.0 x 105	no

Table 8: Exclusivity study results.

510(k) Summary – LRT BAL Application K191967 Rev 3.0

{24}------------------------------------------------

ª For A. aphrophilus, cross-reactivity to H. influenzae was observed down to a concentration of 2.0 x 10° CFUmL (1/2 tests positive). For a concentration of 1.0 x 10 CFU/mL (equivalent to 5x LoD of H. influenzae), cross-reactivity was not observed any more. 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° CFU/mL (2/2 tests positive, equivalent to 2x LoD of H. influenzae). Sequence comparison by BLAST for H. influenzae shows a 3' mismatch to

{25}------------------------------------------------

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, although only predicted with reduced sensitivity.

C For H. parainfluenzae, cross-reactivity to H. influenzae was observed only at the tested worst-case concentration of 1.5 x 107 CFU/mL (2/4 tests positive, equivalent to 750x LoD of H. influenzae). Additional tests at 2.0 x 100 CFU/mL (equivalent to 10x LoD of H. influenzae), cross-reactivity was not observed any more.

d Tested as DNA extract (in copies/mL).

e Tested as DNA or RNA extract (in copies/mL).

Table 9 lists predicted possible cross-reactivity of applicable LRT BAL panel microorganisms based on in silico analysis, exclusivity wet testing and cases observed during the prospective or archived study.

Table 9: In silico cross-reactivity prediction, results of exclusivity wet testing and cross-reactive specimens observed for the prospective and archived study.

Close Neighbor Strain a	Cross-Reactivity Prediction(in-silico analysis)	Wet Testing Result	Cross-ReactionsObserved in ClinicalStudy(N = 1,408 prosp. orarch. specimens)
Citrobacter freundii		-	-
Citrobacter braakii	Detection predicted at higher thanLoD concentrations (certainstrains) / Detection not predicted(other strains) c	-	-
Citrobacter pasteurii	Detection predicted at higher thanLoD concentrations	-	-
Citrobacter werkmannii	Detection predicted at higher thanLoD concentrations (certainstrains) / Detection not predicted(other strains) c	-	-
Citrobacter youngae	Detection predicted at LoD (certainstrains) / Detection predicted athigher than LoD concentrations(other strains) c	-	1
Kluyvera georgiana	Detection predicted at higher thanLoD concentrations	-	-
Citrobacter koseri	Detection not predicted	negativeATCC 27156	-
Escherichia coli
Shigella dysenteriae b	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 thanLoD concentrations	positiveATCC 33390	5
Haemophilus parahaemolyticus	Detection not predicted	negativeATCC 10014	-
Haemophilus parainfluenzae	Detection not predicted	positive at worst-caseconcentration onlyATCC 33392	1
Haemophilus aegvoticus b	Detection predicted at LoD

{26}------------------------------------------------

Aggregatibacter actino-mycetemcomitans	Detection not predicted	negativeATCC 33384	-
Aggregatibacter aphrophilus	Detection predicted at higher thanLoD concentrations (certainstrains) / Detection not predicted(other strains) c	positiveATCC 19415	3
Aggregatibacter segnis	Detection predicted at higher thanLoD concentrations	-	-
Klebsiella oxytoca
Klebsiella michiganensis	Detection predicted at LoD (certainstrains) / Detection predicted athigher than LoD concentrations(other strains) c	-	-
Klebsiella pneumoniae
Klebsiella quasivariicola	Detection predicted at higher thanLoD concentrations	-	-
Staphylococcus aureus
Staphylococcus argenteus b	Detection predicted at LoD	-	-
CNS:S. epidermidisS. capitisS. lugdunensisS. haemolyticusS. saprophyticus	Detection not predicted	negativeATCC 51625ATCC 27840ATCC 43809ATCC 29970ATCC 15305	-
Streptococcus pneumoniae
other Streptococcus spp.:S. agalactiaeS. anginosusS. dysgalactiaeS. gordoniiS. intermediusS. mitisS. mutansS. oralisS. parasanguinisS. pseudopneumoniaeS. pyogenesS. salivariusS. sanguinis	Detection not predicted	negativeATCC 13813ATCC 33397ATCC 43078ATCC 10558ATCC 27335ATCC 49456ATCC 25175ATCC 35037ATCC 15912ATCC BAA-960ATCC 12344ATCC 7073ATCC 10556	-

4 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. nickelliarans; Escherichia coli: E. marnotae; Staphylococcus aureus: S. simiae; Stenotrophomonas maltophilia: S. nitritreducens, S. daejeonensis, S. acidaminiphila, S. koreensis, S. rhizophila, Xanthomonas spp.).

b Clinical relevance unlikely for respiratory infections.

& Predictions for available strains distribute into different categories.

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Interference Testing

Interference testing had already been established with the Unyvero LRT Application (predicate device), which relies on the same DNA extraction procedures and chemistry as the LRT BAL Application. Results are included below for reference. Possible interfering substances, such as respiratory medications, antibiotics, sample storage media, sample liquefying agent (DTT), lysis buffer and endogenous substances blood and human DNA were tested. Pools of panel analytes were analyzed with and without interfering substances added at concentrations recommended in the CLSI guideline 'Interference Testing in Clinical Chemistry'. Representative analytes were pooled and added to PBS as surrogate matrix containing potentially interfering substances at concentrations as indicated in Table 10. No interference was observed at tested concentrations.

	Interferent	Test Concentration	InterferenceObserved
	Guaifenesin	$1.5 x 10^{-2}$ M	no
respiratory drugs	Dextromethorphan	$3.7 x 10^{-6}$ M	no
	Acetyl-Cystein	$1.0 x 10^{-2}$ M	no
	Salbutamol	$4.0 x 10^{-6}$ M	no
	Carbocystein	$2.8 x 10^{-3}$ M	no
	Ambroxol	$8.0 x 10^{-4}$ M	no
	Beclomethason	$7.0 x 10^{-4}$ M	no
	Theophyllin	$2.2 x 10^{-4}$ M	no
	Ampicillin	$1.5 x 10^{-4}$ M	no
	Cefuroxime	$1.4 x 10^{-3}$ M	no
	Erythromycin	$8.2 x 10^{-5}$ M	no
	Ciprofloxacin	$3.0 x 10^{-5}$ M	no
antibiotics	Amikacin	$1.4 x 10^{-4}$ M	no
	Imipenem	$5.0 x 10^{-4}$ M	no
	Clindamycin	$8.9 x 10^{-5}$ M	no
	Trimethoprim	$1.4 x 10^{-4}$ M	no
	Sulfamethoxazole	$1.6 x 10^{-3}$ M	no

lavage samplemedia	Ringer-Lactate solution	100%	no
	Ringer solution	100%	no
inhalation agent(NaCl)	sodium chloride	5% w/v	no
lysis buffer	lysis buffer DTT, 90% v/v	lysis buffer: 90% v/v (final conc. inlysis tube) or 80% v/v (for addedsample), DTT: 40 mM (final conc.in lysis tube) or 35 mM (for addedsample)	no
	EDTA blood	100% v/v	no
sample	human placenta DNA	1 µg/µL	no
components	fish sperm DNA	4 µg/µL	no
	mucin (pig stomach, type II)	20 mg/mL	no

	Table 10: Tested possible interfering substances (M: test concentration in mol/L).

{28}------------------------------------------------

Reproducibility

Assay reproducibility was established with artificial samples (viable strains spiked in ARM surrogate matrix) with a representative strain panel (incl. Gram-positive, Gram-negative and 'atypical' microorganisms) covering the following LRT BAL panel analytes: Acinetobacter spp., C. pneumoniae, C. freundii, H. influenzae, K. pneumoniae (comprising LRT BAL panel antibiotic resistance markers ctx-M, ndm, and tem), M. morganii, Proteus spp. and, S. aureus (comprising LRT BAL panel antibiotic resistance marker mecA). Reproducibility testing was performed at a moderate concentration (2.5x - 10x LoD of the corresponding target analyte), at a low concentration (1x - 4x LoD of the corresponding target analyte), and with all analytes negative (surrogate matrix only) on three different Unyvero systems. Each Unyvero system mimicked a different clinical setting, consisted of one Unyvero Cockpit, three Unyvero Lysators, up to six Unyvero Analyzers, and was operated by one of three dedicated operators with different levels of work experience.

Each operator performed 3 'moderate', 3 'low', and 3 'negative' replicates per test shift on his/her Unyvero system (maximum: 18 replicates per test day). Each operator performed a minimum of 30 replicates per concentration level (90 replicates in total per Unyvero system, 270 replicates in total for all three Unyvero systems with 90 replicates for each concentration level) distributed over a minimum of five test days with sample identities blinded to the operator. Samples on individual Analyzer slots were rotated (e.g., 'moderate' followed by 'low', followed by 'negative' etc.) to achieve a nonconsecutive testing schedule for each sample type between test shifts. 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 at all test concentration levels.

Tables 11 to 13 summarize the reproducibility results for each concentration level, along with the agreement rates with the expected result and 95% confidence intervals (95% CI, calculated using the Wilson score method).

{29}------------------------------------------------

Table 11: Reproducibility study results (agreement with expected result) for concentration level 'moderate', 5x LoD per test analyte or as indicated.

moderate	x-foldLoD	UnyveroSystem/Operator	Agreement with Expected Result
			# Pos./# Exp.	%	95% CI
Acinetobacter baumanniiATCC 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 pneumoniaeATCC 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 freundiiATCC 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 influenzaeATCC 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 pneumoniaeNCTC 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
		total	92/92	100.0	96.0 - 100.0
ctx-MNCTC 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
ndmNCTC 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
temNCTC 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 morganiiATCC 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 vulgarisATCC 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 aureusNCTC 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
mecANCTC 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
		UnyveroSystem/Operator	Agreement with Expected Result
low	x-foldLoD		# Pos./# Exp.	%	95% CI
Acinetobacter baumanniiATCC 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 pneumoniaeATCC 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 freundiiATCC 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 influenzaeATCC 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
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 aATCC 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
negative	UnyveroSystem/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 freundiiATCC 8090	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
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
mec ANCTC 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.

{30}------------------------------------------------

Table 12: Reproducibility study results (agreement with expected result) for concentration level 10w', 2x LoD per analyte or as indicated.

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% Cl 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.

{31}------------------------------------------------

Table 13: Reproducibility study results (agreement with expected result) for negative samples.

ª Reduced number of available results due to invalid analyte results.

For three cartridge runs, unexpected positive results were reported with weak signals close to the assay thresholds on different test days and different operators/test systems (2x E. coli, for one 'moderate' and one 'low' test sample, 1x oxa-23, for one 'moderate' test sample).

{32}------------------------------------------------

Fresh-Frozen Study

Assay reproducibility between fresh samples and samples stored frozen was established with artificial samples (viable strains spiked in pooled native negative BAL specimens as test matrix) with the representative strain panel as described for the reproducibility study above. Testing was performed at a moderate concentration (2.5x - 10x LoD of the corresponding target analyte, at least 10 replicates per condition), at a low concentration (1x - 4x LoD of the corresponding target analyte, at least 30 replicates per condition) and with all target analytes negative (at least 10 replicates per condition).

Pair-wise tests with the LRT BAL Application were performed for samples tested within 2 hrs after test sample preparation (Time 0) and samples tested after freezing below - 70 ℃ for at least 1 day and thawing immediately before testing.

Tables 14 to 16 summarize the fresh-frozen test results for each of the tested concentration levels (moderate, low, negative) for both storage conditions (tested frozen), along with the agreement rates with the expected result and 95% confidence intervals (95% CI, calculated using the Wilson score method).

Comparable agreement rates with the expected result were obtained for all test analytes for both test conditions.

{33}------------------------------------------------

Table 14: Fresh-frozen study test results for concentration level 'moderate', approx. 5x LoD per test analyte.

	x-foldLoD	TestCondition	Agreement with ExpectedResult
moderate			# Pos./# Exp.	%	95% CI
Acinetobacter baumanniiATCC 19606	5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
Chlamydia pneumoniaeATCC VR-2282	5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
Citrobacter freundiiATCC 8090	5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
Haemophilus influenzaeATCC 33391	5	fresh	10/11	90.9	62.3 - 98.4
		frozen	11/11	100.0	74.1 - 100.0
Klebsiella pneumoniaeNCTC 13443	2.5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
ctx-MNCTC 13443	10	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
ndmNCTC 13443	5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
temNCTC 13443	5	fresh	10/11	90.9	62.3 - 98.4
		frozen	11/11	100.0	74.1 - 100.0
Morganella morganiiATCC 25830	5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
Proteus vulgarisATCC 29905	5	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
Staphylococcus aureusNCTC 12493	8.3	fresh	10/10 a	100.0	72.3 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
mecANCTC 12493	3	fresh	11/11	100.0	74.1 - 100.0
		frozen	11/11	100.0	74.1 - 100.0
total (all analytes)		fresh	129/131	98.5	94.6 - 99.6
		frozen	132/132	100.0	97.2 - 100.0

ª Reduced number of available results due to invalid analyte results.

{34}------------------------------------------------

	Table 15: Fresh-frozen study test results for concentration level 'low', approx. 2x LoD per test analyte.
--	-----------------------------------------------------------------------------------------------------------	--	--

	x-foldLoD	TestCondition	Agreement with ExpectedResult
low			# Pos./# Exp.	%	95% CI
Acinetobacter baumanniiATCC 19606	2	fresh	31/32	96.9	84.3-99.4
		frozen	31/31	100.0	89.0-100.0
Chlamydia pneumoniaeATCC VR-2282	2	fresh	32/32	100.0	89.3- 100.0
		frozen	31/31	100.0	89.0-100.0
Citrobacter freundiiATCC 8090	2	fresh	31/32	96.9	84.3-99.4
		frozen	30/31	96.8	83.8-99.4
Haemophilus influenzaeATCC 33391	2	fresh	29/31	93.5	79.3-98.2
		frozen	28/30 a	93.3	78.7-98.2
Klebsiella pneumoniaeNCTC 13443	1	fresh	31/32	96.9	84.3-99.4
		frozen	31/31	100.0	89.0 - 100.0
ctx-MNCTC 13443	4	fresh	32/32	100.0	89.3 - 100.0
		frozen	31/31	100.0	89.0-100.0
ndmNCTC 13443	2	fresh	31/32	96.9	84.3-99.4
		frozen	31/31	100.0	89.0-100.0
temNCTC 13443	2	fresh	30/31	96.8	83.8-99.4
		frozen	29/30 a	96.7	83.3-99.4
Morganella morganiiATCC 25830	2	fresh	32/32	100.0	89.3-100.0
		frozen	31/31	100.0	89.0-100.0
Proteus vulgarisATCC 29905	2	fresh	32/32	100.0	89.3-100.0
		frozen	30/31	96.8	83.8-99.4
Staphylococcus aureusNCTC 12493	3	fresh	32/32	100.0	89.3 - 100.0
		frozen	31/31	100.0	89.0-100.0
mecANCTC 12493	1.3	fresh	32/32	100.0	89.3- 100.0
		frozen	31/31	100.0	89.0-100.0
total (all analytes)		fresh	375/382	98.2	96.3-99.1
		frozen	365/370	98.6	96.9-99.4

ª Reduced number of available results due to invalid analyte results.

{35}------------------------------------------------

				Table 16: Fresh-frozen study test results for samples negative for all analytes

	Test	Agreement with ExpectedResult
negative	Condition	# Neg./# Exp.	%	95% CI
Acinetobacter baumannii	fresh	10/10	100.0	72.3 - 100.0
ATCC 19606	frozen	11/11	100.0	74.1 - 100.0
Chlamydia pneumoniae	fresh	10/10	100.0	72.3 - 100.0
ATCC VR-2282	frozen	11/11	100.0	74.1 - 100.0
Citrobacter freundii	fresh	10/10	100.0	72.3 - 100.0
ATCC 8090	frozen	11/11	100.0	74.1 - 100.0
Haemophilus influenzae	fresh	10/10	100.0	72.3 - 100.0
ATCC 33391	frozen	11/11	100.0	74.1 - 100.0
Klebsiella pneumoniae	fresh	10/10	100.0	72.3 - 100.0
NCTC 13443	frozen	11/11	100.0	74.1 - 100.0
ctx-M	fresh	10/10	100.0	72.3 - 100.0
NCTC 13443	frozen	11/11	100.0	74.1 - 100.0
ndm	fresh	10/10	100.0	72.3 - 100.0
NCTC 13443	frozen	11/11	100.0	74.1 - 100.0
tem	fresh	10/10	100.0	72.3 - 100.0
NCTC 13443	frozen	11/11	100.0	74.1 - 100.0
Morganella morganii	fresh	10/10	100.0	72.3 - 100.0
ATCC 25830	frozen	11/11	100.0	74.1 - 100.0
Proteus vulgaris	fresh	10/10	100.0	72.3 - 100.0
ATCC 29905	frozen	11/11	100.0	74.1 - 100.0
Staphylococcus aureus	fresh	9/9 a	100.0	70.1 - 100.0
NCTC 12493	frozen	11/11	100.0	74.1 - 100.0
mecA	fresh	10/10	100.0	72.3 - 100.0
NCTC 12493	frozen	11/11	100.0	74.1 - 100.0
	fresh	119/119	100.0	96.9 - 100.0
total (all analytes)	frozen	132/132	100.0	97.2 - 100.0

a Reduced number of available results due to invalid analyte results.

Sample Stability Study

Assay reproducibility between fresh samples and samples stored refrigerated for at least 24 hrs was established with artificial samples using the same experimental approach as for the fresh-frozen study.

Pair-wise tests with the LRT BAL Application were performed for samples tested within 2 hrs after test sample preparation (Time 0) and samples tested after at total storage of at least 24 hrs including at least 2 hrs at a worst-case ambient temperature of 30 °C and at least 22 hrs stored in a refrigerator.

Tables 17 to 19 summarize the sample stability test results for each of the tested concentration levels (moderate, low, negative) for both test conditions (tested freshly, tested after storage), along with the agreement rates with the expected result and 95% confidence intervals (95% CI, calculated using the

510(k) Summary - LRT BAL Application K191967 Rev 3.0

{36}------------------------------------------------

Wilson score method). Comparable agreement rates with the expected result were obtained for all test analytes for both test conditions.

