Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on the biological and chemical processes of the test (DNA isolation, PCR, array hybridization) and the automation of these steps by the system components. There is no mention of AI or ML being used for data analysis, interpretation, or result generation. The "Cockpit" component is described as providing the user interface and automatically generating and displaying results, which is typical for automated systems and does not inherently imply AI/ML.

Therapeutic

No.

The device is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from microorganisms and antibiotic resistance markers. It is indicated as an aid in the diagnosis of lower respiratory tract infection, meaning it helps in identifying the cause of a disease, rather than treating or preventing it.

Diagnostic

Yes

The device is intended as an aid in the diagnosis of lower respiratory tract infection. It performs a qualitative nucleic acid multiplex test to detect and identify nucleic acid sequences from microorganisms and antibiotic resistance markers to provide diagnostic information.

SaMD (Software as a Medical Device)

No

The device description clearly outlines multiple hardware components including cartridges, sample tubes, a Lysator, and an Analyzer, which are integral to the device's function. While there is a "Cockpit" component that provides a user interface and generates results, the overall system is not software-only.

IVD (In Vitro Diagnostic)

Based on the provided text, the device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states it's a "qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms and antibiotic resistance markers in endotracheal aspirates from adult hospitalized patients with suspected lower respiratory tract infections." This describes a test performed in vitro (outside the body) on a biological specimen (endotracheal aspirates) to provide information for diagnosis.
Device Description: The description details the components of the test kit (cartridge, sample tube, master mix, etc.) and the system (Lysator, Analyzer, Cockpit) used to perform the test. These are all components of an in vitro diagnostic system.
Performance Studies: The performance studies involve testing biological specimens (tracheal aspirates) and comparing the results to other laboratory methods (culture, PCR/sequencing). This is characteristic of the validation process for an IVD.

The definition of an IVD is a medical device that is used to perform tests on samples such as blood, urine, or tissue to detect diseases or other conditions. The Unyvero LRT Application fits this definition perfectly.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Unyvero LRT Application is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms and antibiotic resistance markers in endotracheal aspirates from adult hospitalized patients with suspected lower respiratory tract infections.

The Unyvero LRT 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 tracheal aspirates commonly contain colonizing microorganisms, detection of Unyvero LRT microbial targets does not indicate that the microorganism is the cause of the disease. Unyvero positive results do not rule out co-infection with microorganisms not detected by the Unyvero LRT Application. 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 resistance markers cannot be definitively linked to specific microorganisms, and may be present in organisms that are not detected by the Unyvero LRT Application such as organisms present as colonizing or normal flora.

Microbiology cultures of aspirates should be performed to obtain isolates for species identification and antimicrobial susceptibility testing, to differentiate quantities of identified microorganisms as well as normal flora present in the specimen and to identify potential microorganisms not targeted by the Unyvero LRT Application.

Product codes (comma separated list FDA assigned to the subject device)

QBH

Device Description

The Unyvero LRT Application is a qualitative test that includes specimen processing, genomic bacterial DNA isolation and purification, multiplex PCR and array hybridization and detection. The Unyvero LRT Application performed using the Unyvero System detects specific nucleic acid sequences from microorganisms and resistance markers in tracheal aspirates collected from patients with signs and symptoms of lower respiratory infection.

The Unyvero LRT Application consists of the following components:

Unyvero LRT Cartridge: Contains DNA isolation and purification reagents, a DNA isolation column, eight separate PCR chambers with eight corresponding detection arrays. The Cartridge also contains fluorescently-labeled primers, hybridization and wash buffers and oligonucleotide probes for detection of targeted PCR products using array hybridization technology.
Unyvero T1 Sample Tube: Contains glass beads and buffers to lyse bacteria and liquefy the sample.
Unyvero T1 Sample Tube Cap (with Internal Control): Contains proteinase K and a synthetic internal control gene for process monitoring. The T1 Sample Tube Cap seals the Unyvero Sample Tube after which the internal control is combined with each patient specimen. The internal control DNA sequence does not have significant homology to targeted sequences and is amplified independently in each of the eight PCR chambers and the amplified internal control product is hybridized on each array.
Unyvero M1 Master Mix: Contains reagents for DNA amplification.
Unyvero T1 Transfer Tool: The Transfer tool can be used to transfer viscous specimens from the primary sample container to the Unyvero Sample Tube.

The Unyvero System consists of the following components:

Unyvero Lysator: The Lysator lyses the specimen and can process up to four specimens simultaneously in four separate slots.
Unyvero Analyzer: The Analyzer automates DNA purification, amplification and detection. Each Analyzer can simultaneously process up to two Unyvero Cartridges with each slot available using random access.
Unyvero Cockpit: The Cockpit provides the main user interface for the Unyvero System, guides the user through the steps to run the Unvvero LRT Application and automatically generates and displays test results. The Cockpit is equipped with a high-resolution touch screen and a barcode reader.
Unyvero Sample Tube Holder: The Sample Tube holder holds the Sample Tube securely while the specimen is transferred into the Sample Tube.