Table 17: Sample stability study test results for concentration level 'moderate', approx. 5x LoD per test
analyte, and a storage time of at least 24 hrs.

moderate	x-foldLoD	TestCondition	Agreement with ExpectedResult
			# Pos./# Exp.	%	95% CI
Acinetobacter baumanniiATCC 19606	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Chlamydia pneumoniaeATCC VR-2282	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Citrobacter freundiiATCC 8090	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Haemophilus influenzaeATCC 33391	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Klebsiella pneumoniaeNCTC 13443	2.5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
ctx-MNCTC 13443	10	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
ndmNCTC 13443	5	fresh	9/10	90.0	59.6 - 98.2
		24 hrs	11/11	100.0	74.1 - 100.0
temNCTC 13443	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Morganella morganiiATCC 25830	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Proteus vulgarisATCC 29905	5	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
Staphylococcus aureusNCTC 12493	8.3	fresh	9/9 a	100.0	70.1 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
mecANCTC 12493	3	fresh	10/10	100.0	72.3 - 100.0
		24 hrs	11/11	100.0	74.1 - 100.0
total (all analytes)		fresh	118/119	99.2	95.4 - 99.9
		24 hrs	132/132	100.0	97.2 - 100.0

ª Reduced number of available results due to invalid analyte results.

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Table 18: Sample stability study test results for concentration level 'low', approx. 2x LoD per test analyte, and a storage time of at least 24 hrs.

	x-foldLoD	TestCondition	Agreement with ExpectedResult
low			# Pos./# Exp.	%	95% CI
Acinetobacter baumanniiATCC 19606	2	fresh24 hrs	31/3231/32	96.996.9	84.3 - 99.484.3 - 99.4
Chlamydia pneumoniaeATCC VR-2282	2	fresh24 hrs	32/3232/32	100.0100.0	89.3 - 100.089.3 - 100.0
Citrobacter freundiiATCC 8090	2	fresh24 hrs	31/3229/32	96.990.6	84.3 - 99.475.8 - 96.8
Haemophilus influenzaeATCC 33391	2	fresh24 hrs	29/3131/31 a	93.5100.0	79.3 - 98.289.0 - 100.0
Klebsiella pneumoniaeNCTC 13443	1	fresh24 hrs	31/3232/32	96.9100.0	84.3 - 99.489.3 - 100.0
ctx-MNCTC 13443	4	fresh24 hrs	32/3232/32	100.0100.0	89.3 - 100.089.3 - 100.0
ndmNCTC 13443	2	fresh24 hrs	31/3231/32	96.996.9	84.3 - 99.484.3 - 99.4
temNCTC 13443	2	fresh24 hrs	30/3130/31 a	96.896.8	83.8 - 99.483.8 - 99.4
Morganella morganiiATCC 25830	2	fresh24 hrs	32/3231/32	100.096.9	89.3 - 100.084.3 - 99.4
Proteus vulgarisATCC 29905	2	fresh24 hrs	32/3230/30	100.0100.0	89.3 - 100.088.7 - 100.0
Staphylococcus aureusNCTC 12493	3	fresh24 hrs	32/3232/32	100.0100.0	89.3 - 100.089.3 - 100.0
mecANCTC 12493	1.3	fresh24 hrs	32/3232/32	100.0100.0	89.3 - 100.089.3 - 100.0
total (all analytes)		fresh24 hrs	375/382373/380	98.298.2	96.3 - 99.196.2 - 99.1

ª Reduced number of available results due to invalid analyte results.

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Table 19: Sample stability study test results for samples negative for all analytes for a total storage time of at least 24 hrs.

	TestCondition	Agreement with ExpectedResult# Neg./# Exp.	%	95% CI
Acinetobacter baumannii	fresh	11/11	100.0	74.1 - 100.0
ATCC 19606	24 hrs	11/11	100.0	74.1 - 100.0
Chlamydia pneumoniae	fresh	11/11	100.0	74.1 - 100.0
ATCC VR-2282	24 hrs	11/11	100.0	74.1 - 100.0
Citrobacter freundii	fresh	11/11	100.0	74.1 - 100.0
ATCC 8090	24 hrs	11/11	100.0	74.1 - 100.0
Haemophilus influenzae	fresh	11/11	100.0	74.1 - 100.0
ATCC 33391	24 hrs	11/11	100.0	74.1 - 100.0
Klebsiella pneumoniae	fresh	11/11	100.0	74.1 - 100.0
NCTC 13443	24 hrs	11/11	100.0	74.1 - 100.0
ctx-M	fresh	10/10 a	100.0	72.3 - 100.0
NCTC 13443	24 hrs	11/11	100.0	74.1 - 100.0
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 a	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

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807.92 (b)(2): Brief Description of Clinical Data

Introduction

Clinical performance of the LRT BAL Application was established by a prospective study cohort using 1,016 lavage specimens that had been collected at nine US clinical sites from hospitalized patients suspected for lower respiratory infections, by an archived study comprising 392 confirmed positive lavage specimens predominantly collected at US clinical sites, as well as by a contrived study using artificial positive samples to augment for rare LRT BAL panel analytes.

Performance characteristics of LRT BAL antibiotic resistance markers was evaluated for the prospective study cohort. Furthermore, antibiotic resistance markers detected by LRT BAL were correlated to resistance phenotypes of corresponding strain isolates using antimicrobial susceptibility test (AST) results collected for the prospective and archived study cohorts. Finally, rare antibiotic resistance markers were also included for contrived study testing.

A. Prospective Study with Frozen Specimens

Specimen Cohort: Inclusion/Exclusion Criteria, Demographics

BAL and mini-BAL specimens from hospitalized patients, age 18 or older, suspected of a lower respiratory infection, were eligible for the prospective study. Specimens had been collected at nine US clinical sites between June 2015 and July 2016 for a previous clinical study. Specimens were stored frozen within 24 hrs after arrival of the specimen in the lab.

Demographic information for prospective specimens included into the LRT BAL prospective study is described in Table 20.

Included Specimens	1,016
Sex
male	રુજરાત રાજ્યના સ્વરૂપ રાજ્યના મધ્યમાં આવેલું એક ગામના લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં મુખ્યત્વે ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં મુખ્
female	418
Age
18 - 30 years	69
31 - 60 years	367
> 60 years	ર્સ્ક્રિ0
Clinical Setting
ICU	ર્સ્ટર
ward	રા I

Table 20: Demographic patient data for the LRT BAL prospective study.

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Reference Methods – Microorganisms

For 'typical' microorganisms, LRT BAL Application results were compared to standard-of-care (SoC) culture results as the reference. SoC culture results had been reported either qualitatively or quantitatively. For quantitative cultures, a reporting threshold of 103 CFU/mL or higher for mini-BAL specimens and of 104 CFU/mL or higher for BAL specimens was applied, following recommendations by the Infectious Diseases Society of America (IDSA) and the American Thoracic Society (ATS). For microorganisms Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionella 'atypical' pneumophila and Pneumocystis jirovecii, no common SoC reference test was performed. Instead, SoC testing had only been performed on special request by the physician for a limited subset of patients.

In addition, LRT BAL Application results for 'typical' microorganisms were compared to a composite comparator as a second reference method. This composite comparator consists of the results of SoC culture combined with a molecular multiplex PCR comparator assay for which all positive PCR results were followed by bi-directional sequencing. For the composite comparator, any specimen that was positive by either SoC culture and/or positive by PCR and bi-directional sequencing was considered positive for the respective microorganism. Any specimen that was negative for both SoC culture and PCR was considered negative (Table 21A). Validated comparator PCR assays with analytical sensitivities in a similar range to the corresponding LRT BAL assays were included into the multiplex PCR comparator assay. These primers target different genetic loci than those used for the LRT BAL Application.

For 'atypical' microorganisms, a combination of two different validated PCR comparator assays was used as composite comparator, each targeting different genetic loci compared to the corresponding LRT BAL assays. As shown in Table 21B, specimens that were positive for either of the PCR/sequencing comparator assays were considered positive and specimens that were negative for both PCR/sequencing assays were considered negative.

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Table 21: Result determination algorithms for the composite comparator: (A) for 'typical' microorganisms, (B) for 'atypical' microorganisms.

composite comparator result('typical' microorganisms)		Comparator 2:PCR assay(included in multiplex PCR)
		positiveresult	negativeresult
Comparator 1:SoC (culture)	positiveresult	composite result:positive	composite result:positive
	negativeresult	composite result:positive	composite result:negative

Bcomposite comparator result('atypical' microorganisms)		Comparator 2:PCR assay 2(included in multiplex PCR)
	positiveresult	negativeresult
Comparator 1:PCR assay 1(included inmultiplex PCR)	positiveresult	composite result:positive	composite result:positive
	negativeresult	composite results:positive	composite result:negative

Reference Methods – Antibiotic Resistance Markers

PCR assays corresponding to each LRT BAL antibiotic resistance marker assay were included into the multiplex PCR comparator assay as a (single) molecular reference. Positive PCRs were followed by bi-directional sequencing.

Cultured isolates had been collected for positive prospective specimens whenever possible. Isolates were regrown and evaluated by MALDI-TOF to confirm strain identities and by whole genome sequencing using a next generation sequencing (NGS) approach to screen for presence or absence of LRT BAL panel antibiotic resistance markers. A few isolates that failed to grow were evaluated by PCR/bi-directional sequencing from frozen isolate stocks to confirm identity and to screen for presence or absence of such markers.

Phenotypic AST results for positive specimens as reported by SoC culture were collected to correlate antibiotic resistance markers detected by the LRT BAL Application to resistance phenotypes.

Discrepant Result Analysis

False positive discrepant results were analyzed by performing singleplex PCRs/bi-directional sequencing using primer pairs targeting different genetic loci compared to the corresponding LRT BAL assays on specimen DNA extracts for each discrepant LRT BAL analyte.

Microorganism Results - Comparison to SoC Culture Reference, PPA/NPA

For the prospective clinical study cohort, 1,016 previously collected specimens were tested with the LRT BAL Application. For 'typical' microorganisms, LRT BAL results were compared to the SoC culture result as reference (Table 22) to determine true positives (TP), false negatives (FN), false

510(k) Summary - LRT BAL Application K191967 Rev 3.0

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positives (FP) and true negatives (TN), as well as positive percent agreements (PPA, TP / (TP + FN), negative percent agreements (NPA, TN / (TN + FP)), positive values (PPV, TP / (TP + FP)) and negative predictive values (NPV, TN / (TN + FN), calculated together with their two-sided 95% confidence intervals (95% CI), determined according to the Wilson score method.

	TP	FN	FPc, d	TN	PPA [%](95% CI)	NPA [%](95% CI)	PPV [%](95% CI)	NPV [%](95% CI)
Acinetobacter spp.	10	1	11	993	90.9(62.3 - 98.4)	98.9(98.0 - 99.4)	47.6(28.3 - 67.6)	99.9(99.4 - 100.0)
Citrobacter freundii	1	0	3	1,011	100.0(20.7 - 100.0)	99.7(99.1 - 99.9)	25.0(4.6 - 69.9)	100.0(99.6 - 100.0)
Enterobacter cloacaecomplex	13	4 e	7	991	76.5(52.7 - 90.4)	99.3(98.6 - 99.7)	65.0(43.3 - 81.9)	99.6(99.0 - 99.8)
Escherichia coli	17	1	30	968	94.4(74.2 - 99.0)	97.0(95.7 - 97.9)	36.2(24.0 - 50.5)	99.9(99.4 - 100.0)
Haemophilusinfluenzae	8	1	48	958	88.9(56.5 - 98.0)	95.2(93.7 - 96.4)	14.3(7.4 - 25.7)	99.9(99.4 - 100.0)
Klebsiella oxytoca	6	1	8	1,001	85.7(48.7 - 97.4)	99.2(98.4 - 99.6)	42.9(21.4 - 67.4)	99.9(99.4 - 100.0)
Klebsiellapneumoniae b	20	4 f	10	982	83.3(64.1 - 93.3)	99.0(98.2 - 99.5)	66.7(48.8 - 80.8)	99.6(99.0 - 99.8)
Klebsiellavariicola b	0	2	2	1,012	0.0(0.0 - 65.8)	99.8(99.3 - 99.9)	0.0(0.0 - 65.8)	99.8(99.3 - 99.9)
Moraxellacatarrhalis	2	0	13	997	100.0(34.2 - 100.0)	98.7(97.8 - 99.2)	13.3(3.7 - 37.9)	100.0(99.6 - 100.0)
Morganella morganii	0	0	3	1,009	NA	99.7(99.1 - 99.9)	0.0(0.0 - 56.1)	100.0(99.6 - 100.0)
Proteus spp.	4	0	6	1,006	100.0(51.0 - 100.0)	99.4(98.7 - 99.7)	40.0(16.8 - 68.7)	100.0(99.6 - 100.0)
Pseudomonasaeruginosa	69	3	43	900	95.8(88.5 - 98.6)	95.4(93.9 - 96.6)	61.6(52.4 - 70.1)	99.7(99.0 - 99.9)
Serratia marcescens	12	0	5	998	100.0(75.8 - 100.0)	99.5(98.8 - 99.8)	70.6(46.9 - 86.7)	100.0(99.6 - 100.0)
Staphylococcusaureus	63	8	41	904	88.7(79.3 - 94.2)	95.7(94.2 - 96.8)	60.6(51.0 - 69.4)	99.1(98.3 - 99.6)
Stenotrophomonasmaltophilia	19	2	22	972	90.5(71.1 - 97.4)	97.8(96.7 - 98.5)	46.3(32.1 - 61.3)	99.8(99.3 - 99.9)
Streptococcuspneumoniae	3	0	10	1,003	100.0(43.9 - 100.0)	99.0(98.2 - 99.5)	23.1(8.2 - 50.3)	100.0(99.6 - 100.0)

Table 22: Prospective study (N = 1,016 specimens 3), comparison to SoC microbiology culture results.

a Observed invalid LRT BAL analyte results: Acinetobacter spp. (1), E. cloacae complex (1), H. influenzae (1), M. catarrhalis (4), M. morganii (4), P. aeruginosa (1), S. marcescens (1) and S. maltophilia (1).

b K. variicola is typically reported by culture as K. pneumoniae K. variicola from K. pneumoniae strain isolates were sequenced. For a few cases, for which no isolates were provided, sequencing was performed from specimen DNA extracts instead. Two of 26 cases were identified as K. variicola and confirmed by both isolate and DNA extract sequencing. Results for K. pneumoniae and K. variicola performance are calculated based on the species identified by sequencing.

° Note that many of the FP detections are confirmed when comparator reference that includes molecular reference assays (see section 'Comparison to Composite Comparator Reference').

4 Specimens with false positive LRT BAL results were analyzed with molecular assays (PCR/bi-directional sequencing) using specimen DNA extracts for presence of the corresponding microorganism: presence was confirmed in 11 of 11 cases for Acinetobacter sp., 3 of 3 cases for C. freundii, 7 of 7 cases for E. cloaca complex, 18 of 30 cases for H. influenzae, 7 of 8 cases for K. oxytoca, 5 of 10 cases for K. pneumoniae, 2 of 2 cases for M. catarrhalis, 3 of 3 cases for M. morganii, 6 of 6 cases for Proteus spp., 41 of 43 cases for S. marcescens, 31 of 41 cases for S. nureus, 21 of 22 cases for S. maltophilia, and 10 of 10 cases for S. pneumoniae. For cases that were not confirmed for H. influenzae, sequencing

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results identified H. haemolyticus (3x), H. parainfluenzae (1x) and Aggregatibacter aphrophilus (1x). For all other cases, PCRs did not amplify sufficient amounts for sequencing.

6 Two of the four specimens that were FN for E. cloacae complex contained multiple host microorganisms identified by culture, and/or LRT BAL. For one specimen, Acinetobacter spp. was additionally reported by both LRT BAL and culture. For the other specimen, S. maltophilia was additionally reported by LRT BAL only.

I Two of the four specimens that were FN for K. pneumoniae contained multiple host microorganisms identified by culture and/or the molecular reference testing. For one FN specimen, P. aeruginosa was additionally reported by both LRT BAL and culture. For the other specimen, E. cloaca complex was additionally reported by both LRT BAL and culture and S. maltophilia was additionally reported by LRT BAL only.

As no common SoC reference method was performed for the 'atypical' microorganisms reported by the LRT BAL panel, Table 23 lists positive and negative detections by the LRT BAL Application, compared to the subset of cases for which a SoC test was performed on special request by the physician. For C. pneumoniae, only one SoC test with a negative result was performed. For the three other 'atypical' microorganisms, the following PPA/NPA estimates when compared against SoC tests were observed: L. pneumophila PPA 0/1 (0%), NPA 237/237 (100%); M. pneumoniae PPA 0/0, NPA 28/28 (100%); P. jirovecii PPA 5/5 (100%), NPA 99/100 (99%).

	SOC Test(tested on request only)		LRT BAL result
			positive	negative
	not tested	1,015 (99.9%)	0	1,015
Chlamydia pneumoniae	tested	1 (0.1%)	0	1
	positive result	0	0	0
	negative result	1	0	1
	not tested	778 (76.6%)	1	776 a
Legionella pneumophila	tested	238 (23.4%)	0	238
	positive result	1	0	1 b
	negative result	237	0	237
	not tested	988 (97.2%)	6	982
Mycoplasma pneumoniae	tested	28 (2.8%)	0	28
	positive result	0	0	0
	negative result	28	0	28
	not tested	911 (89.7%)	16	895
Pneumocystis jirovecii	tested c	105 (10.3%)	6	99
	positive result	5	5 d	0
	negative result	100	1 e	99

Table 23: Summary of LRT BAL results for 'atypical' microorganisms for the prospective study.

4 Reduced number of available LRT BAL results due to one invalid analyte result.

b L. pneumophila was only reported positive by culture after 13 days.

6 Applied Pneumocystis SoC methods for 105 specimens), IFA (29 specimens), IFA (29 specimens), and PCR (13 specimens).

d Positive by DFA (three specimens) or IFA (two specimens).

e Negative by DFA, confirmed positive by molecular reference tests.

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Microorganisms - Stratification by Qualitative and Quantitative Culture Result

For all prospective specimens with positive culture results, true positive and false negative LRT BAL results were stratified by the semi-quantitative result reported by qualitative SoC culture (categories: rare, few, moderate, numerous) or by quantitative SoC culture (categories: 103 - < 104, 104 - < 105, 105 - < 106, > 106, in CFU/mL) as shown in Table 24.

	Qualitative Culture Result				Quantitative Culture Result
	Category	Total	TP	FN	PPA [%]	Category	Total	FN	PPA [%]
	rare	0	0	0	NA	$10^3$ - $10^4$	1	0	100.0
	few	2	2	0	100.0	$10^4$ - $10^5$	5	0	100.0
Acinetobacter spp.	moderate	1	0	1	0.0	$10^5$ - $10^6$	1	0	100.0
	numerous	1	1	0	100.0	> $10^6$	0	0	NA
	rare	0	0	0	NA	$10^3$ - $10^4$	0	0	NA
	few	1	1	0	100.0	$10^4$ - $10^5$	0	0	NA
Citrobacter freundii	moderate	0	0	0	NA	$10^5$ - $10^6$	0	0	NA
	numerous	0	0	0	NA	> $10^6$	0	0	NA
	rare	1	0	1	0.0	$10^3$ - $10^4$	1	1	0.0
Enterobacter	few	1	1	0	100.0	$10^4$ - $10^5$	6	1	83.3
cloacae complex	moderate	6	5	1	83.3	$10^5$ - $10^6$	1	0	100.0
	numerous	1	1	0	100.0	> $10^6$	0	0	NA
	rare	4	3	1	75.0	$10^3$ - $10^4$	0	0	NA
	few	2	2	0	100.0	$10^4$ - $10^5$	5	0	100.0
Escherichia coli	moderate	1	1	0	100.0	$10^5$ - $10^6$	3	0	100.0
	numerous	3	3	0	100.0	> $10^6$	0	0	NA
	rare	0	0	0	NA	$10^3$ - $10^4$	0	0	NA
Haemophilus	few	0	0	0	NA	$10^4$ - $10^5$	6	1	83.3
influenzae	moderate	2	2	0	100.0	$10^5$ - $10^6$	1	0	100.0
	numerous	0	0	0	NA	> $10^6$	0	0	NA
	rare	0	0	0	NA	$10^3$ - $10^4$	1	0	100.0
	few	2	2	0	100.0	$10^4$ - $10^5$	3	1	66.7
Klebsiella oxytoca	moderate	1	1	0	100.0	$10^5$ - $10^6$	0	0	NA
	numerous	0	0	0	NA	> $10^6$	0	0	NA
	rare	1	0	1	0.0	$10^3$ - $10^4$	4	1	75.0
Klebsiella	few	4	3	1	75.0	$10^4$ - $10^5$	11	0	100.0
pneumoniae	moderate	1	0	1	0.0	$10^5$ - $10^6$	2	0	100.0
	numerous	1	1	0	100.0	> $10^6$	0	0	NA
	rare	0	0	0	NA	$10^3$ - $10^4$	0	0	NA
Klebsiella variicola	few	0	0	0	NA	$10^4$ - $10^5$	1	1	0.0
	moderate	0	0	0	NA	$10^5$ - $10^6$	0	0	NA
	numerous	1	0	1	0.0	> $10^6$	0	0	NA
Moraxella	rare	0	0	0	NA	$10^3$ - $10^4$	0	0	NA
catarrhalis	few	0	0	0	NA	$10^4$ - $10^5$	0	0	NA
	moderate	0	0	0	NA	$10^5$ - $10^6$	0	0	NA
	numerous	2	2	0	100.0	> $10^6$	0	0	NA
	rare	0	0	0	NA	$10^3$ - $10^4$	0	0	NA
Morganella	few	0	0	0	NA	$10^4$ - $10^5$	0	0	NA
morganii	moderate	0	0	0	NA	$10^5$ - $10^6$	0	0	NA
	numerous	0	0	0	NA	> $10^6$	0	0	NA
Proteus spp.	rare	0	0	0	NA	$10^3$ - $10^4$	1	0	100.0
	Qualitative Culture Result				Quantitative Culture Result
	Category	Total	TP	FN	PPA [%]	Category	Total	TP	FN	PPA [%]
	few	0	0	0	NA	104 - < 105	0	0	0	NA
	moderate	3	3	0	100.0	105 - < 106	0	0	0	NA
	numerous	0	0	0	NA	> 106	0	0	0	NA
	rare	7	5	2	71.4	103 - < 104	5	5	0	100.0
Pseudomonas	few	13	12	1	92.3	104 - < 105	19	19	0	100.0
aeruginosa a	moderate	16	16	0	100.0	105 - < 106	4	4	0	100.0
	numerous	7	7	0	100.0	> 106	0	0	0	NA
	rare	0	0	0	NA	103 - < 104	4	4	0	100.0
	few	1	1	0	100.0	104 - < 105	4	4	0	100.0
Serratia marcescens	moderate	2	2	0	100.0	105 - < 106	1	1	0	100.0
	numerous	0	0	0	NA	> 106	0	0	0	NA
	rare	8	4	4	50.0	103 - < 104	5	4	1	80.0
Staphylococcus	few	11	10	1	90.9	104 - < 105	18	17	1	94.4
aureus a	moderate	11	10	1	90.9	105 - < 106	10	10	0	100.0
	numerous	7	7	0	100.0	> 106	0	0	0	NA
	rare	1	1	0	100.0	103 - < 104	1	0	1	0.0
Stenotrophomonas	few	1	1	0	100.0	104 - < 105	6	6	0	100.0
maltophilia	moderate	6	6	0	100.0	105 - < 106	0	0	0	NA
	numerous	6	5	1	83.3	> 106	0	0	0	NA
	rare	0	0	0	NA	103 - < 104	0	0	0	NA
Streptococcus	few	0	0	0	NA	104 - < 105	0	0	0	NA
pneumoniae	moderate	2	2	0	100.0	105 - < 106	1	1	0	100.0
	numerous	0	0	0	NA	> 106	0	0	0	NA

Table 24: Prospective study, comparison to SoC culture, stratification by qualitative or quantitative culture result.

510(k) Summary – LRT BAL Application K191967 Rev 3.0

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↑ One P. aeruginosa and one S. aureus case were reported positive by SoC culture without any qualitative information, and were therefore not included in this table.

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Microorganism Results – Comparison to SoC Culture Reference, Concordance Rates for the Prospective Study

For 212 specimens, at least one LRT BAL panel microorganism was reported by both the LRT BAL Application and SoC; for 21 specimens, a positive SoC result was missed by LRT BAL. For 632 specimens, both the LRT BAL Application and SoC reported a negative result (no growth or normal/mixed flora result). For 151 specimens, the LRT BAL Application reported a positive result while SoC was negative. For 65 of these 151 specimens, SoC reported normal/mixed flora, while for 81 of 151 specimens, no growth was reported (Table 25).

	Positive SoC Result(N = 233)	Negative SoC Results(N = 783)
LRT BALResult	Microorganism(s)Reported	No Growth	Normal/MixedFlora	NA a	Total
any positive(N = 363)	212	81	65	5	151
all negative(N = 653)	21	358	258	16	632

Table 25: Comparison of positive and negative SoC results to LRT BAL.

a Presence or absence of flora not reported.

For 1,016 prospective specimens, the LRT BAL Application reported 363 specimens with at least one LRT BAL panel microorganism. For 113 specimens, multi-detections with two or more LRT BAL panel microorganisms were reported. SoC culture reported 233 specimens with at least one LRT BAL panel microorganism. For 40 specimens, multi-detections with two or more LRT BAL panel microorganisms were reported (Table 26).

Table 26: Numbers of detected LRT BAL panel microorganisms as reported by LRT BAL or SoC.

Number of DetectedMicroorganisms	LRT BAL		SoC
	# specimens	[%]	# specimens	[%]
0	653	64.3	783	77.1
any positive	363	35.7	233	22.9
1	250	24.6	194	19.1
2	78	7.7	31	3.1
3	19	1.9	5	0.5
4	7	0.7	3	0.3
5	7	0.7	0	0.0
6	2	0.2	0	0.0

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Table 27 lists all LRT BAL results compared to SoC culture results stratified into the following categories:

category 1: concordant result for both LRT BAL and SoC

. category 1a: both negative,
category 1b: both positive with a fully concordant result, .

category 2: LRT BAL reported additional microorganisms compared to SoC

category 2a: for specimens with a negative SoC result, ●
category 2b: for specimens with a positive SoC result, .

category 3: LRT BAL reported fewer microorganisms compared to SoC

. category 3a: for specimens with a negative LRT BAL result,
category 3b: for specimens with a positive LRT BAL result, ●

category 4: LRT BAL and SoC report discordant results with different microorganisms

category 4a: LRT BAL and SoC reportings share at least one concordant microorganism, .
category 4b: LRT BAL and SoC results are fully discordant.

When comparing LRT BAL to SoC culture results, an overall concordance rate of 75.9% (632 negative specimens (62.2%), 139 positive specimens (13.7%)) was observed; discrepancies were mostly due to LRT BAL detection of additional microorganisms. For 214 of all specimens (21.1%), LRT BAL reported one or more additional microorganisms compared to SoC. 151 of these specimens (14.9%) were reported fully negative by SoC, while for 63 specimens (6.2%) at least one LRT BAL panel microorganism was reported by SoC. For 25 of all specimens (2.5%), one or more positive SoC results were not reported by LRT BAL. 21 of these specimens (2.1%) were reported fully negative by LRT BAL, while four specimens (0.4%) were reported with at least one positive LRT BAL result. For 0.4% of all specimens, LRT BAL and SoC reported different microorganisms. Two of these specimens (0.2%) shared at least one concordant microorganism result, while for four specimens (0.4%) results were fully discordant.

LRT BAL Result	SoC Result	# Cases	%
Category 1: concordant LRT BAL and SoC results		concordant results:75.9%
Category 1a: both negative		632	62.2
Category 1b: concordant positive results		139	13.7
Acinetobacter spp.	Acinetobacter spp.	4
Enterobacter cloacae complex	Enterobacter cloacae complex	4
Escherichia coli	Escherichia coli	10
Haemophilus influenzae	Haemophilus influenzae	5
Klebsiella pneumoniae	Klebsiella pneumoniae	6
Moraxella catarrhalis	Moraxella catarrhalis	1
Pneumocystis jirovecii	Pneumocystis jirovecii	5
Pseudomonas aeruginosa	Pseudomonas aeruginosa	31
Serratia marcescens	Serratia marcescens	5
Staphylococcus aureus	Staphylococcus aureus	39
Stenotrophomonas maltophilia	Stenotrophomonas maltophilia	8
Streptococcus pneumoniae	Streptococcus pneumoniae	1
LRT BAL Result	SoC Result	# Cases	%
Acinetobacter spp.,Citrobacter freundii	Acinetobacter spp.,Citrobacter freundii	1
Acinetobacter spp.,Stenotrophomonas maltophilia	Acinetobacter spp.,Stenotrophomonas maltophilia	1
Enterobacter cloacae complex,Staphylococcus aureus	Enterobacter cloacae complex,Staphylococcus aureus	1
Escherichia coli,Klebsiella oxytoca	Escherichia coli,Klebsiella oxytoca	1
Klebsiella oxytoca,Staphylococcus aureus	Klebsiella oxytoca,Staphylococcus aureus	1
Klebsiella pneumoniae,Pseudomonas aeruginosa	Klebsiella pneumoniae,Pseudomonas aeruginosa	1
Proteus spp.,Pseudomonas aeruginosa	Proteus spp.,Pseudomonas aeruginosa	1
Pseudomonas aeruginosa,Staphylococcus aureus	Pseudomonas aeruginosa,Staphylococcus aureus	5
Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Pseudomonas aeruginosa,Stenotrophomonas maltophilia	3
Serratia marcescens,Staphylococcus aureus	Serratia marcescens,Staphylococcus aureus	1
Staphylococcus aureus,Stenotrophomonas maltophilia	Staphylococcus aureus,Stenotrophomonas maltophilia	3
Proteus spp.,Pseudomonas aeruginosa,Staphylococcus aureus	Proteus spp.,Pseudomonas aeruginosa,Staphylococcus aureus	1
Category 2: LRT BAL detects additional microorganisms		additional detections:21.1 %
Category 2a: negative SoC result		151	14.9
Acinetobacter spp.	negative	3
Citrobacter freundii	negative	1
Enterobacter cloacae complex	negative	4
Escherichia coli	negative	14
Haemophilus influenzae	negative	24
Klebsiella oxytoca	negative	2
Klebsiella pneumoniae	negative	3
Legionella pneumophila	negative	1
Moraxella catarrhalis	negative	3
Mycoplasma pneumoniae	negative	4
Pneumocystis jirovecii	negative	9
Pseudomonas aeruginosa	negative	27
Staphylococcus aureus	negative	21
Stenotrophomonas maltophilia	negative	2
Streptococcus pneumoniae	negative	6
Enterobacter cloacae complex,Staphylococcus aureus	negative	1
Escherichia coli,Klebsiella pneumoniae	negative	1
Escherichia coli,Serratia marcescens	negative	1
Haemophilus influenzae,Moraxella catarrhalis	negative	3
Haemophilus influenzae,Mycoplasma pneumoniae	negative	1
Haemophilus influenzae,Staphylococcus aureus	negative	1
Haemophilus influenzae,Streptococcus pneumoniae	negative	3
LRT BAL Result	SoC Result	# Cases	%
Proteus spp.
Klebsiella pneumoniae,Staphylococcus aureus	negative	1
Moraxella catarrhalis,Pneumocystis jirovecii	negative	1
Moraxella catarrhalis,Streptococcus pneumoniae	negative	1
Pneumocystis jirovecii,Staphylococcus aureus	negative	1
Pseudomonas aeruginosa,Staphylococcus aureus	negative	6
Staphylococcus aureus,Stenotrophomonas maltophilia	negative	1
Enterobacter cloacae complex,Haemophilus influenzae,Moraxella catarrhalis	negative	1
Escherichia coli,Haemophilus influenzae,Staphylococcus aureus	negative	1
Klebsiella oxytoca,Pseudomonas aeruginosa,Staphylococcus aureus	negative	1
Enterobacter cloacae complex,Haemophilus influenzae,Moraxella catarrhalis,Morganella morganii,Staphylococcus aureus,Stenotrophomonas maltophilia	negative	1
Category 2b: positive result for both LRT BAL and SoC
		63	6.2
Acinetobacter spp.,Klebsiella pneumoniae	Klebsiella pneumoniae	1
Acinetobacter spp.,Pseudomonas aeruginosa	Pseudomonas aeruginosa	1
Enterobacter cloacae complex,Mycoplasma pneumoniae	Enterobacter cloacae complex	1
Escherichia coli,Haemophilus influenzae	Escherichia coli	1
Escherichia coli,Pseudomonas aeruginosa	Pseudomonas aeruginosa	1
Escherichia coli,Serratia marcescens	Escherichia coli	1
Escherichia coli,Staphylococcus aureus	Escherichia coli	1
Escherichia coli,Staphylococcus aureus	Staphylococcus aureus	1
Haemophilus influenzae,Klebsiella oxytoca	Klebsiella oxytoca	1
Haemophilus influenzae,Moraxella catarrhalis	Haemophilus influenzae	1
Haemophilus influenzae,Pseudomonas aeruginosa	Pseudomonas aeruginosa	3
Haemophilus influenzae,Staphylococcus aureus	Haemophilus influenzae	1
Haemophilus influenzae,Staphylococcus aureus	Staphylococcus aureus	2
Klebsiella oxytoca,Klebsiella pneumoniae	Klebsiella pneumoniae	1
Klebsiella oxytoca,	Klebsiella oxytoca	1
LRT BAL Result	SoC Result	# Cases	%
Klebsiella oxytoca,Stenotrophomonas maltophilia	Klebsiella oxytoca	1
Klebsiella pneumoniae,Pseudomonas aeruginosa	Klebsiella pneumoniae	1
Klebsiella pneumoniae,Staphylococcus aureus	Klebsiella pneumoniae	1
Klebsiella pneumoniae,Staphylococcus aureus	Staphylococcus aureus	1
Pneumocystis jirovecii,Pseudomonas aeruginosa	Pseudomonas aeruginosa	1
Pneumocystis jirovecii,Stenotrophomonas maltophilia	Stenotrophomonas maltophilia	2
Proteus spp.,Staphylococcus aureus	Proteus spp.	1
Pseudomonas aeruginosa,Serratia marcescens	Pseudomonas aeruginosa	1
Pseudomonas aeruginosa,Staphylococcus aureus	Pseudomonas aeruginosa	1
Pseudomonas aeruginosa,Staphylococcus aureus	Staphylococcus aureus	2
Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Pseudomonas aeruginosa	3
Staphylococcus aureus,Stenotrophomonas maltophilia	Staphylococcus aureus	1
Acinetobacter spp.,Klebsiella pneumoniae,Staphylococcus aureus	Klebsiella pneumoniae,Staphylococcus aureus	1
Acinetobacter spp.,Klebsiella pneumoniae,Stenotrophomonas maltophilia	Klebsiella pneumoniae	1
Acinetobacter spp.,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Acinetobacter spp.,Pseudomonas aeruginosa	1
Enterobacter cloacae complex,Klebsiella pneumoniae,Moraxella catarrhalis	Enterobacter cloacae complex	1
Enterobacter cloacae complex,Klebsiella pneumoniae,Pseudomonas aeruginosa	Enterobacter cloacae complex	1
Escherichia coli,Morganella morganii,Pseudomonas aeruginosa	Pseudomonas aeruginosa	1
Escherichia coli,Pneumocystis jirovecii,Pseudomonas aeruginosa	Escherichia coli	1
Escherichia coli,Pseudomonas aeruginosa,Serratia marcescens	Pseudomonas aeruginosa,Serratia marcescens	1
Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus	Staphylococcus aureus	1
Haemophilus influenzae,Moraxella catarrhalis,Streptococcus pneumoniae	Moraxella catarrhalis,Streptococcus pneumoniae	1
Klebsiella oxytoca,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Pseudomonas aeruginosa	1
Klebsiella pneumoniae,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Klebsiella pneumoniae,Pseudomonas aeruginosa	1
LRT BAL Result	SoC Result	# Cases	%
Pseudomonas aeruginosa,Serratia marcescens,Stenotrophomonas maltophilia	Pseudomonas aeruginosa	1
Pseudomonas aeruginosa,Serratia marcescens,Stenotrophomonas maltophilia	Pseudomonas aeruginosa,Serratia marcescens	1
Acinetobacter spp.,Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa	Klebsiella pneumoniae,Pseudomonas aeruginosa	1
Acinetobacter spp.,Escherichia coli,Klebsiella pneumoniae,Stenotrophomonas maltophilia	Acinetobacter spp.	1
Acinetobacter spp.,Escherichia coli,Proteus spp.,Pseudomonas aeruginosa	Pseudomonas aeruginosa	1
Enterobacter cloacae complex,Escherichia coli,Klebsiella oxytoca,Stenotrophomonas maltophilia	Enterobacter cloacae complex,Escherichia coli,Klebsiella oxytoca	1
Escherichia coli,Haemophilus influenzae,Pneumocystis jirovecii,Staphylococcus aureus	Staphylococcus aureus	1
Haemophilus influenzae,Proteus spp.,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Pseudomonas aeruginosa	1
Pseudomonas aeruginosa,Serratia marcescens,Staphylococcus aureus,Stenotrophomonas maltophilia	Pseudomonas aeruginosa,Serratia marcescens,Staphylococcus aureus	1
Acinetobacter spp.,Enterobacter cloacae complex,Haemophilus influenzae,Klebsiella pneumoniae,Streptococcus pneumoniae	Enterobacter cloacae complex,Klebsiella pneumoniae,Streptococcus pneumoniae	1
Citrobacter freundii,Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa,Serratia marcescens	Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa,Serratia marcescens	1
Enterobacter cloacae complex,Escherichia coli,Klebsiella oxytoca,Klebsiella variicola,Proteus spp.	Enterobacter cloacae complex	1
Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa,Serratia marcescens,Stenotrophomonas maltophilia	Klebsiella pneumoniae,Pseudomonas aeruginosa,Serratia marcescens,Stenotrophomonas maltophilia	1
Escherichia coli,Morganella morganii,Proteus spp.,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	Pseudomonas aeruginosa	1
Haemophilus influenzae,Klebsiella pneumoniae,	Haemophilus influenzae,Klebsiella pneumoniae,	1
LRT BAL Result	SoC Result	# Cases	%
Klebsiella variicola,Proteus spp.,Staphylococcus aureus	Proteus spp.,Staphylococcus aureus
Haemophilus influenzae,Klebsiella pneumoniae,Moraxella catarrhalis,Pneumocystis jirovecii,Serratia marcescens	Serratia marcescens	1
Acinetobacter spp.,Escherichia coli,Klebsiella pneumoniae,Proteus spp.,Pseudomonas aeruginosa,Staphylococcus aureus	Klebsiella pneumoniae	1
Category 3: LRT BAL detects fewer microorganisms
Category 3a: LRT BAL negative			fewer detections:2.5 %
		21	2.1
negative	Enterobacter cloacae complex	2
negative	Escherichia coli	1
negative	Klebsiella oxytoca	1
negative	Klebsiella pneumoniae	2
negative	Klebsiella variicola	1
negative	Legionella pneumophila	1
negative	Pneumocystis jirovecii	3
negative	Pseudomonas aeruginosa	3
negative	Staphylococcus aureus	5
negative	Stenotrophomonas maltophilia	2
Category 3b: positive result for both LRT BAL and SoC
		4	0.4
Acinetobacter spp.	Acinetobacter spp.,Enterobacter cloacae complex	1
Pseudomonas aeruginosa	Klebsiella pneumoniae,Pseudomonas aeruginosa	1
Stenotrophomonas maltophilia	Haemophilus influenzae,Stenotrophomonas maltophilia	1
Acinetobacter spp.,Enterobacter cloacae complex	Acinetobacter spp.,Enterobacter cloacae complex,Klebsiella variicola	1
Category 4 LRT BAL and SoC detect different microorganisms
Category 4a: partially concordant positive results			discordantmicroorganism results:0.6%
		2	0.2
Citrobacter freundii,Pseudomonas aeruginosa	Pseudomonas aeruginosa,Staphylococcus aureus	1
Enterobacter cloacae complex,Klebsiella oxytoca,Stenotrophomonas maltophilia	Enterobacter cloacae complex,Klebsiella pneumoniae	1
Category 4b: fully discordant results
		4	0.4
Haemophilus influenzae	Staphylococcus aureus	1
Pseudomonas aeruginosa	Acinetobacter spp.	1
Pseudomonas aeruginosa	Staphylococcus aureus	1