Other materials required but not provided:

Pipette capable of dispensing 180uL
DNase/RNase free, aerosol resistant pipette tips
1mL Luer-Lock svringe

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

lower respiratory tract, tracheal aspirates

Indicated Patient Age Range

Adult hospitalized patients

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Prospective Study:
A total of 860 tracheal aspirate specimens were prospectively collected from patients with signs and symptoms of lower respiratory infection and were tested with the LRT assay within 24 hours of specimen collection. Specimens excluded from the performance analyses included 38 specimens that did not meet the specimen inclusion/exclusion criteria, 161 specimens that generated non-reportable results (e.g., fully invalid results or instrument failures) and 58 specimens that generated partially invalid results (invalid results in one or more PCR chambers). Altogether, a total number of 603 evaluable prospectively tested specimens were included in the performance analyses. Gram stains (quality screening) were performed for the majority of specimens tested in the study.

Reference/comparator methods used in the prospective study included standard of care (SoC) tracheal aspirate culture and validated comparator PCR assays. All positive comparator PCR results were followed with bi-directional sequencing. Validation of each comparator PCR assay included demonstration of similar LoDs and inclusivity to the Unyvero LRT Application.

Retrospective Clinical Study, Archived Specimens:
For the retrospective clinical study, 266 previously frozen tracheal aspirate specimens were tested at US study sites with the Unyvero LRT Application. Four specimens were excluded for not meeting specimen inclusion criteria. 32 specimens were excluded due to non-reportable results and 19 specimens with reportable results were excluded due to partially valid results. The remaining 211 evaluable US study specimens were supplemented with 158 specimens collected at other US or European sites and tested in-house at Curetis. Altogether, results from a total number of 369 evaluable archived specimens were included in the performance analyses. All specimens were selected based on positive standard of care results which included culture for 'typical' analytes and other test methods for 'atypical' analytes. All historical positive results were confirmed with validated PCR/bi-directional sequencing assays prior to inclusion in the study.

Clinical Study, Contrived Specimens:
For low prevalence microorganism and resistance marker analytes, the prospective and archived studies were supplemented with contrived specimens. Each contrived specimen was prepared in a unique natural tracheal aspirate specimen matrix. All aspirate specimen matrices were prescreened to ensure that they were negative for all Unyvero LRT analytes prior to use in the study. Specimens were spiked with pools of microorganisms at two concentrations: low positive (2x LoD or lower) and moderate positive (typically 3-10x LoD). Microorganisms evaluated in the contrived study were C. pneumoniae, C. freundii, K. oxytoca, K. variicola, L. pneumophila, M. catarrhalis, M. morganii, and M. pneumoniae. LRT Application resistance markers evaluated in the contrived study were ctx-M, oxa-23, oxa-24, oxa-48, oxa-58, kpc, vim, and ndm. Testing of contrived specimens was performed at three sites in the United States as well as in-house at Curetis.

Up to five different strains for each analyte were used to prepared contrived specimens with the total number of specimens ranging from 21 to 50 for each microorganism or resistance marker.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Study Type: Clinical Studies (Prospective Study, Retrospective Clinical Study (Archived Specimens), Clinical Study (Contrived Specimens))
Sample Size:

Prospective Study: 603 evaluable specimens
Retrospective Clinical Study (Archived Specimens): 369 evaluable archived specimens
Clinical Study (Contrived Specimens): Ranged from 21 to 50 for each microorganism or resistance marker.

Standalone Performance: Not explicitly stated as standalone performance study, but results of the Unyvero LRT Application are provided.

Key Results:

Prospective Study (comparison to reference culture):

Acinetobacter spp.: PPA 100.0% (10/10), NPA 97.1% (576/592)
Citrobacter freundii: PPA 25.0% (1/4), NPA 99.7% (597/599)
Enterobacter cloacae complex: PPA 93.3% (14/15), NPA 99.0% (582/588)
Escherichia coli: PPA 95.8% (23/24), NPA 96.2% (557/579)
Haemophilus influenzae: PPA 100.0% (8/8), NPA 97.3% (579/595)
Klebsiella oxytoca: PPA 66.7% (4/6), NPA 98.2% (586/597)
Klebsiella pneumoniae: PPA 87.5% (21/24), NPA 97.1% (562/579)
Klebsiella variicola: PPA 100.0% (2/2), NPA 99.7% (599/601)
Moraxella catarrhalis: PPA 33.3% (1/3), NPA 98.0% (588/600)
Morganella morganii: PPA 100.0% (1/1), NPA 98.5% (593/602)
Proteus spp.: PPA 90.9% (10/11), NPA 96.8% (573/592)
Pseudomonas aeruginosa: PPA 92.8% (64/69), NPA 96.3% (514/534)
Serratia marcescens: PPA 100.0% (11/11), NPA 97.6% (578/592)
Staphylococcus aureus: PPA 97.6% (81/83), NPA 92.5% (481/520)
Stenotrophomonas maltophilia: PPA 96.2% (25/26), NPA 95.3% (550/577)
Streptococcus pneumoniae: PPA 77.8% (7/9), NPA 99.0% (588/594)