Table 27: Comparison of LRT BAL and SoC results, full listing.

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510(k) Summary – LRT BAL Application K191967 Rev 3.0

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Microorganism Results – Comparison to Composite Comparator Reference

LRT BAL results from the prospective clinical study were also compared to a composite comparator reference consisting of SoC culture results combined with a validated molecular test (PCR/sequencing, being part of a multiplex PCR assay). For 'atypical' microorganisms (for which SoC testing was not performed for all specimens), the composite comparator reference consists of two validated molecular tests for each microorganism (PCR/sequencing, also being part of the multiplex PCR assay). Table 28 summarizes comparison results together with their 95% confidence intervals.

	TP	FN	FP	TN	PPA [%](95% CI)	NPA [%](95% CI)	PPV [%](95% CI)	NPV [%](95% CI)
Acinetobacter spp.	16	2	5	992	88.9(67.2 - 96.9)	99.5(98.8 - 99.8)	76.2(54.9 - 89.4)	99.8(99.3 - 99.9)
Chlamydiapneumoniae b	0	0	0	1,016	NA	100.0(99.6 - 100.0)	NA	100.0(99.6 - 100.0)
Citrobacter freundii	1	0	3	1,011	100.0(20.7 - 100.0)	99.7(99.1 - 99.9)	25.0(4.6 - 69.9)	100.0(99.6 - 100.0)
Enterobacter cloacaecomplex	18	4	2	991	81.8(61.5 - 92.7)	99.8(99.3 - 99.9)	90.0(69.9 - 97.2)	99.6(99.0 - 99.8)
Escherichia coli	28	6	19	963	82.4(66.5 - 91.7)	98.1(97.0 - 98.8)	59.6(45.3 - 72.4)	99.4(98.7 - 99.7)
Haemophilusinfluenzae	20	2	36	957	90.9(72.2 - 97.5)	96.4(95.0 - 97.4)	35.7(24.5 - 48.8)	99.8(99.2 - 99.9)
Klebsiella oxytoca	8	2	6	1,000	80.0(49.0 - 94.3)	99.4(98.7 - 99.7)	57.1(32.6 - 78.6)	99.8(99.3 - 99.9)
Klebsiellapneumoniae a, c	21	4	9	981	84.0(65.3 - 93.6)	99.1(98.3 - 99.5)	70.0(52.1 - 83.3)	99.6(99.0 - 99.8)
Klebsiellavariicola a, c	0	2	1	1,012	0.0(0.0 - 65.8)	99.9(99.4 - 100.0)	0.0(0.0 - 79.3)	99.8(99.3 - 99.9)
Legionellapneumophila b	1	0	0	1,014	100.0(20.7 - 100.0)	100.0(99.6 - 100.0)	100.0(20.7 - 100.0)	100.0(99.6 - 100.0)
Moraxellacatarrhalis	12	4	3	993	75.0(50.5 - 89.8)	99.7(99.1 - 99.9)	80.0(54.8 - 93.0)	99.6(99.0 - 99.8)
Morganella morganii	3	0	0	1,009	100.0(43.9 - 100.0)	100.0(99.6 - 100.0)	100.0(43.9 - 100.0)	100.0(99.6 - 100.0)
Mycoplasmapneumoniae b	3	0	3	1,010	100.0(43.9 - 100.0)	99.7(99.1 - 99.9)	50.0(18.8 - 81.2)	100.0(99.6 - 100.0)
Pneumocystisjirovecii b	20	5	2	989	80.0(60.9 - 91.1)	99.8(99.3 - 99.9)	90.9(72.2 - 97.5)	99.5(98.8 - 99.8)
Proteus spp.	10	0	0	1,006	100.0(72.3 - 100.0)	100.0(99.6 - 100.0)	100.0(72.3 - 100.0)	100.0(99.6 - 100.0)
Pseudomonasaeruginosa	100	7	12	896	93.5(87.1 - 96.8)	98.7(97.7 - 99.2)	89.3(82.2 - 93.8)	99.2(98.4 - 99.6)
Serratia marcescens	15	1	2	997	93.8(71.7 - 98.9)	99.8(99.3 - 99.9)	88.2(65.7 - 96.7)	99.9(99.4 - 100.0)
Staphylococcusaureus	80	16	24	896	83.3(74.6 - 89.5)	97.4(96.1 - 98.2)	76.9(68.0 - 84.0)	98.2(97.2 - 98.9)
Stenotrophomonasmaltophilia	34	7	7	967	82.9(68.7 - 91.5)	99.3(98.5 - 99.7)	82.9(68.7 - 91.5)	99.3(98.5 - 99.7)
Streptococcuspneumoniae	11	0	2	1,003	100.0(74.1 - 100.0)	99.8(99.3 - 99.9)	84.6(57.8 - 95.7)	100.0(99.6 - 100.0)

Table 28: Prospective study (N = 1,016 specimens 4), comparison to composite comparator.

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a Observed invalid LRT BAL analyte results: Acinetobacter spp. (1), C. freundii (1), E. cloacae complex (1), M. influenzae (1), M. catarrhalis (4), M. morganii (4), P. aeruginosa (1) and S. maltophilia (1). For one SoC negative specimen, K. pneumoniae and K. variicola were simultaneously reported by the composite comparator, but were negative by SoC culture. As it was not feasible to resolve whether these analytes exceeded the molecular reporting threshold, both were set to 'invalid'. For this specimen, LRT BAL had reported a positive result for K. variicola and a negative result for K. pneumoniae.

b 'Atypical' microorganisms C. pneumoniae, L. pneumoniae, and P. jirovecii were compared to two independent molecular tests (PCR/bi-directional sequencing) as composite comparator.

C K. variicola is typically reported by culture as K. pneumoniae. To discriminate K. variicola from K. pneumoniae, provided K. pneumoniae strain isolates were sequenced. For a few cases, no isolate was provided and sequencing was performed from specimen DNA extracts instead. Two of 27 cases were identified as K. variicola and confirmed by both isolate and DNA extract sequencing. Results for K. pneumoniae and K. variicola performance are calculated based on the species identified by sequencing.

d For two of five FN P. jirovecii cases, repeat indicating specimen degradation during prolonged storage as likely reason for observed failures.

Antibiotic Resistance Marker Results – Comparison to Molecular Reference

Performance characteristics of LRT BAL panel antibiotic resistance markers were evaluated for the prospective study (1,016 specimens) by comparing to corresponding molecular reference assays to determine PPAs (TP / (TP + FN)) and NPAs (FN / (FN + FP)) together with 95% confidence intervals. For each antibiotic resistance marker, one PCR assay was performed followed by bi-directional sequencing.

Antibiotic resistance marker results are only reported by the LRT BAL Application if at least one corresponding host microorganism is simultaneously detected. Results for antibiotic resistance markers detected without a positive corresponding host microorganism result are masked on the results screen. Table 29 reports antibiotic resistance markers as displayed on the LRT BAL results screen, i.e., all antibiotic resistance markers for which a corresponding host microorganism was simultaneously detected. Note that PPA/NPA performance data are driven by LRT BAL microorganism results.

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Table 29: Comparison of antibiotic resistance markers to corresponding molecular reference assays (PCR/sequencing) for the prospective study (N=1,016 specimens) for markers as displayed and reported on the LRT BAL results screen (number of detected host microorganisms for each marker in brackets).

	TP	FN	FP c, d	TN	PPA [%](95% CI)	NPA [%](95% CI)
ctx-M(N = 208)	8	1 b	1	197	88.9(56.5 - 98.0)	99.5(97.2 - 99.9)
kpc(N = 208)	3	0	1	204	100.0(43.9 - 100.0)	99.5(97.3 - 99.9)
mecA(N = 104)	22	0	25 e	57	100.0(85.1 - 100.0)	69.5(58.9 - 78.4)
ndm(N = 208)	1	0	0	207	100.0(20.7 - 100.0)	100.0(98.2 - 100.0)
oxa-23(N = 21)	3	0	0	18	100.0(43.9 - 100.0)	100.0(82.4 - 100.0)
oxa-24(N = 21)	3	0	1	16	100.0(43.9 - 100.0)	94.1(73.0 - 99.0)
oxa-48(N = 112)	1	0	0	111	100.0(20.7 - 100.0)	100.0(96.7 - 100.0)
oxa-58(N = 21)	0	0	0	21	NA	100.0(84.5 - 100.0)
tem(N = 56)	9	0	7 f	40	100.0(70.1 - 100.0)	85.1(72.3 - 92.6)
vim(N = 208)	0	0	1	207	NA	99.5(97.3 - 99.9)

a Observed invalid LRT BAL analyte results: ctx-M (1) and oxa-24 (1).

b One false negative result belongs to the cx-M9 subgroup that is not covered by the LRT BAL Application (targeted against ctx-M1 subgroup).

6 Note that molecular reference assays use cutoffs to achieve analytical sensitivities in a similar range to LRT BAL. Negative results by molecular reference assays do not presence of the antibiotic resistance marker in the specimen at a low concentration, nor its possible clinical relevance. Please refer to section of Detected Antibiotic Resistance Markers to Strain Genotypes and Phenotypes', p. 68 for genotypic and phenotypic correlation of reported antibiotic resistance markers.

d Specimens with false positive LRT BAL results were analyzed with additional molecular assays (PCR/bi-directional sequencing) using specimen DNA extracts for presence of antibiotic resistance markers. Presence of antibiotic resistance markers was confirmed in 0 of 1 case for ctx-M, 1 of 1 case of kpc, 10 of 25 cases for mecA, 6 of 7 cases for tem and 1 of 1 case for vin. Cross-reactivity to other off-panel analytes has not been observed. For all not amplify sufficient amounts for sequencing.

6 Note that Staphylococci other than S. aureus commonly harbor mecA results can, therefore, be due to the presence of this marker in such microorganisms.

4 Although tem is reported by LRT BAL for H. influenzae only, other Gram-negative microorganisms (e.g., Enterobacteriaceae, Acinetobacter spp., P. aeruginosa) can harbor tem results can, therefore, be due to the presence of this marker in such microorganisms.

Antibiotic Resistance Marker Results - Agreement Rates to Positive and Negative Microorganisms as Detected by Culture/Isolate Sequencing or Composite Comparator Testing

In order to determine agreement rates of antibiotic resistance markers reported by LRT BAL to positive and negative reference results, a comparison to the following references was performed: (A) SoC culture result for microorganisms, combined with sequencing results/marker screenings of provided strain isolates, and (B) composite comparator result for microorganisms and molecular reference results (PCR/sequencing) for antibiotic resistance markers. Note that an isolate was not provided for all SoC positive microorganisms, thereby limiting the dataset for comparison (A). Comparison (B) evaluates the entire specimen cohort, including all positive LRT BAL results that were negative by 510(k) Summary - LRT BAL Application K191967 Rev 3.0 Page 52 of 87

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SoC culture. Molecular reference assays use cutoffs that were set to achieve an analytical assay sensitivity in a comparable range to LRT BAL.

For both comparisons, the following agreement rates are evaluated for ctx-M, kpc, ndm, and vim (for Enterobacteriaceae, Acinetobacter spp. and/or P. aeruginosa as possible host microorganisms), for oxa-48 (for Enterobacteriaceae), for tem (for H. influenzae), and for mecA (for S. aureus) in Tables 30 - 38:

1. Agreement of one (or more) detected host microorganisms in combination with an antibiotic resistance marker (Org+/Res+) between LRT BAL and reference methods (A) or (B).
1. Agreement of one (or more) detected host microorganisms without reported antibiotic resistance marker (Org+/Res-) between LRT BAL and reference methods (A) or (B).
1. Agreement of a negative result for one (or more) host microorganisms (Org-) between LRT BAL and reference methods (A) and (B).

As presence of mecA is expected to directly correlate with cefoxitin/oxacillin resistance in S. aureus strains (MRSA, methicillin-resistant S. aureus), while the absence of mecA is expected to correlate with susceptible S. aureus strains (MSSA, methicillin-sensitive S. aureus), mecA was furthermore compared to the MRSA/MSSA status of the isolate as reported by SoC culture as a third reference (C) in Table 38:

1. Agreement of a detected S. aureus in combination with a detected mecA as reported by LRT BAL (Org+/Res+) to S. aureus reported with an MRSA phenotype by SoC culture (Org+/MRSA).
1. Agreement of a detected S. aureus together with a negative result for mecA (Org+/Res-) as reported by LRT BAL to S. aureus reported with an MSSA phenotype by SoC culture (Org+/MSSA).
1. Agreement of a negative result for S. aureus between LRT BAL and SoC culture.

No agreement tables are included for oxa-58 for which positive results were reported neither by the LRT BAL Application, nor by the reference methods.

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Table 30: Agreement rates for oxa-23 between LRT BAL and reference methods A (culture) and B (composite comparator) for Acinetobacter spp. as host microorganism.

A		Culture Result for Acinetobacter spp. +Isolate Sequencing/Marker Screening for oxa-23
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	1	0	1	1	3
	Org+/Res-	0	7	1	10	18
	Org-	0	0	1	993	994
	Total	1	7	3	1,004	1,015 a
Acinetobacter spp./oxa-23		rate [%]	positivity		95% CI
Agreement (Org+/Res+)		100.0	1/1		20.7 - 100.0
Agreement (Org+/Res-)		100.0	7/7		64.6 - 100.0
Agreement (Org-)		98.9	993/1,004		98.0 - 99.4

B	Acinetobacter spp.oxa-23			Composite Comparator Result for Acinetobacter spp. +Mol. Reference (PCR/Seq.) for oxa-23
		Org+/Res+	Org+/Res-	Org-	Total
LRT BALResult	Org+/Res+	3	0	0	3
	Org+/Res-	0	13	5	18
	Org-	0	2	992	994
	Total	3	15	997	1,015 a
		Acinetobacter spp./oxa-23	rate [%]	positivity	95% CI
		Agreement (Org+/Res+)	100.0	3/3	43.9 - 100.0
		Agreement (Org+/Res-)	86.7	13/15	62.1 - 96.3
		Agreement (Org-)	99.5	992/997	98.8 - 99.8

ª One invalid LRT BAL result for Acinetobacter spp.

{58}------------------------------------------------

Table 31: Agreement rates for oxa-24 between LRT BAL and reference methods A (culture) and B (composite comparator) for Acinetobacter spp. as host microorganism.

A	Culture Result for Acinetobacter spp. +Isolate Sequencing/Marker Screening for oxa-24
	Acinetobacter spp.oxa-24	Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	3	0	0	1	4
	Org+/Res-	0	4	2	10	16
	Org-	0	0	1	993	994
Total		3	4	3	1,004	1,014 a
Acinetobacter spp./oxa-24		rate [%]		positivity	95% CI
Agreement (Org+/Res+)		100.0		3/3	43.9 - 100.0
Agreement (Org+/Res-)		100.0		4/4	51.0 - 100.0
Agreement (Org-)		98.9		993/1,004	98.0 - 99.4

B	Acinetobacter spp.oxa-24	Composite Comparator Result for Acinetobacter spp. +Mol. Reference (PCR/Seq.) for oxa-24
		Org+/Res+	Org+/Res-	Org-	Total
	Org+/Res+	3	0	1	4
LRT BALResult	Org+/Res-	0	12	4	16
	Org-	1	1	992	994
	Total	4	13	997	1,014 a
	Acinetobacter spp./oxa-24		rate [%]	positivity	95% CI
	Agreement (Org+/Res+)		75.0	3/4	30.1 - 95.4
	Agreement (Org+/Res-)		92.3	12/13	66.7 - 98.6
	Agreement (Org-)		99.5	992/997	98.8 - 99.8

ª One invalid LRT BAL result for Acinetobacter spp. and one for oxa-24.