Prospective Study (comparison to composite comparator):

Acinetobacter spp.: PPA 95.8% (23/24), NPA 99.3% (575/579)
Chlamydia pneumoniae: PPA na (0/0), NPA 100.0% (603/603)
Citrobacter freundii: PPA 16.7% (1/6), NPA 99.7% (595/597)
Enterobacter cloacae complex: PPA 94.4% (17/18), NPA 99.5% (582/585)
Escherichia coli: PPA 97.4% (37/38), NPA 98.6% (557/565)
Haemophilus influenzae: PPA 88.2% (15/17), NPA 98.6% (577/585)
Klebsiella oxytoca: PPA 77.8% (7/9), NPA 98.7% (586/594)
Klebsiella pneumoniae: PPA 90.9% (30/33), NPA 98.8% (562/569)
Klebsiella variicola: PPA 100.0% (2/2), NPA 99.7% (599/601)
Legionella pneumophila: PPA 100.0% (2/2), NPA 100.0% (601/601)
Moraxella catarrhalis: PPA 52.2% (12/23), NPA 99.8% (579/580)
Morganella morganii: PPA 85.7% (6/7), NPA 99.3% (592/596)
Mycoplasma pneumoniae: PPA 100.0% (2/2), NPA 99.8% (600/601)
Proteus spp.: PPA 96.0% (24/25), NPA 99.1% (573/578)
Pseudomonas aeruginosa: PPA 89.4% (76/85), NPA 98.5% (510/518)
Serratia marcescens: PPA 87.5% (21/24), NPA 99.3% (575/579)
Staphylococcus aureus: PPA 91.6% (109/119), NPA 97.7% (473/484)
Stenotrophomonas maltophilia: PPA 89.3% (50/56), NPA 99.6% (545/547)
Streptococcus pneumoniae: PPA 62.5% (10/16), NPA 99.5% (584/587)

Archived study performance, 'typical' microorganisms:

Acinetobacter spp.: PPA 100.0% (18/18), NPA 98.3% (344/350c)
Citrobacter freundii: PPA 100.0% (2/2), NPA 98.3% (354/360)
Enterobacter cloacae complex: PPA 96.0% (24/25), NPA 97.9% (329/336)
Escherichia coli: PPA 97.4% (37/38), NPA 94.5% (311/329)
Haemophilus influenzae: PPA 87.5% (21/24), NPA 96.7% (326/337)
Klebsiella oxytoca: PPA 100.0% (17/17), NPA 97.1% (337/347)
Klebsiella pneumoniae: PPA 96.6% (28/29), NPA 95.0% (321/338)
Klebsiella variicola: PPA 100.0% (9/9), NPA 99.4% (356/358)
Moraxella catarrhalis: PPA 100.0% (9/9), NPA 97.4% (336/345)
Morganella morganii: PPA 100.0% (1/1), NPA 98.0% (346/353)
Proteus spp.: PPA 96.7% (29/30), NPA 96.2% (325/338)
Pseudomonas aeruginosa: PPA 92.3% (48/52), NPA 92.3% (286/310)
Serratia marcescens: PPA 97.1% (34/35), NPA 97.9% (326/333)
Staphylococcus aureus: PPA 92.3% (72/78), NPA 90.6% (259/286)
Stenotrophomonas maltophilia: PPA 97.1% (33/34), NPA 90.9% (298/328)
Streptococcus pneumoniae: PPA 91.3% (21/23), NPA 98.2% (335/341)

Archived study performance, 'atypical' microorganisms:

Chlamydia pneumoniae: PPA na (0/0)
Legionella pneumophila: PPA 100.0% (2/2)
Mycoplasma pneumoniae: PPA na (0/0)

Contrived specimen testing:

Chlamydia pneumoniae: PPA 100.0% (21/21), NPA 100.0% (303/303)
Citrobacter freundii: PPA 86.0% (43/50), NPA 99.6% (236/237)
Klebsiella oxytoca: PPA 89.3% (25/28), NPA 99.7% (294/295)
Klebsiella variicola: PPA 96.4% (27/28), NPA 99.7% (295/296)
Legionella pneumophila: PPA 100.0% (49/49), NPA 100.0% (247/247)
Moraxella catarrhalis: PPA 96.0% (48/50), NPA 100.0% (245/245)
Morganella morganii: PPA 95.9% (47/49), NPA 100.0% (245/245)
Mycoplasma pneumoniae: PPA 100.0% (50/50), NPA 100.0% (274/274)
ctx-M: PPA 94.0% (47/50), NPA 95.3% (183/192)
kpc: PPA 98.0% (48/49), NPA 100.0% (245/245)
ndm: PPA 96.6% (28/29), NPA 90.1% (237/263)
oxa-23: PPA 96.4% (27/28), NPA 99.3% (139/140)
oxa-24: PPA 100.0% (50/50), NPA 100.0% (90/90)
oxa-48: PPA 98.0% (48/49), NPA 100.0% (205/205)
oxa-58: PPA 100.0% (30/30), NPA 100.0% (138/138)
vim: PPA 95.9% (47/49), NPA 100.0% (234/234)

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Key metrics provided are PPA (Positive Percent Agreement) and NPA (Negative Percent Agreement), along with their 95% Confidence Intervals (CI).

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not Found

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

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.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Unyvero LRT Application and Unyvero System DECISION SUMMARY

A. DEN Number:

DEN170047

B. Purpose for Submission: De Novo request for evaluation of automatic class III designation for the Unyvero Lower Respiratory Tract (LRT) Application and the Unyvero System.
C. Measurand: DNA sequences of the following organisms and antibiotic resistance markers:

Microorganism Targets	Antibiotic Resistance Marker Targets
Acinetobacter spp.	ctx-M ( bla CTX-M, subgroup 1 only)
Chlamydia pneumoniae	kpc ( bla KPC)
Citrobacter freundii	mecA
Escherichia coli	ndm ( bla NDM)
Enterobacter cloacae complex	oxa-23 ( bla OXA-23)
Haemophilus influenzae	oxa-24 ( bla OXA-24)
Klebsiella oxytoca	oxa-48 ( bla OXA-48)
Klebsiella pneumoniae	oxa-58 ( bla OXA-58)
Klebsiella variicola	tem ( bla TEM)
Legionella pneumophila	vim ( bla VIM)
Moraxella catarrhalis
Morganella morganii
Mycoplasma pneumoniae
Proteus spp.
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Stenotrophomonas maltophilia
Streptococcus pneumoniae

D. Type of Test:

Qualitative nucleic acid amplification assay

E. Applicant:

Curetis GmbH

F. Proprietary and Established Names:

Unyvero Lower Respiratory Tract (LRT) Application Unyvero System

G. Regulatory Information:

1

1. Regulation section:

21 CFR 866.3985

1. Classification:
  Class II (Special Controls)
1. Product code:
  QBH
1. Panel:
  83-Microbiology

H. Indications for use:

1. Indications for use:
  The Unyvero LRT Application is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms and antibiotic resistance markers in endotracheal aspirates from adult hospitalized patients with suspected lower respiratory tract infections.

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 complex c	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 pneumoniae d	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	-
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	-

ª Acinetobacter spp. includes: A. baumannii, A. calcoaceticus, A. haemolyticus, A. junii, A.

2

lwoffii, A. nosocomialis, A. parvus A. pittii, (detected by LRT Application) and A. ursingii (not detected by LRT Application). b ctx-M1 subgroup. & Enterobacter cloacae complex includes: E. asburiae, E. cloacae, E. hormaechei, E. kobei, E. ludwigii, and E. xiangfangensis. d Klebsiella pneumoniae includes two variants: K. pneumoniae (variant 1), and K. quasipneumoniae (variant 2) e Proteus spp. includes P. hauseri, P. mirabilis, P. penneri and P. vulgaris

The Unyvero LRT 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 tracheal aspirates commonly contain colonizing microorganisms, detection of Unyvero LRT microbial targets does not indicate that the microorganism is the cause of the disease. Unyvero positive results do not rule out co-infection with microorganisms not detected by the Unyvero LRT Application. 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 resistance markers cannot be definitively linked to specific microorganisms, and may be present in organisms that are not detected by the Unyvero LRT Application such as organisms present as colonizing or normal flora.

Microbiology cultures of aspirates should be performed to obtain isolates for species identification and antimicrobial susceptibility testing, to differentiate quantities of identified microorganisms as well as normal flora present in the specimen and to identify potential microorganisms not targeted by the Unyvero LRT Application.

1. Special conditions for use statement(s):
- . For in vitro diagnostic use only
- . For prescription use only

Limitations:

. Antimicrobial resistance can occur via mechanisms other than the resistance markers detected by the Unyvero LRT Assay. Negative results for the LRT resistance markers do not indicate antimicrobial susceptibility of detected organisms.
. Detected resistance markers cannot be definitively linked to specific microorganism(s), and may be present in organisms that are not detected by the Unyvero LRT assay, including colonizing flora. Standard culture of tracheal aspirates and subsequent testing of the isolated microorganism(s) is necessary to definitively link antimicrobial resistance with a specific microorganism.
Detection of antibiotic resistance markers in a specimen may not correlate with phenotypic gene expression. Resistance marker results should be used in

3

conjunction with final culture and antimicrobial susceptibility testing results.