{59}------------------------------------------------

Table 32: Agreement rates for ctx-M between LRT BAL and reference methods A (culture) and B (composite comparator) for combined possible host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

A	Combined Hostsctx-M	Culture Result for Host Microorganism(s) +Isolate Sequencing/Marker Screening for ctx-M
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	3 d	4b, e	1b, f	1	9
	Org+/Res-	0	92	32	74	198
	Org-	0	3	8	794	805
	Total	3 a	99	41	869	1,012c
	Combined Hosts/ ctx-M		rate [%]	positivity	95% CI
	Agreement (Org+/Res+)		100.0	3/3	43.9 - 100.0
	Agreement (Org+/Res-)		92.9	92/99	86.1 - 96.5
	Agreement (Org-)		91.4	794/869	89.3 - 93.1

Combined Hostsctx-M		Composite Comparator Result for Host Microorganism(s) +Mol. Reference (PCR/Seq.) for ctx-M
		Org+/Res+	Org+/Res-	Org-	total
LRT BALResult	Org+/Res+	8	1	0	9
	Org+/Res-	1	161	36	198
	Org-	0	20	785	805
	total	9 a	182	821	1,012 c
Combined Hosts/ctx-M		rate [%]	positivity	95% CI
Agreement (Org+/Res+)		88.9	8/9	56.5 - 98.0
Agreement (Org+/Res-)		88.5	161/182	83.0 - 92.3
Agreement (Org-)		95.6	785/821	94.0 - 96.8

a Specimens with multiple detected possible host microorganisms by culture: 1 of 3, by the composite comparator: 6 of 9.

b For one 'Org+/Res-' and for one 'Org+/No Isolate' case, presence of the ctx-M gene was confirmed for an off-panel isolate of Providencia stuartii.

6 Four invalid LRT BAL ctx-M results.

d SoC reported the following possible corresponding host microorganisms for three Org+/Res+ cases: E. coli, K. pneumoniae, K. pneumoniae/P. aeruginosa.

& SoC reported the following possible corresponding host microorganisms for four Org+Res- cases: E. coli, P. aeruginosa (2x), K. pneumoniae/P. aeruginosa/S. marcescens.

f SoC reported the following possible corresponding host microorganisms for one Org+/No Isolate case: K. pneumoniae.

{60}------------------------------------------------

Table 33: Agreement rates for kpc between LRT BAL and reference methods A (culture) and B (composite comparator) for combined possible host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

A	Combined Hostskpc	Culture Result for Host Microorganism(s) +Isolate Sequencing/Marker Screening for kpc
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	1 b	2 c	1 d	0	4
	Org+/Res-	0	96	32	76	204
	Org-	0	3	8	797	808
	Total	1 a	101	41	873	1,016
Combined Hosts/ kpc		rate [%]		positivity	95% CI
Agreement (Org+/Res+)		100.0		1/1	20.7 - 100.0
Agreement (Org+/Res-)		95.0		96/101	88.9 - 97.9
Agreement (Org-)		91.3		797/873	89.2 - 93.0

B	Combined Hostskpc		Composite Comparator Result for Host Microorganism(s) +Mol. Reference (PCR/Seq.) for kpc
			Org+/Res+	Org+/Res-	Org-	Total
LRT BALResult	kpc	Org+/Res+	3	1	0	4
		Org+/Res-	0	167	37	204
		Org-	0	20	788	808
		Total	3 a	188	825	1,016
	Combined Hosts/ kpc		rate [%]	positivity	95% CI
	Agreement (Org+/Res+)		100.0	3/3	43.9 - 100.0
	Agreement (Org+/Res-)		88.8	167/188	83.5 - 92.6
	Agreement (Org-)		95.5	788/825	93.9 - 96.7

a Specimens with multiple detected possible host microorganisms by culture: 0 of 1, by the composite comparator: 2 of 3.

b SoC reported the following possible corresponding host microorganisms for one Org+/Res+ case: E. cloacae complex.

SoC reported the following possible corresponding host microorganisms for two Org+/Res- cases: Acinetobacter spp., K. pneumoniae/P. aeruginosa.

d SoC reported the following possible corresponding host microorganisms for one Org+/No Isolate case: K. pneumoniae.

{61}------------------------------------------------

Table 34: Agreement rates for ndm between LRT BAL and reference methods A (culture) and B (composite comparator) for combined possible host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

A	Combined Hostsndm		Culture Result for Host Microorganism(s) +Isolate Sequencing/Marker Screening for ndm
		Org+/Res+	Org+/Res-	Org+/-No Isolate	Org-	Total
LRT BALResult	Org+/Res+	1 c	0	0	0	1
	Org+/Res-	0	98	33	76	207
	Org-	0	3	8	796	807
	Total	1 a	101	41	872	1,015 b
Combined Hosts/ ndm		rate [%]		positivity	95% CI
Agreement (Org+/Res+)		100.0		1/1	20.7 - 100.0
Agreement (Org+/Res-)		97.0		98/101	91.6 - 99.0
Agreement (Org-)		91.3		796/872	89.2 - 93.0

B	Combined Hostsndm		Composite Comparator Result for Host Microorganism(s) +Mol. Reference (PCR/Seq.) for ndm
			Org+/Res+	Org+/Res-	Org-	Total
LRT BALResult		Org+/Res+	1	0	0	1
		Org+/Res-	0	170	37	207
		Org-	0	20	787	807
	Total		1 a	190	824	1,015 b
	Combined Hosts/ ndm		rate [%]	positivity	95% CI
	Agreement (Org+/Res+)		100.0	1/1	20.7 - 100.0
	Agreement (Org+/Res-)		89.5	170/190	84.3 - 93.1
	Agreement (Org-)		95.5	787/824	93.9 - 96.7

ª Specimens with multiple detected possible host microorganisms by culture: 1 of 1, by the composite comparator: 1 of 1.

b One invalid LRT BAL result for ndm.

· SoC reported the following possible corresponding host microorganisms for one Org+Res+ case: K. pneumoniae/P. aeruginosa.

{62}------------------------------------------------

Table 35: Agreement rates for vim between LRT BAL and reference methods A (culture) and B (composite comparator) for combined possible host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

A	Combined Hostsvim	Culture Result for Host Microorganism(s) +Isolate Sequencing/Marker Screening for vim
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	0	1 a	0	0	1
	Org+/Res-	0	98	33	76	207
	Org-	0	3	8	797	808
	Total	0	102	41	873	1,016
Combined Hosts/ vim			rate [%]	positivity	95% CI
Agreement (Org+/Res+)			NA	0/0	NA
Agreement (Org+/Res-)			96.1	98/102	90.3 - 98.5
Agreement (Org-)			91.3	797/873	89.2 - 93.0

B	Combined Hostsvim	Org+/Res+	Org+/Res-	Org-	Total
LRT BALResult	Org+/Res+	0	1	0	1
	Org+/Res-	0	170	37	207
	Org-	0	20	788	808
	Total	0	191	825	1,016
	Combined Hosts/ vim	rate [%]	positivity	95% CI
	Agreement (Org+/Res+)	NA	0/0	NA
	Agreement (Org+/Res-)	89.0	170/191	83.8 - 92.7
	Agreement (Org-)	95.5	788/825	93.9 - 96.7

a SoC reported the following possible corresponding host microorganisms for one Org+/Res- case: P. aeruginosa.

{63}------------------------------------------------

Table 36: Agreement rates for oxa-48 between LRT BAL and reference methods A (culture) and B (composite comparator) for combined possible host microorganisms: Enterobacteriaceae.

A	Combined Hostsoxa-48	Culture Result for Host Microorganism(s) +Isolate Sequencing/Marker Screening for oxa-48
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BAL Result	Org+/Res+	1c	0	0	0	1
	Org+/Res-	0	51	13	47	111
	Org-	0	4	6	890	900
	Total	1a	55	19	937	1,012b
Combined Hosts/ oxa-48		rate [%]		positivity	95% CI
Agreement (Org+/Res+)		100.0		1/1	20.7 - 100.0
Agreement (Org+/Res-)		92.7		51/55	82.7 - 97.1
Agreement (Org-)		95.0		890/937	93.4 - 96.2

Combined Hostsoxa-48		Composite Comparator Result for Host Microorganism(s) +Mol. Reference (PCR/Seq.) for oxa-48
	Org+/Res+	Org+/Res-	Org-	Total
LRT BAL	Org+/Res+	1	0	0	1
Result	Org+/Res-	0	84	27	111
	Org-	0	14	886	900
	Total	1 a	98	913	1,012 b
Combined Hosts/ oxa-48			rate [%]	positivity	95% CI
Agreement (Org+/Res+)			100.0	1/1	20.7 - 100.0
Agreement (Org+/Res-)			85.7	84/98	77.4 - 91.3
Agreement (Org-)			97.0	886/913	95.7 - 98.0

ª Specimens with multiple detected possible host microorganisms by culture: 0 of 1, by the composite comparator: 0 of 1. b Four invalid LRT BAL results for oxa-48.

SoC reported the following possible corresponding host microorganisms for one one Org+/Res+ case: K. pneumoniae/P. aeruginosa.

{64}------------------------------------------------

Table 37: Agreement rates for tem between LRT BAL and reference methods A (culture) and B (composite comparator) for H. influenzae as host microorganism.

A	H. influenzaetem	Culture Result for H. influenzae +Isolate Sequencing/Marker Screening for tem
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	2	0	1	13	16
	Org+/Res-	0	3	2	35	40
	Org-	0	0	1	958	959
	Total	2	3	4	1,006	1,015 a
H. influenzae/tem		rate [%]	positivity	95% CI
Agreement (Org+/Res+)		100.0	2/2	34.2 - 100.0
Agreement (Org+/Res-)		100.0	3/3	43.9 - 100.0
Agreement (Org-)		95.2	958/1,006	93.7 - 96.4

B	H. influenzaetem	Composite Comparator Result for H. influenzae +Mol. Reference (PCR/Seq.) for tem
		Org+/Res+	Org+/Res-	Org-	Total
LRT BALResult	Org+/Res+	6	0	10	16
	Org+/Res-	0	14	26	40
	Org-	0	2	957	959
	Total	6	16	993	1,015 a
	H. influenzae/tem	rate [%]	positivity	95% CI
	Agreement (Org+/Res+)	100.0	6/6	61.0 - 100.0
	Agreement (Org+/Res-)	87.5	14/16	64.0 - 96.5
	Agreement (Org-)	96.4	957/993	95.0 - 97.4

ª One LRT BAL run with an invalid result for both H. influenzae and tem.

{65}------------------------------------------------

Table 38: Agreement rates for mecA between LRT BAL and reference methods A (culture) and B (composite comparator) and C (phenotypic resistance as reported by AST for the culture isolate) for S. aureus as host microorganism.

A	S. aureusmecA	Culture Result for S. aureus +Isolate Sequencing/Marker Screening for mecA
		Org+/Res+	Org+/Res-	Org+/No Isolate	Org-	Total
LRT BALResult	Org+/Res+	17	4	9	17	47
	Org+/Res-	1	23	9	24	57
	Org-	3	2	3	904	912
	Total	21	29	21	945	1,016
	S. aureus/mecA		rate [%]		positivity	95% CI
	Agreement (Org+/Res+)			81.0	17/21	60.0 - 92.3
	Agreement (Org+/Res-)			79.3	23/29	61.6 - 90.2
	Agreement (Org-)			95.7	904/945	94.2 - 96.8

B	S. aureusmecA	Composite Comparator Result for S. aureus +Mol. Reference (PCR/Seq.) for mecA
		Org+/Res+	Org+/Res-	Org-	Total
LRT BALResult	Org+/Res+	20	18 a	9	47
	Org+/Res-	0	42	15	57
	Org-	5	11	896	912
	Total	25	71	920	1,016
	S. aureus/mecA		rate [%]	positivity	95% CI
	Agreement (Org+/Res+)		80.0	20/25	60.9 - 91.1
	Agreement (Org+/Res-)		59.2	42/71	47.5 - 69.8
	Agreement (Org-)		97.4	896/920	96.1 - 98.2

	S. aureusmecA	Culture Result for S. aureus +and Cefoxitin/Oxacillin AST Results
		Org+/MRSA	Org+/MSSA	Org+/No AST	Org-	Total
LRT BALResult	Org+/Res+	23 b	6	1	17	47
	Org+/Res-	1	27	5	24	57
	Org-	3	4	1	904	912
	Total	27	37	7	945	1,016
S. aureus/MRSA status		rate [%]		positivity	95% CI
Agreement (Org+/Res+)		85.1		23/27	67.5 - 94.1
Agreement (Org+/Res-)		73.0		27/37	57.0 - 84.6
Agreement (Org-)		95.7		904/945	94.2 - 96.8

4 18 cases were determined as Org+/Res- negative by the molecular reference assay using cutoffs set to achieve an analytical sensitivity in a comparable range to LRT BAL. For 10 of these 18 cases, presence of mecA was confirmed by alternate molecular tests (PCR/sequencing).

b One strain was reported by the clinical site as MRSA, although this was not supported by the provided oxacillin Kirby-Bauer zone diameter. Independent cefoxitin AST confirmed the MRSA phenotype for this strain.

{66}------------------------------------------------

B. Archived Study

Specimen Cohort: Inclusion/Exclusion Criteria, Demographics

Lavage specimens from patients, age 18 or older, suspected of a lower respiratory infection, with at least one LRT BAL panel microorganism reported positive by standard-of-care (SoC) were eligible for the archived study. Specimens were collected at 11 US clinical sites between 2015 and 2019, complemented with few specimens collected from other sites. In contrast to the prospective study, where specimens were only from hospitalized patients), specimens from outpatients or from patients with an unknown status were also accepted. Confirmatory testing (PCR/bi-directional sequencing using two independent PCR assays per analyte) to exclude specimens that may have degraded during storage was performed for the reported SoC results prior to the study. Only specimens with at least one confirmed SoC result were included.

The archived cohort included 197 specimens from a previous study. These specimens were supplemented with 195 specimens meeting inclusion criteria that had been collected over time to achieve a total set of 392 archived study specimens.

Demographic information for specimens included into the LRT BAL archived study is described in Table 39.

Included Specimens	392
Sex a
male	198
female	122
Age b
18 - 30 years	18
31 - 60 years	106
> 60 years	190
Clinical Setting c
ICU	112
ward	101
in-patient (not specified)	69
out-patient	32

Table 39: Demographic patient data for the LRT BAL archived study.

a Not reported: 72 patients.

b Not reported: 78 patients.

6 Not reported: 78 patients.

{67}------------------------------------------------

Reference Methods

For all archived specimens, LRT BAL Application microorganism results were compared to confirmed positive results from SoC microbiology culture (for 'typical' microorganism) or other SoC methods (for 'atypical' microorganisms) as reference. SoC culture results were reported either qualitatively or quantitatively. For quantitative cultures, a reporting threshold of 102 CFU/mL or higher for mini-BAL specimens and of 10ª CFU/mL or higher for BAL specimens was applied, following recommendations by the Infectious Diseases Society of America (IDSA) and the American Thoracic Society (ATS).

Evaluation of collected isolates and AST results, as well as discrepant result resolution was performed as for the prospective study.

Microorganism Results – Comparison to confirmed SoC reference, PPA/NPA

LRT BAL Application results were compared to confirmed SoC results as reference (Tables 40 and 41) to determine true positives (TP), false negatives (FP) and true negatives (FP) and true negatives (TN) and to calculate positive percent agreements (PPA, TP / (TP + FN)) and negative percent agreements (NPA, TN / (TN + FP)), together with their two-sided 95% confidence intervals (95% CI), determined according to the Wilson score method.

Note that SoC testing for 'atypical' microorganisms was only performed on special request by the physician. Other specimens besides the ones collected because of a positive SoC result for any of the 'atypical' microorganisms, were typically not tested, and, therefore, the NPA cannot be determined.

	TP	FN	FP b	TN	PPA [%](95% CI)	NPA [%](95% CI)
Acinetobacter spp.	18	0	3	371	100.0(82.4 - 100.0)	99.2(97.7 - 99.7)
Citrobacter freundii	5	0	5	382	100.0(56.6 - 100.0)	98.7(97.0 - 99.4)
Enterobacter cloacaecomplex	15	4 c	0	373	78.9(56.7 - 91.5)	100.0(99.0 - 100.0)
Escherichia coli	46	3	17	326	93.9(83.5 - 97.9)	95.0(92.2 - 96.9)
Haemophilusinfluenzae	50	0	21	321	100.0(92.9 - 100.0)	93.9(90.8 - 95.9)
Klebsiella oxytoca	16	1	6	369	94.1(73.0 - 99.0)	98.4(96.6 - 99.3)
Klebsiellapneumoniae a	29	2	10	351	93.5(79.3 - 98.2)	97.2(95.0 - 98.5)
Klebsiellavariicola a	2	0	4	386	100.0(34.2 - 100.0)	99.0(97.4 - 99.6)
Moraxellacatarrhalis	21	0	9	362	100.0(84.5 - 100.0)	97.6(95.5 - 98.7)
Morganella morganii	1	0	0	391	100.0(20.7 - 100.0)	100.0(99.0 - 100.0)
Proteus spp.	15	0	7	370	100.0(79.6 - 100.0)	98.1(96.2 - 99.1)

Table 40: Archived study (N = 392), comparison to confirmed SoC results, 'typical' microorganisms.

510(k) Summary - LRT BAL Application K191967 Rev 3.0

{68}------------------------------------------------

Pseudomonasaeruginosa	54	2	2	334	96.4(87.9 - 99.0)	99.4(97.9 - 99.8)
Serratia marcescens	23	2	3	364	92.0(75.0 - 97.8)	99.2(97.6 - 99.7)
Staphylococcusaureus	56	2	21	313	96.6(88.3 - 99.1)	93.7(90.6 - 95.9)
Stenotrophomonasmaltophilia	37	3	10	342	92.5(80.1 - 97.4)	97.2(94.9 - 98.4)
Streptococcuspneumoniae	34	1	5	352	97.1(85.5 - 99.5)	98.6(96.8 - 99.4)

ª K. variicola is typically reported by culture as K. pneumoniae K. variicola from K. pneumoniae, K. pneumoniae strain isolates, if available, were sequenced. If no isolate was provided, sequencing was performed from specimen instead. Results for K. pneumoniae and K. variicola performance are calculated based on the species identified by sequencing.

6 Specimens with false positive LRT BAL results were analyzed with molecular assays (PCR bi-directional sequencing) using specimen DNA extracts for presence or absence of microorganisms was confirmed in 3 of 3 cases for Acinetobacter spp., 4 of 5 cases for C. freundii (one confirmed case was reported as C. youngae by SoC), 15 of 17 cases for H. influenzae, 6 of 6 cases for K. axytoca, 7 of 10 cases for K. variicola, 8 of 9 cases for M. catarrhalis, 6 of 7 cases for Proteus spp., 2 of 2 cases for P. aeruginosa, 1 of 3 cases for S. marcescens, 18 of 10 cases for S. maltophilia, and 4 of 5 cases for S. pneumoniae. For confirmed for H. influenzae, sequencing results identified H. haemolyticus (2x). A. aphropilus (2x). For all other cases, PCRs did not amounts for sequencing.

6 Three of the four specimens that were FN for E. cloacae complex contains identified by culture, and or LRT BAL. For one specimen. Klebsiella oxytoca was additionally reported by both LRT BAL and culture. For the two other specimens, Proteus spp. or E. coli, respectively, were additionally reported by LRT BAL only.

Table 41: Archived study (N = 392), comparison to confirmed SoC results, 'atypical' microorganisms.
-----------------------------------------------------------------------------------------------------	--	--	--

	TP	FN	add.apos.	add.neg.	PPA [%](95% CI)
Chlamydia pneumoniae	0	0	0	392	NA
Legionella pneumophila	17	2	0	373	89.5(68.6 - 97.1)
Mycoplasma pneumoniae	5	1	3	383	83.3(43.7 - 97.0)
Pneumocystis jirovecii	16	3	13	360	84.2(62.4 - 94.5)

4 Specimens with additional positive ('add. pos.') LRT BAL results were analyzed with molecular assays (PCR/b-directional sequencing) using specimen DNA extracts for presence of microorganisms: presence was confirmed in 2 of 3 cases for M. pneumoniae, and 10 of 13 cases for P. jirovecii. For all other cases, PCRs did not amplify sufficient amounts for sequencing.

For all archived specimens, true positive and false negative LRT BAL results were stratified by the semi-quantitative result reported by qualitative SoC culture (categories: rare, few, moderate, numerous) or by quantitative SoC culture (categories: 103 - < 105, 105 - < 105, 105 - < 106, > 106, in CFU/mL) as shown in Table 42.

{69}------------------------------------------------

	Qualitative Culture Result a					Quantitative Culture Result a
	Category	Total	TP	FN	PPA [%]		Category	Total	TP	FN	PPA [%]
	rare	0	0	0	NA		103 - < 104	3	3	0	100.0
	few	3	3	0	100.0		104 - < 105	4	4	0	100.0
Acinetobacter spp.	moderate	2	2	0	100.0		105 - < 106	2	2	0	100.0
	numerous	3	3	0	100.0		> 106	0	0	0	NA
	rare	0	0	0	NA		103 - < 104	0	0	0	NA
	few	0	0	0	NA		104 - < 105	3	3	0	100.0
Citrobacter freundii	moderate	1	1	0	100.0		105 - < 106	1	1	0	100.0
	numerous	0	0	0	NA		> 106	0	0	0	NA
	rare	0	0	0	NA		103 - < 104	1	0	1	0.0
Enterobacter	few	2	2	0	100.0		104 - < 105	2	1	1	50.0
cloacae complex	moderate	3	2	1	66.7		105 - < 106	1	1	0	100.0
	numerous	6	6	0	100.0		> 106	0	0	0	NA
	rare	7	6	1	85.7		103 - < 104	0	0	0	NA
	few	12	12	0	100.0		104 - < 105	4	4	0	100.0
Escherichia coli	moderate	6	6	0	100.0		105 - < 106	3	3	0	100.0
	numerous	10	10	0	100.0		> 106	0	0	0	NA
	rare	1	1	0	100.0		103 - < 104	0	0	0	NA
Haemophilus	few	8	8	0	100.0		104 - < 105	8	8	0	100.0
influenzae	moderate	7	7	0	100.0		105 - < 106	6	6	0	100.0
	numerous	13	13	0	100.0		> 106	3	3	0	100.0
	rare	2	2	0	100.0		103 - < 104	1	1	0	100.0
	few	5	4	1	80.0		104 - < 105	1	1	0	100.0
Klebsiella oxytoca	moderate	0	0	0	NA		105 - < 106	2	2	0	100.0
	numerous	2	2	0	100.0		> 106	0	0	0	NA
	rare	1	0	1	0.0		103 - < 104	0	0	0	NA
Klebsiella	few	10	10	0	100.0		104 - < 105	3	3	0	100.0
pneumoniae	moderate	9	8	1	88.9		105 - < 106	2	2	0	100.0
	numerous	1	1	0	100.0		> 106	0	0	0	NA
	rare	0	0	0	NA		103 - < 104	0	0	0	NA
	few	1	1	0	100.0		104 - < 105	0	0	0	NA
Klebsiella variicola	moderate	1	1	0	100.0		105 - < 106	0	0	0	NA
	numerous	0	0	0	NA		> 106	0	0	0	NA
	rare	0	0	0	NA		103 - < 104	1	1	0	100.0
Moraxella	few	3	3	0	100.0		104 - < 105	2	2	0	100.0
catarrhalis	moderate	5	5	0	100.0		105 - < 106	2	2	0	100.0
	numerous	6	6	0	100.0		> 106	1	1	0	100.0
	rare	0	0	0	NA		103 - < 104	1	1	0	100.0
Morganella	few	0	0	0	NA		104 - < 105	0	0	0	NA
morganii	moderate	0	0	0	NA		105 - < 106	0	0	0	NA
	numerous	0	0	0	NA		> 106	0	0	0	NA
	rare	4	4	0	100.0		103 - < 104	0	0	0	NA
Proteus spp.	few	2	2	0	100.0		104 - < 105	1	1	0	100.0
	moderate	2	2	0	100.0		105 - < 106	2	2	0	100.0
	numerous	2	2	0	100.0		> 106	0	0	0	NA
	rare	6	4	2	66.7		103 - < 104	2	2	0	100.0
Pseudomonas	few	7	7	0	100.0		104 - < 105	4	4	0	100.0
aeruginosa	moderate	12	12	0	100.0		105 - < 106	7	7	0	100.0
	numerous	7	7	0	100.0		> 106	0	0	0	NA
Qualitative Culture Result a						Quantitative Culture Result a
Category	Total	TP	FN	PPA [%]		Category	Total	TP	FN	PPA [%]
few	4	3	1	75.0		104 - < 105	2	2	0	100.0
moderate	1	1	0	100.0		105 - < 106	8	8	0	100.0
numerous	2	2	0	100.0		> 106	0	0	0	NA
rare	6	5	1	83.3		103 - < 104	0	0	0	NA
Staphylococcusaureus	few	14	13	1	92.9		104 - < 105	6	6	0	100.0
	moderate	7	7	0	100.0		105 - < 106	6	6	0	100.0
	numerous	9	9	0	100.0		> 106	1	1	0	100.0
Stenotrophomonasmaltophilia	rare	3	2	1	66.7		103 - < 104	1	1	0	100.0
	few	18	16	2	88.9		104 - < 105	4	4	0	100.0
	moderate	6	6	0	100.0		105 - < 106	1	1	0	100.0
	numerous	4	4	0	100.0		> 106	0	0	0	NA
Streptococcuspneumoniae	rare	2	1	1	50.0		103 - < 104	0	0	0	NA
	few	4	4	0	100.0		104 - < 105	4	4	0	100.0
	moderate	5	5	0	100.0		105 - < 106	6	6	0	100.0
	numerous	11	11	0	100.0		> 106	2	2	0	100.0

Table 42: Archived study, comparison to confirmed SoC results, stratification by qualitative or quantitative SoC culture result.

510(k) Summary – LRT BAL Application K191967 Rev 3.0

{70}------------------------------------------------

3 One Acinetobacter spp., 4 E. cloacae complex, 7. E. coli, 4 H. influenzae, 4 K. oxytoca, 5 K. pneumoniae, 1 M. catarrhalis, 2 Poteus spp. 11 P. aeruginosa, 4 S. marcescens, 9 S. maltophilia and 1 S. pneumoniae case were reported as positive by SoC culture without qualitative or quantitative results, and were therefore not included in this table.

Antibiotic Resistance Markers

Antibiotic resistance markers reported by LRT BAL for the archived study are listed in Table 43. Note that antibiotic resistance markers are only reported and displayed on the Unyvero results screen, if a corresponding host microorganism was simultaneously detected. Results for ctx-M, kpc, ndm, and vim are reported for LRT BAL positive specimens for Enterobacteriaceae, Acinetobacter spp., and P. aeruginosa (N = 212); results for oxa-48 are reported for LRT BAL positive specimens for Enterobacteriaceae (N = 166); results for oxa-23, oxa-24, and oxa-58 are reported for LRT BAL positive specimens for Acinetobacter spp. (N = 21); results for tem are reported for LRT BAL positive specimens for H. influenzae (N = 71); and results for mecA are reported for LRT BAL positive specimens for S. aureus (N = 77). Only specimens with antibiotic resistance markers reported positive by LRT BAL were tested for such markers by molecular reference assays (PCR/sequencing).

{71}------------------------------------------------

	Positive a	Negative
ctx-M(N = 212)	14	198
kpc(N = 212)	2	210
mecA(N = 77)	44	33
ndm(N = 212)	0	212
oxa-23(N = 21)	4	17
oxa-24(N = 21)	3	18
oxa-48(N = 166)	0	166
oxa-58(N = 21)	1	20
tem(N = 71)	24	47
vim(N = 212)	0	212

Table 43: Archived study (N = 392), listing of antibiotic resistance marker results as reported by LRT BAL.

ª Specimens with false positive LRT BAL results with molecular assays (PCR/vi-directional sequencing) using specimen DNA extracts for presence or absence of microorganisms was confirmed in 14 of 14 cases for ctx-M, 2 of 2 cases for kpc, 34 of 44 cases for mech, 4 of 4 cases for oxa-24, 1 of 1 case for oxa-24, 1 of 1 case for oxa-58, and 22 of 24 cases for tem. For all other cases, PCRs did not amplify sufficient amounts for sequencing.

Correlation of Detected Antibiotic Resistance Markers to Strain Genotypes and Phenotypes

As described in the previous sections, isolates that had been collected for most of the prospective and part of archived study specimens were screened for presence of antibiotic resistance markers of the LRT BAL panel. Furthermore, corresponding AST results were collected to predict resistance phenotypes of such isolates using one or more of the drug assays listed in Table 44. AST results were reported as MIC values or zone diameters (for Kirby-Bauer tests). Strain phenotypes across all test sites were standardized according to breakpoints listed in CLSI guidance M100S (Performance Standards for Antimicrobial Susceptibility Testing, 26th Edition 2016). 'Intermediate' calls were regarded as 'resistant' calls for this study. For the analysis, any strain was regarded as 'resistant' if at least one of the corresponding drugs resulted in an 'intermediate' or 'resistant' call. Any strain was regarded as 'susceptible' if a 'sensitive' call was obtained for all applicable tested drugs.

{72}------------------------------------------------

Antibiotic Resistance Marker(s)	Associated Resistance	AST Assay
tem	penicillins	H. influenzae :ampicillin, cefinase
ctx-M	3rd generation cephalosporins,cefepime	Enterobacteriaceae :cefotaxime, ceftazidime, ceftriaxoneAcinetobacter spp. :cefotaxime, ceftazidime, ceftriaxone, cefepimeP. aeruginosa :ceftazidime, cefepime
kpc, ndm, oxa-48, vim	carbapenems	Enterobacteriaceae :meropenem, ertapenem, imipenem
kpc, ndm, oxa-23, oxa-24, oxa-58,vim	carbapenems	Acinetobacter spp.meropenem, imipenem
kpc, ndm, vim	carbapenems	P. aeruginosa :meropenem, imipenem
mecA	oxacillin/cefoxitin	S. aureus :oxacillin, cefoxitin

Table 44: AST assays used for evaluation of antibiotic resistance markers detected by LRT BAL.

Antibiotic resistance markers reported by LRT BAL were correlated to available isolates for:

1. Genotypic agreement of reported antibiotic resistance markers to presence of such markers in cultured isolates for a specific specimen.
1. Phenotypic agreement of reported antibiotic resistance markers with associated phenotypic culture results to provided isolates as determined by antimicrobial susceptibility tests (AST).

Note that antibiotic resistance marker results are only reported by the LRT BAL Application if a corresponding host microorganism is simultaneously reported. Otherwise, such results are masked on the Unyvero results screen. Antibiotic resistance marker results reported together with a corresponding host microorganism may not be linked to this organism, but may have originated from other on-panel or off-panel microorganisms, e.g., mecA may not only originate from S. aureus, but also originate from coagulase-negative Staphylococci present in respiratory flora; tem may not only originate from H. influenzae, but also from other on-panel or off-panel Enterobacteriaceae. If multiple corresponding microorganisms are simultaneously reported for a certain antibiotic resistance marker, one or more than one of these microorganisms could be the host of such marker.

Genotypic and phenotypic agreements were evaluated for all relevant resistance phenotypes including all applicable specimens from the prospective and archived study combined (Tables 45 to 49).

{73}------------------------------------------------

Oxacillin/Cefoxitin Resistance for S. aureus (mecA)

Table 45: Correlation of positive mec A results reported by LRT BAL for the prospective ('p') or archived ('a') study to available S. aureus isolates (genotypic agreement, 'yes': presence of mecA confirmed by sequencing of isolate, 'no': mecA not confirmed, 'not prov.': no isolate provided) and to AST results indicating oxacillin/cefoxitin resistance for isolates with confirmed mecA (phenotypic agreement; R: resistant phenotype, S: sensitive phenotype, NA: AST results not reported).

# Cases			LRT BAL Result(relevant hosts only)	SoC Result(relevant hosts only)	GenotypicAgreement(for availableisolates)	PhenotypicAgreement(for isolateswith conf.mecA)	Comment
total	p	a	S. aureus	S. aureus	yes	Ra	-
4	4	-	S. aureus	S. aureus	no	-	-

3 One prospective strain was reported by the clinical site as MRSA, although this was not supported by the provided oxacillin Kirby-Bauer zone diameter. An independent cefoxitin AST confirmed the MRSA phenotype for this strain. For another archived strain, no AST results were reported by the clinical site. The MRSA phenotype was determined by an independent cefoxitin AST instead.

Penicillin Resistance for H. influenzae (tem)

Table 46: Correlation of positive tem results reported by LRT BAL for the prospective ('p') or archived ('a') study to available H. influenzae isolates (genotypic agreement, 'yes': presence of tem confirmed by sequencing of isolate, 'no': tem not confirmed, 'not prov.': no isolate provided) and to AST results indicating penicillin resistance for isolates with confirmed tem (phenotypic agreement: R: resistant phenotype, S: sensitive phenotype, NA: AST results not reported).

Genotypic Agreement for Specimens with Available Isolate: 4/4, 100.0% (95% CI: 51.0 - 100.0)
Phenotypic Agreement for Isolates with Confirmed Marker: 4/4, 100.0% (95% CI: 51.0 - 100.0)
# Cases			LRT BAL Result(relevant hosts only)	SoC Result(relevant hosts only)	GenotypicAgreement(for availableisolates)	PhenotypicAgreement(for isolateswith conf. tem)	Comment
total	p	a	(relevant hosts only)	(relevant hosts only)	(for available isolates)	(for isolates with conf. tem)
4	2	2	H. influenzae	H. influenzae	yes	R	-

{74}------------------------------------------------

Third Generation Cephalosporin Resistance for Enterobacteriaceae, Acinetobacter spp., and P. aeruginosa (ctx-M)

Table 47: Correlation of positive ctx-M results reported by LRT BAL for the prospective ('p') or archived ('a') study to available isolates of Enterobacteriaceae, Acinetobacter spp., or P. aeruginosa (genotypic agreement, 'yes': presence of ctx-M confirmed by sequencing of isolate, 'no': ctr-M not confirmed, 'not prov.': no isolate provided) and to AST results indicating third generation cephalosporin resistance for isolates with confirmed ctx-M (phenotypic agreement; R: resistant phenotype, S: sensitive phenotype, NA: AST results not reported).

Study	LRT BAL Result(relevant hosts only)	SoC Result(relevant hosts only)	GenotypicAgreement(for availableisolates)	PhenotypicAgreement(for isolateswith conf. ctx-M)	Comment
p	Acinetobacter spp.E. coliK. pneumoniaeP. aeruginosa	-K. pneumoniaeP. aeruginosa	--yesno	-R-	-
p	K. pneumoniaeP. aeruginosa	K. pneumoniaeP. aeruginosa	yesno	R-	-
p	E. coliP. aeruginosa	E. coli-	yes-	R-	-
p	E. coli[off-panel]	E. coliProvidencia stuartii	noyes	-R	-
p	Acinetobacter spp.E. coliK. pneumoniaeProteus spp.P. aeruginosa[off-panel]	--K. pneumoniae--Providencia stuartii	--(not. prov.)--yes	-----NA	-
p	E. coliK. pneumoniaeP. aeruginosaS. marcescens	-K. pneumoniaeP. aeruginosaS. marcescens	(not. prov.)no(not. prov.)	----	-
p	E. coliP. aeruginosa	-P. aeruginosa	-no	--	-
p	E. coliM. morganiiProteus spp.P. aeruginosa	---P. aeruginosa	---no	----	-

{75}------------------------------------------------

Carbapenem Resistance for Enterobacteriaceae, Acinetobacter spp., and P. aeruginosa (kpc, ndm, oxa-48, vim)

Table 48: Correlation of positive results for kpc, ndm, vim reported by LRT BAL for the prospective ('p') or archived ('a') study to available isolates of Enterobacteriaceae, Acinetobacter spp., or P. aeruginosa, or positive results for oxa-48 to available Enterobacteriaceae isolates (genotypic agreement, 'yes'; presence of any of these markers confirmed by sequencing, 'no': presence not confirmed, 'not prov.': no isolate provided) and to AST results indicating carbapenem resistance for isolates with confirmed marker (phenotypic agreement; R: resistant phenotype, S: sensitive phenotype, NA: AST results not reported).

Genotypic Agreement for Specimens with at Least One Available Isolate: 3/5, 60.0% (95% CI: 23.1 - 88.2)
Phenotypic Agreement for Isolates with Confirmed Marker: 2/2, 100.0% (95% CI: 34.2 - 100.0)
Study	Marker	LRT BAL Result (relevant hosts only)	SoC Result (relevant hosts only)	Genotypic Agreement (for available isolates)	Phenotypic Agreement (for isolates with conf. marker)	Comment
p	kpc	E. cloacae complexK. pneumoniaeP. aeruginosa	E. cloacae complex	yes	R	-
p	ndm,oxa-48	Acinetobacter spp.E. coliK. pneumoniaeP. aeruginosa	K. pneumoniaeP. aeruginosa	yes (both)	R	-
p	kpc	Acinetobacter spp.E. coliK. pneumoniae	Acinetobacter spp.	no	-	-
p	kpc	K. pneumoniaeP. aeruginosa	-	yes	NA	subclinical isolate a
p	kpc	K. pneumoniaeP. aeruginosa	K. pneumoniaeP. aeruginosa	(not prov.)no	-	AST: resistant
p	vim	Proteus spp.P. aeruginosa	P. aeruginosa	-no	-	-

a For K. pneumoniae, SoC reported a concentration < 10' CFUmL (considered a negative, 'subclinical' result below the reporting threshold for mini-BAL specimens).