. Detected microorganisms may be indicative of colonizing or normal respiratory flora and may not be the causative agent of pneumonia.
(b) (4)
The LRT Application is a qualitative test and does not provide a quantitative ● value for the microorganisms or antibiotic resistance markers detected in the specimen. Compared to standard of care culture methods, the Unyvero LRT Application is not able to assess the presence or amount of normal flora in the specimen or whether the detected microorganism/antibiotic resistance marker is present in predominating amounts as compared to other microorganisms in the specimen. The LRT Application also does not assess if detected microorganisms are present at clinically relevant concentrations, or are present as colonizing flora.
. When three or more microorganisms are detected for a specimen, the user is advised to wait for microbiology culture results to verify the predominant microorganism(s) and to assess for the presence of normal respiratory flora.
Bacterial nucleic acids may be present in the specimen independent of organism ● viability. Detection of organism nucleic acid target(s) does not imply that the corresponding organisms are the causative agents for clinical symptoms.
. A negative result for the 'atypical' microorganisms (C. pneumoniae, L. pneumophila, and M. pneumoniae) does not exclude the presence of this microorganism in the patient specimen. A positive result should be evaluated in the overall context of the patient's clinical condition and other laboratory results being part of the standard-of-care routine.
. Due to the small number of positive prospective and archived specimens for certain microorganisms and antibiotic resistance markers, performance characteristics for C. pneumoniae, C. freundii, K. oxytoca, K. variicola, L. pneumophila, M. catarrhalis, M. morganii, M. pneumoniae, and antibiotic resistance markers ctx-M, kpc, ndm, oxa-23, oxa-24, oxa-48, oxa-58, and vim were established primarily using contrived clinical specimens.
. Based on in-silico analysis and inclusivity wet testing, certain clinically relevant species for some LRT panel microorganism analytes are not detected or detected with reduced sensitivity: Acinetobacter ursingii detection is predicted at reduced sensitivity for the Acinetobacter spp. assay, Enterobacter asburiae detection is predicted at reduced sensitivity for the Enterobacter cloacae complex assay.
Subtypes included in ctx-M subgroups ctx-M2, ctx-M8, ctx-M9 (ctx-M14), ctx-. M25 and ctx-M45 were not evaluated and are not predicted to be detected by the LRT assay based on in-silico analysis.
. The mecC variant is not detected by Unyvero LRT and for mecC positive Staphylococcus aureus strains, the assay will generate negative results for mecA.
. Based on in-silico analysis and exclusivity wet testing. the following LRT panel microorganism assays are expected to cross-react with closely related clinically relevant species: Citrobacter freundii (cross-reactive to C. braakii, Kluyvera georgiana), Escherichia coli (cross-reactive to E. albertii, E. fergusonii and

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Shigella spp. (S. dysenteriae, S. sonnei, S. boydii)), Haemophilus influenzae (cross-reactive to H. haemolyticus), Klebsiella oxytoca (cross-reactive to K. michiganensis).

4. Special instrument requirements:

The Unyvero LRT Application is performed on the Unyvero System which includes the Unyvero Lysator, Unyvero Analyzer and Unyvero Cockpit.

I. Device Description:

The Unyvero LRT Application is a qualitative test that includes specimen processing, genomic bacterial DNA isolation and purification, multiplex PCR and array hybridization and detection. The Unyvero LRT Application performed using the Unyvero System detects specific nucleic acid sequences from microorganisms and resistance markers in tracheal aspirates collected from patients with signs and symptoms of lower respiratory infection.

The Unyvero LRT Application consists of the following components:

Unyvero LRT Cartridge: Contains DNA isolation and purification reagents, a DNA . isolation column, eight separate PCR chambers with eight corresponding detection arrays. The Cartridge also contains fluorescently-labeled primers, hybridization and wash buffers and oligonucleotide probes for detection of targeted PCR products using array hybridization technology.
Unyvero T1 Sample Tube: Contains glass beads and buffers to lyse bacteria and liquefy the sample.
. Unyvero T1 Sample Tube Cap (with Internal Control): Contains proteinase K and a synthetic internal control gene for process monitoring. The T1 Sample Tube Cap seals the Unyvero Sample Tube after which the internal control is combined with each patient specimen. The internal control DNA sequence does not have significant homology to targeted sequences and is amplified independently in each of the eight PCR chambers and the amplified internal control product is hybridized on each array.
. Unyvero M1 Master Mix: Contains reagents for DNA amplification.
. Unyvero T1 Transfer Tool: The Transfer tool can be used to transfer viscous specimens from the primary sample container to the Unyvero Sample Tube.

The Unyvero System consists of the following components:

. Unyvero Lysator: The Lysator lyses the specimen and can process up to four specimens simultaneously in four separate slots.

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Unyvero Analyzer: The Analyzer automates DNA purification, amplification and detection. Each Analyzer can simultaneously process up to two Unyvero Cartridges with each slot available using random access.
Unyvero Cockpit: The Cockpit provides the main user interface for the Unyvero System, guides the user through the steps to run the Unvvero LRT Application and automatically generates and displays test results. The Cockpit is equipped with a high-resolution touch screen and a barcode reader.
Unyvero Sample Tube Holder: The Sample Tube holder holds the Sample Tube securely while the specimen is transferred into the Sample Tube.

Other materials required but not provided:

Pipette capable of dispensing 180uL
DNase/RNase free, aerosol resistant pipette tips
1mL Luer-Lock svringe ●

J. Standard/Guidance Document Referenced:

CLSI EP07-A2. Interference Testing in Clinical Chemistry: Approved Guideline, 2nd ● edition.
CLSI EP17-A2. Evaluation of Detection Capability for Clinical Laboratory ● Measurement Procedures; Approved Guideline - 2nd edition.
CLSI M100, Performance Standards for Antimicrobial Susceptibility Testing, 26th ● Edition.

K. Test Principle:

The Unyvero LRT Application is a qualitative PCR-based assay that detects DNA sequences of microorganisms and associated resistance markers in tracheal aspirate specimens. The assay includes specimen processing (lysis), DNA extraction and isolation, multiplex PCR with eight parallel multiplex endpoint PCR reactions, and qualitative detection of amplified DNA products using hybridization arrays.

A tracheal aspirate specimen is first pipetted into the Unyvero Sample Tube and closed with the Unyvero Sample Tube Cap. Closing the sample tube automatically adds the lysis reagent and the internal control gene template to the specimen. The sample tube which fits into the Unyvero Lysator only if closed with the Unyvero sample tube cap is then placed on the Lysator. After the specimen is lysed in the Lysator, the Sample Tube and Master Mix are loaded into the Unyvero LRT Cartridge which is then inserted into the position assigned by the Unyvero Analyzer for automated processing and analysis.

In the Unyvero LRT Cartridge, the remainder of the testing steps are automated by the Unyvero Analyzer. The lysed specimen is further processed and then transferred onto a

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(b) DNA purification column for nucleic acid (b) (4) DNA is transferred to a chamber, where mixing with the Master Mix takes place. This mixture is distributed into eight separate PCR reaction chambers each containing multiple primer pairs. After amplification, PCR products are hybridized to the corresponding array probes. Each array has been manufactured (b) (4) (b) (4)

(b) (4)

Result data are transferred

to the Unyvero Cockpit for visualization and result printout. A test run is completed after approximately 4.5 hours, and results for panel microorganisms and associated antibiotic resistance markers are displayed on the Unyvero Cockpit screen.

L. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Reproducibility:

The reproducibility of the Unyvero LRT Application was assessed using a representative panel of samples prepared in artificial respiratory matrix (ARM, adapted from Dinesh S. D.1) which was used as a surrogate for tracheal aspirate matrix. In a separate analytical study, the Unyvero LRT Application was shown to demonstrate equivalent performance for samples prepared in ARM matrix and samples prepared in natural tracheal aspirate matrix (See Section L.2.b below).

Samples for the reproducibility study were prepared with whole-organism preparations for the following LRT panel analytes: E. coli/tem. P. geruginosa, K. pneumoniae, M. morganii, S. maltophilia and S. aureus/mecA. Sample #1 (REPRO 1) was inoculated with each organism at moderate positive concentrations (~5 x LoD) and Sample #2 (REPRO 2) was inoculated with each organism at low positive concentrations (~1.7 x LoD). For the mec A target, the REPRO 1 and REPRO2 panel members had concentrations of 12.5 x LoD and 4.2 x LoD respectively. For the tem target, the REPRO1 and REPRO2 panel members had concentrations of 6.2 and 2.1 x LoD respectively. Test runs were independently performed by three different operators. Each operator was assigned one of three Unyvero systems, each consisting of 1 Unyvero Cockpit, 2 Unyvero Lysators and at least 4 Unyvero Analyzers. A total of 90 replicate samples were tested for each panel member with each operator testing samples in triplicate on a minimum of five testing days. Positive and negative controls were also tested daily by each operator. The organisms and concentrations used to prepare study samples are presented in Table 1 below. Study results are presented in Table 2.

Table 1: Microorganisms for Reproducibility Study ATCC Strain | LoD (CFU/mL) | Repro1/x-fold LoD | Repro2/x-fold LoD Analytes

1 Dinesh, Artificial Sputum Medium. Protocol Exchange (2010)

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E. coli	35218	7.50E+04	3.75E+05	5.0	1.28E+05	1.7
K. pneumoniae V2	700603	5.00E+04	2.50E+05	5.0	8.50E+04	1.7
M. morganii	25830	5.00E+05	2.50E+06	5.0	8.50E+05	1.7
P. aeruginosa	10145	5.00E+04	2.50E+05	5.0	8.50E+04	1.7
S. aureus	33591	5.00E+06	2.50E+07	5.0	8.50E+06	1.7
S. maltophilia	13637	3.00E+04	1.50E+05	5.0	5.10E+04	1.7
mec A	33591	2.00E+06	2.50E+07	12.5	8.50E+06	4.3
tem	35218	6.00E+04	3.75E+05	6.3	1.28E+05	2.1