{76}------------------------------------------------

Carbapenem Resistance for Acinetobacter spp. (oxa-23, oxa-24, oxa-58)

Table 49: Correlation of positive results for oxa-24, oxa-58, reported by LRT BAL for the prospective ('p') or archived ('a') study to available Acinetobacter spp. isolates (genotypic agreement, 'yes': presence of any of these markers confirmed by sequencing, 'no': presence not confirmed, 'not prov.': no isolate provided) and to AST results indicating carbapenem resistance for isolates with confirmed marker (phenotypic agreement; R: resistant phenotype, S: sensitive phenotype, NA: AST results not reported).

Genotypic Agreement for Specimens with at Least One Available Isolate: 6/6, 100.0% (95% CI: 61.0 - 100.0)
Phenotypic Agreement for Isolates with Confirmed Marker: 6/6, 100.0% (95% CI: 61.0 - 100.0)
Study	Marker	LRT BAL Result (relevant hosts only)	SoC Result (relevant hosts only)	Genotypic Agreement (for available isolates)	Phenotypic Agreement (isolates with conf. marker)	Comment
p	oxa-23	Acinetobacter spp.	Acinetobacter spp.	yes	R	-
a	oxa-23	Acinetobacter spp.	Acinetobacter spp.	yes	R	-
p	oxa-24	Acinetobacter spp.	Acinetobacter spp.	yes	R	-
p	oxa-24	Acinetobacter spp.	Acinetobacter spp.	yes	R a	-
a	oxa-23	Acinetobacter spp.	Acinetobacter spp.	yes	R	-
p	oxa-24	Acinetobacter spp.	Acinetobacter spp.	yes	R	-

4 For one prospective strain, a resistant phenotype to meropenem was reported by the clinical site, although this was not supported by the provided Kirby-Bauer zone diameter. Independent AST for meropenem and imipenem confirmed resistance to this strain.

C. Contrived Study

The clinical performance evaluation of rare LRT BAL analytes was supplemented by a contrived study, i.e., by testing artificial clinical specimens that were prepared by spiking pooled microorganisms at concentrations close to their respective analyte LoD or at clinically relevant concentrations into unique negative lavage specimens. LRT BAL microorganisms, for which, due to limited specimen numbers, a PPA of 90% or higher with a lower bound of the two-sided 95% confidence interval of 80% or higher was not demonstrated for the clinical performance evaluation using archived specimens, were selected for contrived specimen testing (C. pneumoniae, C. freundli, E. cloacae complex, K. oxytoca, K. pneumoniae, K. variicola, L. pneumophila, M. morganii, M. pneumoniae, P. jirovecii, Proteus spp., S. marcescens). In addition, all low prevalence LRT BAL antibiotic resistance markers (ctx-M, kpc, ndm, oxa-24, oxa-48, oxa-58, vim) were also included. For each target analyte a total of 60 contrived specimens with up to five different spiked reference strains was tested at a low concentration (2x LoD, 30 specimens) and at two moderate concentrations (3.5x LoD and 5x LoD, 15 specimens each) spanning the clinically relevant concentration range. For P. jirovecii, a positive clinical specimen was used for spiking, and all 60 contrived specimens were tested at a 2x LoD concentration.

{77}------------------------------------------------

Agreement rates to the expected results were determined for each target analyte and stratified by actual test concentrations Tables 50 and 51 show agreement rates for expected positive results (positive results / expected positive results) and expected negative results / number of expected negative results) together with two-sided 95% confidence intervals (95% CI) for included LRT BAL microorganisms or antibiotic resistance markers, respectively.

	Agreement with Expected Results
	# Pos./# Exp.	%	95% CI	# Neg./# Exp.	%	95% CI
Chlamydia pneumoniae	57/60	95.0	86.3 - 98.3	180/180	100.0	97.9 - 100.0
6.4 x 102/2x LoD	29/30	96.7	83.3 - 99.4
ATCC 53592	5/6
ATCC VR-1310	12/12
ATCC VR-2282	12/12
1.1 x 103/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 53592	3/3
ATCC VR-1310	6/6
ATCC VR-2282	6/6
1.6 x 103/5x LoD	13/15	86.7	62.1 - 96.3
ATCC 53592	2/3
ATCC VR-1310	5/6
ATCC VR-2282	6/6
Citrobacter freundii	59/60	98.3	91.1 - 99.7	168/168	100.0	97.8 - 100.0
1.6 x 105/2x LoD	29/30	96.7	83.3 - 99.4
ATCC 43864	6/6
ATCC 8090	6/6
NCTC 8581	5/6
NRZ-00452	6/6
UCLA C1	6/6
2.8 x 105/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 43864	3/3
ATCC 8090	3/3
NCTC 8581	3/3
NRZ-00452	3/3
UCLA C1	3/3
4.0 x 105/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 43864	3/3
ATCC 8090	3/3
NCTC 8581	3/3
NRZ-00452	3/3
UCLA C1	3/3
Enterobacter cloacae complex	58/60	96.7	88.6 - 99.1	156/156	100.0	97.6 - 100.0
4.0 x 105/2x LoD	28/30	93.3	78.7 - 98.2
ATCC 13047	5/6
ATCC 23373	6/6
ATCC 49141	5/6
ATCC BAA-2468	6/6
NRZ-00239	6/6
7.0 x 105/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13047	3/3
ATCC 23373	3/3
ATCC 49141	3/3
ATCC BAA-2468	3/3
NRZ-00239	3/3
1.0 x 106/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13047	3/3
ATCC 23373	3/3

Table 50: Contrived study results for targeted LRT BAL microorganism analytes.

{78}------------------------------------------------

	Agreement with Expected Results
	# Pos./# Exp.	%	95% CI	# Neg./# Exp.	%	95% CI
ATCC 49141ATCC BAA-2468NRZ-00239	3/33/33/3
Klebsiella oxytoca	56/60	93.3	84.1 - 97.4	168/168	100.0	97.8 - 100.0
2.0 x 104/2x LoD	26/30	86.7	70.3 - 94.7
ATCC 13182	4/6
ATCC 43863	6/6
ATCC 49131	6/6
ATCC 8724	5/6
NCIMB 12819	5/6
3.5 x 104/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13182	3/3
ATCC 43863	3/3
ATCC 49131	3/3
ATCC 8724	3/3
NCIMB 12819	3/3
5.0 x 104/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13182	3/3
ATCC 43863	3/3
ATCC 49131	3/3
ATCC 8724	3/3
NCIMB 12819	3/3
Klebsiella pneumoniae	60/60	100.0	94.0-100.0	96/96	100.0	96.2-100.0
8.0 x 104/2x LoD	30/30	100.0	88.7-100.0
ATCC 13883	6/6
Micromyx 4653	6/6
NCTC 13438	6/6
NCTC 13439	6/6
NCTC 13442	6/6
1.4 x 105/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13883	3/3
Micromyx 4653	3/3
NCTC 13438	3/3
NCTC 13439	3/3
NCTC 13442	3/3
2.0 x 105/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13883	3/3
Micromyx 4653	3/3
NCTC 13438	3/3
NCTC 13439	3/3
NCTC 13442	3/3
Klebsiella variicola	58/60	96.7	88.6-99.1	180/180	100.0	97.9-100.0
4.0 x 104/2x LoD	29/30	96.7	83.3-99.4
ATCC BAA-830	6/6
clinical isolate 1	6/6
clinical isolate 2	6/6
clinical isolate 3	6/6
clinical isolate 4	5/6
7.0 x 104/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC BAA-830	3/3
clinical isolate 1	3/3
clinical isolate 2	3/3
clinical isolate 3	3/3
clinical isolate 4	3/3
1.0 x 105/5x LoD	14/15	93.3	70.2-98.8
ATCC BAA-830	3/3
clinical isolate 1	3/3
clinical isolate 2	clinical isolate 3	2/3	3/3

{79}------------------------------------------------

	Agreement with Expected Results
	# Pos./# Exp.	%	95% CI	# Neg./# Exp.	%	95% CI
clinical isolate 4	3/3
Legionella pneumophila	57/60	95.0	86.3 - 98.3	180/180	100.0	97.9 - 100.0
1.6 x 105/2x LoD	28/30	93.3	78.7 - 98.2
ATCC 33152	6/6
ATCC 33154	5/6
ATCC 33155	5/6
ATCC 33215	6/6
ATCC 35096	6/6
2.8 x 105/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 33152	3/3
ATCC 33154	3/3
ATCC 33155	3/3
ATCC 33215	3/3
ATCC 35096	3/3
4.0 x 105/5x LoD	14/15	93.3	70.2 - 98.8
ATCC 33152	2/3
ATCC 33154	3/3
ATCC 33155	3/3
ATCC 33215	3/3
ATCC 35096	3/3
Morganella morganii	52/60	86.7	75.8 - 93.1	179/179	100.0	97.9 - 100.0
4.0 x 104/2x LoD	23/30	76.7	59.1 - 88.2
ATCC 25829 a	3/6
ATCC 25830	6/6
ATCC 49948	6/6
ATCC 8019	6/6
DSM-46262 a	2/6
7.0 x 104/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 25829	3/3
ATCC 25830	3/3
ATCC 49948	3/3
ATCC 8019	3/3
DSM-46262	3/3
1.0 x 105/5x LoD	14/15	93.3	70.2 - 98.8
ATCC 25829	3/3
ATCC 25830	3/3
ATCC 49948	3/3
ATCC 8019	3/3
DSM-46262	2/3
Mycoplasma pneumoniae	52/60	86.7	75.8 - 93.1	180/180	100.0	97.9 - 100.0
3.2 x 103/2x LoD	23/30	76.7	59.1 - 88.2
ATCC 15293	6/6
ATCC 29085	8/12
ATCC 49894	9/12
5.6 x 103/3.5x LoD	14/15	93.3	70.2 - 98.8
ATCC 15293	2/3
ATCC 29085	6/6
ATCC 49894	6/6
8.0 x 103/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 15293	3/3
ATCC 29085	6/6
ATCC 49894	6/6
Pneumocystis jirovecii	59/60	98.3	91.1 - 99.7	180/180	100.0	97.9 - 100.0
1.0 x 106/2x LoD	59/60	98.3	91.1 - 99.7
positive clinical specimen	59/60
Proteus spp.	60/60	100.0	94.0 - 100.0	180/180	100.0	97.9 - 100.0
1.0 x 104/2x LoD	30/30	100.0	88.7 - 100.0
ATCC 12453	6/6
ATCC 14153	6/6
			Agreement with Expected Results
	# Pos./# Exp.	%	95% CI	# Neg./# Exp.	%	95% CI
ATCC 25933	6/6
ATCC 29906	6/6
ATCC 6380	6/6
1.8 x 104/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 12453	3/3
ATCC 14153	3/3
ATCC 25933	3/3
ATCC 29906	3/3
ATCC 6380	3/3
2.5 x 104/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 12453	3/3
ATCC 14153	3/3
ATCC 25933	3/3
ATCC 29906	3/3
ATCC 6380	3/3
Serratia marcescens	59/60	98.3	91.1 - 99.7	180/180	100.0	97.9 - 100.0
8.0 x 104/2x LoD	29/30	96.7	83.3 - 99.4
ATCC 13880	6/6
ATCC 14756	6/6
ATCC 15365	6/6
ATCC 27117	6/6
DSM-17174	5/6
1.4 x 105/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13880	3/3
ATCC 14756	3/3
ATCC 15365	3/3
ATCC 27117	3/3
DSM-17174	3/3
2.0 x 105/5x LoD	15/15	100.0	79.6 - 100.0
ATCC 13880	3/3
ATCC 14756	3/3
ATCC 15365	3/3
ATCC 27117	3/3
DSM-17174	3/3

{80}------------------------------------------------

ª Morganella morganii strains ATCC 25829 and DSM-46262 strains were retested with pooled negative BAL matrix at 2x LoD both showing 10/10 results.

b Mycoplasma pneumoniae strains ATCC 49894 were retested with pooled negative BAL matrix at 2x LoD showing 19/20 or 18/20 positive results, For three of four unexpected negative cases of ATCC 29085, used unique matrices correlated with reduced signals or false negative results and therefore such failures may be matrix-related.

Table 51: Contrived study results for targeted LRT BAL antibiotic resistance marker analytes.
-----------------------------------------------------------------------------------------------

Agreement with Expected Results
	# Pos./# Exp.	%	95 %CI	# Neg./# Exp.	%	95 %CI
ctx-M	59/60	98.3	91.1 - 99.7	120/120	100.0	96.9 - 100.0
2.0 x 104/2x LoD	30/30	100.0	88.7 - 100.0
JMI 49767	6/6
JMI 50067	6/6
NCTC 13443	6/6
NRZ-00249	6/6
NRZ-00751	6/6
3.5 x 104/3.5x LoD	14/15	93.3	70.2 - 98.8
JMI 49767	3/3
JMI 50067	2/3
NCTC 13443	3/3
NRZ-00249	3/3
	Agreement with Expected Results
	# Pos./# Exp.	%	95 %CI	# Neg./# Exp.	%	95 %CI
NRZ-00751	3/3
5.0 x 104/5x LoD	15/15	100.0	79.6 - 100.0
JMI 49767	3/3
JMI 50067	3/3
NCTC 13443	3/3
NRZ-00249	3/3
NRZ-00751	3/3
kpc	58/60	96.7	88.6 - 99.1	156/156	100.0	97.6 - 100.0
8.0 x 104/2x LoD	29/30	96.7	83.3 - 99.4
Micromyx 4653	6/6
NCTC 13438	6/6
NRZ-00103	6/6
NRZ-00222	5/6
NRZ-00281	6/6
1.4 x 105/3.5x LoD	14/15	93.3	70.2 - 98.8
Micromyx 4653	3/3
NCTC 13438	3/3
NRZ-00103	2/3
NRZ-00222	3/3
NRZ-00281	3/3
2.0 x 105/5x LoD	15/15	100.0	79.6 - 100.0
Micromyx 4653	3/3
NCTC 13438	3/3
NRZ-00103	3/3
NRZ-00222	3/3
NRZ-00281	3/3
ndm	59/60	98.3	91.1 - 99.7	132/132	100.0	97.2 - 100.0
4.0 x 104/2x LoD	29/30	96.7	83.3 - 99.4
ATCC BAA-2468	6/6
JMI 46239	6/6
JMI 49755	5/6
JMI 49831	6/6
NCTC 13443	6/6
7.0 x 104/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC BAA-2468	3/3
JMI 46239	3/3
JMI 49755	3/3
JMI 49831	3/3
NCTC 13443	3/3
1.0 x 105/5x LoD	15/15	100.0	79.6 - 100.0
ATCC BAA-2468	3/3
JMI 46239	3/3
JMI 49755	3/3
JMI 49831	3/3
NCTC 13443	3/3
oxa-23	60/60	100.0	94.0 - 100.0	180/180	100.0	97.9 - 100.0
2.0 x 107/2x LoD	30/30	100.0	88.7 - 100.0
Micromyx 4410	6/6
Micromyx 6148	6/6
Micromyx 6149	6/6
NCTC 13301	6/6
UCLA A5	6/6
3.5 x 107/3.5x LoD	15/15	100.0	79.6 - 100.0
Micromyx 4410	3/3
Micromyx 6148	3/3
Micromyx 6149	3/3
NCTC 13301	3/3
UCLA A5	3/3
	Agreement with Expected Results
	# Pos./# Exp.	%	95 %CI	# Neg./# Exp.	%	95 %CI
Micromyx 4410	3/3
Micromyx 6148	3/3
Micromyx 6149	3/3
NCTC 13301	3/3
UCLA A5	3/3
oxa-24	59/60	98.3	91.1 - 99.7	180/180	100.0	97.9 - 100.0
1.0 x 104/2x LOD	29/30	96.7	83.3 - 99.4
clinical isolate 1	6/6
clinical isolate 2	6/6
NCTC 13302	6/6
NRZ-00449	5/6
UCLA A4	6/6
1.8 x 104/3.5x LOD	15/15	100.0	79.6 - 100.0
clinical isolate 1	3/3
clinical isolate 2	3/3
NCTC 13302	3/3
NRZ-00449	3/3
UCLA A4	3/3
2.5 x 104/5x LOD	15/15	100.0	79.6 - 100.0
clinical isolate 1	3/3
clinical isolate 2	3/3
NCTC 13302	3/3
NRZ-00449	3/3
UCLA A4	3/3
oxa-48	59/60	98.3	91.1 - 99.7	167/167	100.0	97.8 - 100.0
6.0 x 105/2x LoD	29/30	96.7	83.3 - 99.4
ATCC BAA-2523	5/6
NCTC 13442	6/6
NRZ-00176	6/6
NRZ-00472	6/6
NRZ-22060	6/6
1.1 x 106/3.5x LoD	15/15	100.0	79.6 - 100.0
ATCC BAA-2523	3/3
NCTC 13442	3/3
NRZ-00176	3/3
NRZ-00472	3/3
NRZ-22060	3/3
1.5 x 106/5x LoD	15/15	100.0	79.6 - 100.0
ATCC BAA-2523	3/3
NCTC 13442	3/3
NRZ-00176	3/3
NRZ-00472	3/3
NRZ-22060	3/3
oxa-58	59/60	98.3	91.1 - 99.7	180/180	100.0	97.9 - 100.0
1.0 x 105/2x LoD	30/30	100.0	88.7 - 100.0
NCTC 13305	18/18
NRZ-00518	12/12
1.8 x 105/3.5x LoD	14/15	93.3	70.2 - 98.8
NCTC 13305	8/9
NRZ-00518	6/6
2.5 x 105/5x LoD	15/15	100.0	79.6 - 100.0
NCTC 13305	9/9
NRZ-00518	6/6
vim	60/60	100.0	94.0 - 100.0	144/144	100.0	97.4 - 100.0
4.0 x 104/2x LoD	30/30	100.0	88.7 - 100.0
DSM-24600	6/6
NCTC 13437	6/6
NCTC 13439	6/6
NCTC 13440	6/6
		Agreement with Expected Results
	# Pos./# Exp.	%	95 %CI	# Neg./# Exp.	%	95 %CI
NRZ-00452	6/6
7.0 x 104/3.5x LoD	15/15	100.0	79.6 - 100.0
DSM-24600	3/3
NCTC 13437	3/3
NCTC 13439	3/3
NCTC 13440	3/3
NRZ-00452	3/3
1.0 x 105/5x LoD	15/15	100.0	79.6 - 100.0
DSM-24600	3/3
NCTC 13437	3/3
NCTC 13439	3/3
NCTC 13440	3/3
NRZ-00452	3/3

{81}------------------------------------------------

510(k) Summary – LRT BAL Application K191967 Rev 3.0

{82}------------------------------------------------

{83}------------------------------------------------

For other LRT BAL panel analytes not evaluated in the contrived study, the following agreement rates with the expected negative result were observed: 120/120 (100.0%, 95% CI 96.9 - 100.0 for Acinetobacter spp., 200/200 (100.0%, 95% CI 98.1 - 100.0) for E. coli, 228/228 (100.0%, 95% CI 98.3 - 100.0) for H. influenzae, 232/232 (100.0%, 95% CI 98.4 - 100.0) for mecA, 239/239 (100.0%, 95% CI 98.4 - 100.0) for M. catarrhalis, 208/208 (100.0%, 95% CI 98.2 - 100.0) for P. aeruginosa, 179/180 (99.4 %, 95% CI 96.9 - 99.9) for S. aureus, 240/240 (100.0%, 95% CI 98.4 - 100.0) for S. maltophilia, 239/240 (99.6%, 95% Cl 97.7 - 99.9) for S. pneumoniae, and 82/82 (100%, 95% Cl 95.5 - 100.0) for tem.