Table 2: Reproducibility study results, stratified by target, concentration and operator

		REPRO1 (5x LoD)				REPRO 2 (1.7x LoD)
analyte	user	x-fold
LoD	Pos.	[%]	95 % CI	x-
fold
LoD	Pos.	[%]	95 % CI
E. coli
ATCC 35218	user 1	5	30/30	100.0	88.7 - 100.0	1.7	29/29	100.0	88.3 - 100.0
	user 2		30/30	100.0	88.7 - 100.0		29/30	96.7	83.3 - 99.4
	user 3		29/30	96.7	83.3 - 99.4		29/30	96.7	83.3 - 99.4
	all		89/90	98.9	94.0 - 99.8		87/89	97.8	92.2 - 99.4
K. pneumoniae
variant 2
ATCC 700603	user 1	5	21/29	72.4	54.3 - 85.3	1.7	14/30	46.7	30.2 - 63.9
	user 2		25/29	86.2	69.4 - 94.5		15/30	50.0	33.2 - 66.8
	user 3		24/30	80.0	62.7 - 90.5		19/29	65.5	47.3 - 80.1
	all		70/88	79.5	70.0 - 86.7		48/89	53.9	43.6 - 63.9
mecA
(from: S. aureus
ATCC 33591)	user 1	12.5	30/30	100.0	88.7 - 100.0	4.2	25/29	86.2	69.4 - 94.5
	user 2		27/30	90.0	74.4 - 96.5		29/30	96.7	83.3 - 99.4
	user 3		29/30	96.7	83.3 - 99.4		29/30	96.7	83.3 - 99.4
	all		86/90	95.6	89.1 - 98.3		83/89	93.3	86.1 - 96.9
M. morganii
ATCC 25830	user 1	5	28/29	96.6	82.8 - 99.4	1.7a	21/30	70.0	52.1 - 83.3
	user 2		27/29	93.1	78.0 - 98.1		23/30	76.7	59.1 - 88.2
	user 3		28/30	93.3	78.7 - 98.2		24/29	82.8	65.5 - 92.4
	all		83/88	94.3	87.4 - 97.5		68/89	76.4	66.6 - 84.0
P. aeruginosa
ATCC 10145	user 1	5	30/30	100.0	88.7 - 100.0	1.7	30/30	100.0	88.7 - 100.0
	user 2		30/30	100.0	88.7 - 100.0		28/30	93.3	78.7 - 98.2
	user 3		30/30	100.0	88.7 - 100.0		30/30	100.0	88.7 - 100.0
	all		90/90	100.0	95.9 - 100.0		88/90	97.8	92.3 - 99.4
S. aureus
ATCC 33591	user 1	5	28/30	93.3	78.7 - 98.2	1.7	30/30	100.0	88.7 - 100.0
	user 2		30/30	100.0	88.7 - 100.0		26/30	86.7	70.3 - 94.7
	user 3		30/30	100.0	88.7 - 100.0		29/30	96.7	83.3 - 99.4
	all		88/90	97.8	92.3 - 99.4		85/90	94.4	87.6 - 97.6
S. maltophilia
ATCC 13637	user 1	5	30/30	100.0	88.7 - 100.0	1.7	29/30	96.7	83.3 - 99.4
	user 2		28/30	93.3	78.7 - 98.2		28/30	93.3	78.7 - 98.2
	user 3		30/30	100.0	88.7 - 100.0		29/30	96.7	83.3 - 99.4
	all		88/90	97.8	92.3 - 99.4		86/90	95.6	89.1 - 98.3
tem
(from: E. coli
ATCC 35218)	user 1	6.2	29/30	96.7	83.3 - 99.4	2.1	29/29	100.0	88.3 - 100.0
	user 2		29/30	96.7	83.3 - 99.4		22/30	73.3	55.6 - 85.8
	user 3		30/30	100.0	88.7 - 100.0		26/30	86.7	70.3 - 94.7
	all		88/90	97.8	92.3 - 99.4		77/89	86.5	77.9 - 92.1

ª M. morganii was also evaluated at 0.6x LoD in PBS matrix in an initial reproducibility study. Results for this panel member were acceptable based on the microorganism concentration tested (e.g., positivity: 77/88, 87.5% detection for samples prepared at below LoD concentrations).

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Testing of reproducibility samples generated acceptable results for all analytes except for K. pneumoniae and M. morganii for which positivity rates were lower than expected for both the 5x LoD and 1.7x LoD panel members. The reason for these unexpected results was likely due to inaccurate quantitation of organism stocks used for sample preparation. It was noted that testing by different operators/sites generated equivalent results for these analytes (i.e., similarly lower than expected percent positivity at each testing site).