Expected Values

Expected values were determined for the prospective study cohort with 1.016 evaluable BAL specimens collected from hospitalized patients suspected for lower respiratory tract infection at nine US sites between June 2015 and July 2016. The LRT BAL Application reported 363 specimens with at least one positive LRT BAL panel microorganism, including 113 specimens with multi-detections of two or more LRT BAL panel microorganisms.

Table 52 lists expected values for microorganisms; Tables 53 and 54 list expected values for antibiotic resistance markers of the LRT BAL panel (for all specimens and stratified for each host microorganism). Note that antibiotic resistance markers are reported by LRT BAL only for specimens positive for a corresponding host microorganism.

{84}------------------------------------------------

Table 52: Expected values of LRT BAL panel microorganisms as reported for the prospective study stratified by all specimens (N = 1,016), as well as by single-detection (N = 250) or multi-detection specimens (N = 113). Expected value rates are given as percentage of 1,016 prospective study specimens.

Expected Values (N = 1,016 Prospective Specimens)
Microorganism		# Spec.	%	Microorganism		# Spec.	%
	all	21	2.1%		all	15	1.5%
Acinetobacter spp.	single	8	0.8%	Moraxella catarrhalis	single	4	0.4%
	multi	13	1.3%		multi	11	1.1%
	all	0	0.0%		all	3	0.3%
Chlamydia pneumoniae	single	0	0.0%	Morganella morganii	single	0	0.0%
	multi	0	0.0%		multi	3	0.3%
	all	4	0.4%		all	6	0.6%
Citrobacter freundii	single	1	0.1%	Mycoplasma pneumoniae	single	4	0.4%
	multi	3	0.3%		multi	2	0.2%
	all	20	2.0%		all	22	2.2%
Enterobacter cloacae	single	8	0.8%	Pneumocystis jirovecii	single	14	1.4%
complex	multi	12	1.2%		multi	8	0.8%
	all	47	4.6%		all	10	1.0%
Escherichia coli	single	24	2.4%	Proteus spp.	single	0	0.0%
	multi	23	2.3%		multi	10	1.0%
	all	56	5.5%		all	112	11.0%
Haemophilus influenzae	single	30	3.0%	Pseudomonas aeruginosa	single	61	6.0%
	multi	26	2.6%		multi	51	5.0%
	all	14	1.4%		all	17	1.7%
Klebsiella oxytoca	single	2	0.2%	Serratia marcescens	single	5	0.5%
	multi	12	1.2%		multi	12	1.2%
	all	30	3.0%		all	104	10.2%
Klebsiella pneumoniae	single	9	0.9%	Staphylococcus aureus	single	60	5.9%
	multi	21	2.1%		multi	44	4.3%
	all	2	0.2%		all	41	4.0%
Klebsiella variicola	single	0	0.0%	Stenotrophomonas	single	12	1.2%
	multi	2	0.2%	maltophilia	multi	29	2.9%
	all	1	0.1%		all	13	1.3%
Legionella pneumophila	single	1	0.1%	Streptococcus pneumoniae	single	7	0.7%
	multi	0	0.0%		multi	6	0.6%

Table 53: Expected values of LRT BAL panel antibiotic resistance markers as determined by the LRT BAL Application for the prospective study.

Expected Values, All Specimens (N = 1,016)
	# Spec.	%
ctx-M	9	0.9%
kpc	4	0.4%
mecA	47	4.6%
ndm	1	0.1%
oxa-23	3	0.3%
oxa-24	4	0.4%
oxa-48	1	0.1%
oxa-58	0	0.0%
tem	16	1.6%
vim	1	0.1%

{85}------------------------------------------------

	ctx-M	kpc	ndm	oxa-23	oxa-24	oxa-48	oxa-58	vim	tem	mecA
total reported	9	4	1	3	4	1	0	1	16	47
multi host detections	8	4	1	-	-	1	-	1	-	-
Acinetobacter spp.(N = 21)	2/219.5%	2/219.5%	1/214.8%	3/2114.3%	4/2119.0%	-	-	-	-	-
Citrobacter freundii(N = 4)	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacaecomplex (N = 20)	-	1/205.0%	-	-	-	-	-	-	-	-
Escherichia coli(N = 47)	8/4717.0%	1/472.1%	1/472.1%	-	-	1/472.1%	-	-	-	-
Klebsiella oxytoca(N = 14)	-	-	-	-	-	-	-	-	-	-
Klebsiella pneumoniae(N = 30)	4/3013.3%	4/3013.3%	1/303.3%	-	-	1/303.3%	-	-	-	-
Klebsiella variicola(N = 2)	-	-	-	-	-	-	-	-	-	-
Morganella morganii(N = 3)	1/333.3%	-	-	-	-	-	-	-	-	-
Proteus spp.(N = 10)	2/1020.0%	-	-	-	-	-	-	1/1010.0%	-	-
Pseudomonasaeruginosa (N = 112)	7/1126.3%	2/1121.8%	1/1120.9%	-	-	-	-	1/1120.9%	-	-
Serratia marcescens(N = 17)	2/1711.8%	-	-	-	-	-	-	-	-	-
Haemophilus influenzae(N = 56)	-	-	-	-	-	-	-	-	16/5628.6%	-
Staphylococcus aureus(N = 104)	-	-	-	-	-	-	-	-	-	47/10445.2%

Table 54: Expected values of LRT BAL panel antibiotic resistance markers, stratified by reported host microorganism.

{86}------------------------------------------------

Specimens reported as negative, as single detection, or as multi-detection, together with reported antibiotic resistance markers are listed in Table 55. For example, for eight specimens, LRT BAL reported a single detection of Acinetobacter spp. For four of these specimens (50%), an antibiotic resistance marker indicating a possible carbapenem resistance was simultaneously reported, while for four other specimens, no corresponding antibiotic resistance marker was reported. Similarly, for 60 specimens, LRT BAL reported a single detection of S. aureus. For 32 of these specimens (53%), a mecA was simultaneously reported, while for the remaining 28 specimens, mecA was not reported.

Table 55: Rates of negative, single detection and multi-detection specimens together with reported antibiotic resistance markers as detected by the LRT BAL Application for the prospective study (N = 1,016 specimens). Note that resistance marker oxa-58 was not detected in the prospective study cohort.

Microorganism Result	# cases	none	tem	ctx-M	kpc	oxa-23	oxa-24	mecA	ctx-M,mecA	tem ,vim	ctx-M,mecA,oxa-23	ctx-M,ndm,oxa-48	kpc,mecA,oxa-24
Negative	653(64.3%)	-	-	-	-	-	-	-	-	-	-	-	-
Single Organism Detected	250(24.6%)	-	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp.	8	4	-	-	-	1	3	-	-	-	-	-	-
Citrobacter freundii	1	1	-	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex	8	8	-	-	-	-	-	-	-	-	-	-	-
Escherichia coli	24	24	-	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae	30	26	4	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca	2	2	-	-	-	-	-	-	-	-	-	-	-
Klebsiella pneumoniae	9	9	-	-	-	-	-	-	-	-	-	-	-
Legionella pneumophila	1	1	-	-	-	-	-	-	-	-	-	-	-
Moraxella catarrhalis	4	4	-	-	-	-	-	-	-	-	-	-	-
Mycoplasma pneumoniae	4	4	-	-	-	-	-	-	-	-	-	-	-
Pneumocystis jirovecii	14	14	-	-	-	-	-	-	-	-	-	-	-
Pseudomonas aeruginosa	61	61	-	-	-	-	-	-	-	-	-	-	-
Serratia marcescens	5	5	-	-	-	-	-	-	-	-	-	-	-
Staphylococcus aureus	60	32	-	-	-	-	-	28	-	-	-	-	-
Stenotrophomonas maltophilia	12	12	-	-	-	-	-	-	-	-	-	-	-
Streptococcus pneumoniae	7	7	-	-	-	-	-	-	-	-	-	-	-
Two Organisms Detected	78(7.7%)	-	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp.,Citrobacter freundii	1	1	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp., Enterobactercloacae complex	1	1	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp.,Klebsiella pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp., Pseudomonasaeruginosa	1	1	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp.,Stenotrophomonas maltophilia	1	1	-	-	-	-	-	-	-	-	-	-	-
Citrobacter freundii, Pseudomonasaeruginosa	1	1	-	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex,Mycoplasma pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex,Staphylococcus aureus	2	2	-	-	-	-	-	-	-	-	-	-	-
Escherichia coli,Haemophilus influenzae	1	0	1	-	-	-	-	-	-	-	-	-	-
Escherichia coli,Klebsiella oxytoca	1	1	-	-	-	-	-	-	-	-	-	-	-
Microorganism Result	# cases	none	tem	ctx-M	kpc	oxa-23	oxa-24	mecA	ctx-M, mecA	tem, vim	ctx-M, mecA, oxa-23	ctx-M, ndm, oxa-48	kpc, mecA, oxa-24
Escherichia coli,Klebsiella pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Escherichia coli,Pseudomonas aeruginosa	1	0	-	1	-	-	-	-	-	-	-	-	-
Escherichia coli,Serratia marcescens	2	1	-	1	-	-	-	-	-	-	-	-	-
Escherichia coli,Staphylococcus aureus	2	1	-	-	-	-	-	-	1	-	-	-	-
Haemophilus influenzae,Klebsiella oxytoca	1	1	-	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Moraxella catarrhalis	4	3	1	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Mycoplasma pneumoniae	1	0	1	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Pseudomonas aeruginosa	3	1	2	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Staphylococcus aureus	4	2	2	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Streptococcus pneumoniae	3	1	2	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca,Klebsiella pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca,Proteus spp.	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca,Serratia marcescens	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca, Staphylococcus aureus	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca,Stenotrophomonas maltophilia	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella pneumoniae,Pseudomonas aeruginosa	2	1	-	1	-	-	-	-	-	-	-	-	-
Klebsiella pneumoniae,Staphylococcus aureus	3	2	-	-	-	-	-	1	-	-	-	-	-
Moraxella catarrhalis,Pneumocystis jirovecii	1	1	-	-	-	-	-	-	-	-	-	-	-
Moraxella catarrhalis,Streptococcus pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Pneumocystis jirovecii,Pseudomonas aeruginosa	1	1	-	-	-	-	-	-	-	-	-	-	-
Pneumocystis jirovecii,Staphylococcus aureus	1	1	-	-	-	-	-	-	-	-	-	-	-
Pneumocystis jirovecii,Stenotrophomonas maltophilia	2	2	-	-	-	-	-	-	-	-	-	-	-
Proteus spp.,Pseudomonas aeruginosa	1	1	-	-	-	-	-	-	-	-	-	-	-
Proteus spp.,Staphylococcus aureus	1	0	-	-	-	-	-	-	1	-	-	-	-
Pseudomonas aeruginosa, Serratia marcescens	1	1	-	-	-	-	-	-	-	-	-	-	-
Pseudomonas aeruginosa,Staphylococcus aureus	14	4	-	-	-	-	-	-	10	-	-	-	-
Pseudomonas aeruginosa,Stenotrophomonas maltophilia	6	6	-	-	-	-	-	-	-	-	-	-	-
Serratia marcescens,Staphylococcus aureus	1	0	-	-	-	-	-	-	1	-	-	-	-
Staphylococcus aureus,Stenotrophomonas maltophilia	5	3	-	-	-	-	-	-	2	-	-	-	-
Three Organisms Detected	19(1.9%)
Acinetobacter spp.,Klebsiella pneumoniae,	1	0	-	-	-	-	-	-	-	-	-	-	1
		Antibiotic Resistance Marker Result
						Single Marker Detected			Multiple Markers Detected
Microorganism Result	# cases	none	tem	ctx-M	kpc	oxa-23	oxa-24	mecA	ctx-M,mecA	tem,vim	ctx-M,mecA,oxa-23	ctx-M,ndm,oxa-48	kpc,mecA,oxa-24
Acinetobacter spp.,Klebsiella pneumoniae,Stenotrophomonas maltophilia	1	1	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp., Pseudomonasaeruginosa, Stenotrophomonasmaltophilia	1	0	-	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex,Haemophilus influenzae,	1	1	-	-	-	-	-	-	-	-	-	-	-
Moraxella catarrhalis
Enterobacter cloacae complex,Klebsiella oxytoca,	1	1	-	-	-	-	-	-	-	-	-	-	-
Stenotrophomonas maltophilia
Enterobacter cloacae complex,Klebsiella pneumoniae, Moraxella	1	1	-	-	-	-	-	-	-	-	-	-	-
catarrhalis
Enterobacter cloacae complex,Klebsiella pneumoniae,	1	0	-	-	1	-	-	-	-	-	-	-	-
Pseudomonas aeruginosa
Escherichia coli,Haemophilus influenzae,	1	1	-	-	-	-	-	-	-	-	-	-	-
Staphylococcus aureus
Escherichia coli,Morganella morganii,	1	1	-	-	-	-	-	-	-	-	-	-	-
Pseudomonas aeruginosa
Escherichia coli,Pneumocystis jirovecii,	1	0	-	-	1	-	-	-	-	-	-	-	-
Pseudomonas aeruginosa
Escherichia coli,Pseudomonas aeruginosa, Serratia	1	1	-	-	-	-	-	-	-	-	-	-	-
marcescens
Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus	1	1	-	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Moraxella catarrhalis,Streptococcus pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca, Pseudomonasaeruginosa, Staphylococcusaureus	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella oxytoca, Pseudomonasaeruginosa, Stenotrophomonasmaltophilia	1	1	-	-	-	-	-	-	-	-	-	-	-
Klebsiella pneumoniae,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	1	0	-	-	1	-	-	-	-	-	-	-	-
Proteus spp.,Pseudomonas aeruginosa,Staphylococcus aureus	1	0	-	-	-	-	-	-	-	-	-	-	-
Pseudomonas aeruginosa, Serratiamarcescens, Stenotrophomonasmaltophilia	2	2	-	-	-	-	-	-	-	-	-	-	-
Four Organisms Detected	7(0.7%)	-	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp.,Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa	1	0	-	-	-	-	-	-	-	-	-	-	-
Acinetobacter spp.,Escherichia coli,Klebsiella pneumoniae,Stenotrophomonas maltophilia	1	0	-	-	1	-	-	-	-	-	-	-	-
Acinetobacter spp.,Escherichia coli.	1	1	-	-	-	-	-	-	-	-	-	-	-
		Antibiotic Resistance Marker Result
		Single Marker Detected							Multiple Markers Detected
Microorganism Result	# cases	none	tem	ctx-M	kpc	oxa-23	oxa-24	mecA	ctx-M,mecA	tem,vim	ctx-M,mecA,oxa-23	ctx-M,ndm,oxa-48	kpc,mecA,oxa-24
Proteus spp.,Pseudomonas aeruginosa
Enterobacter cloacae complex,Escherichia coli,Klebsiella oxytoca,Stenotrophomonas maltophilia	1	1	-	-	-	-	-	-	-	-	-	-	-
Escherichia coli,Haemophilus influenzae,Pneumocystis jirovecii,Staphylococcus aureus	1	1	-	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae, Proteusspp.,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	1	0	-	-	-	-	-	-	-	1	-	-	-
Pseudomonas aeruginosa, Serratiamarcescens, Staphylococcusaureus, Stenotrophomonasmaltophilia	1	1	-	-	-	-	-	-	-	-	-	-	-
Five Organisms Detected	7(0.7%)
Acinetobacter spp., Enterobactercloacae complex, Haemophilusinfluenzae, Klebsiella pneumoniae,Streptococcus pneumoniae	1	1	-	-	-	-	-	-	-	-	-	-	-
Citrobacter freundii,Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa, Serratiamarcescens	1	1	-	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex,Escherichia coli,Klebsiella oxytoca,Klebsiella variicola,	1	1	-	-	-	-	-	-	-	-	-	-	-
Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa, Serratiamarcescens, Stenotrophomonasmaltophilia	1	0	-	-	-	-	-	-	1	-	-	-	-
Escherichia coli,Morganella morganii,Proteus spp.,Pseudomonas aeruginosa,Stenotrophomonas maltophilia	1	0	-	-	-	1	-	-	-	-	-	-	-
Haemophilus influenzae,Klebsiella pneumoniae, Klebsiellavariicola,Proteus spp.,Staphylococcus aureus	1	1	-	-	-	-	-	-	-	-	-	-	-
Haemophilus influenzae,Klebsiella pneumoniae, Moraxellacatarrhalis, Pneumocystisjirovecii,Serratia marcescens	1	0	-	1	-	-	-	-	-	-	-	-	-
Six Organisms Detected	2(0.2%)
Acinetobacter spp.,Escherichia coli,Klebsiella pneumoniae,Proteus spp.,Pseudomonas aeruginosa,Staphylococcus aureus	1	0	-	-	-	-	-	-	1	-	-	-	-
Microorganism Result	# cases	none	Single Marker Detected					Multiple Markers Detected
			tem	ctx-M	kpc	oxa-23	oxa-24	mecA	ctx-M, mecA	tem, vim	ctx-M, mecA, oxa-23	ctx-M, ndm, oxa-48	kpc, mecA, oxa-24
Haemophilus influenzae, Moraxella catarrhalis, Morganella morganii, Staphylococcus aureus, Stenotrophomonas maltophilia
Total	1,016	938	15	6	3	2	3	44	1	1	1	1	1

510(k) Summary - LRT BAL Application K191967 Rev 3.0

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510(k) Summary – LRT BAL Application K191967 Rev 3.0

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510(k) Summary – LRT BAL Application K191967 Rev 3.0

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807.92 (b)(3): Conclusions from Nonclinical and Clinical Data

The conclusions drawn from the analytical and clinical data demonstrate that the device is safe and effective for its intended use.

Regulation Number and Section

§ 866.3985 Device to detect and identify microorganisms and associated resistance marker nucleic acids directly in respiratory specimens.

(a)
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.(b)
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.