To further evaluate the reproducibility of the Unyvero LRT assay for detection of these two organism targets, ten additional sample replicates for both K. pneumoniae and M. morganii were prepared using freshly grown and quantitated organism suspensions with final sample concentrations of 5x LoD. For both microorganisms, a positivity rate of 90% was observed with 9/10 samples generating positive results.

Out of 14 positive quality control runs with samples containing moderate positive analyte concentrations (~8x LoD). one quality control sample was false negative for K. pneumoniae and one sample was false negative for M. morganii.

Three false positive results were observed during the study; two false positive results in reproducibility test sample runs (S. pneumoniae, vim) and one false positive in a positive control run (M. catarrhalis).

Partially invalid results (one PCR chamber only) were observed in 8/180 (4.4%) reproducibility test runs, 2/14 (1.4%) negative quality control runs, and 0/14 positive quality control runs.

Results from the reproducibility study were further evaluated based on Unyvero signal output levels (RBU) for each target and shown in Table 3 below.

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| analyte | user | x-fold
LoD | Avg
Signal | % CV | Range | x-fold
LoD | Avg
Signal | % CV | Range |
|----------------|--------| | | user 1 | 5 | E. coli | user 2 | | ATCC 35218 | user 3 | | | all | | K. pneumoniae | user 1 | 5 | variant 2 | user 2 | | ATCC 700603 | user 3 | | | all | | mecA | user 1 | 12.5 | (from: | user 2 | | S. aureus | user 3 | | ATCC 33591) | all | | | user 1 | 5 | M. morganii | user 2 | | ATCC 25830 | user 3 | | | all | | | user 1 | 5 | P. aeruginosa | user 2 | | ATCC 10145 | user 3 | | | all | | | user 1 | 5 | S. aureus | user 2 | | ATCC 33591 | user 3 | | | all | | | user 1 | 5 | S. maltophilia | user 2 | | ATCC 13637 | user 3 | | | all | | tem | user 1 | 6.2 | (from: E. coli | user 2 | | ATCC 35218) | user 3 | | | all | ---------------|---------------|--------|---------|---------------|---------------|---------|---------|
| (b) (4) | b) (4) | (b) (4) | 1.7 | (b) (4) | (b) (4) | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 1.7b | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 4.2 | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 1.7 | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 1.7 | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 1.7 | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 1.7 | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |
| (b) (4) | | (b) (4) | 2.1 | (b) (4) | | (b) (4) |
| | | | | | | |
| | | | | | | |
| | | | | | | |

Table 3: Reproducibility study, Quantitative analysis of Unyvero signal levels

b. Linearity/assay reportable range:
Not applicable.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Internal Control: An internal control consisting of a synthetic DNA sequence without homology to LRT microorganism or resistance marker target sequences is separately processed in each of the eight Unyvero LRT Cartridge PCR chambers. The internal control allows for assessment of DNA purification, amplification, array hybridization, and detection steps. The internal control monitors for the presence of PCR inhibitors

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in the specimens and enables the system to detect any failures in the testing process that could potentially result in an incorrect test result. Results generated from Individual PCR chambers are considered valid if successful amplification and detection is completed for either the internal control or any microorganism/resistance marker target in the multiplex PCR reaction.

External Controls

Positive and negative controls were run daily at each of the testing sites during the clinical study. Testing included (b) (4)

External controls are not provided with the Unyvero LRT Application: however. testing of external positive and negative controls are recommended in the assay labeling. Controls may consist of previously characterized positive samples or negative samples spiked with well characterized microorganisms. Previously characterized negative samples may be used as negative controls. External controls should be used in accordance with local, state, and/or federal regulations, accreditation requirements and individual laboratory's quality control policies, as applicable.

Specimen Stability

The Unyvero LRT instructions for use indicates that it is acceptable to store tracheal aspirate specimens at 2-8°C for up to 24 hours prior to testing. To evaluate effects on assay performance for 24 hour refrigerated storage prior to starting an LRT test, an analysis was performed for prospectively tested clinical tracheal aspirate specimens that were tested with the LRT application at various times of storage up to 24 hours. Both qualitative performance (compared to culture) and semi-quantitative Unyvero LRT signal levels for positive analytes were assessed between groups stored for different time periods. Results for specimen which were stored for times close to the 24-hour sample storage time limit did not show a significant difference in clinical performance or a significant difference in assay signal as compared to specimens that were tested immediately after collection.

The sample storage recommendations for the LRT application are consistent with those for traditional culture2 which include storage and transport of tracheal aspirate specimens at 2-8°C for up to 24 hours. The analysis of the clinical specimen results together with the same recommendation for tracheal aspirate culture support the claim

2 Baron, E. J. et al. A guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a). Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. An. 57, (2013).

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of refrigerated storage of tracheal aspirates for up to 24 hours prior to testing with the Unyvero LRT Application.

d. Detection limit:

A study was conducted to determine the Limit of Detection (LoD) of the Unyvero LRT Application for each targeted analyte. Samples were prepared in Artificial Respiratory Matrix (ARM) which was used as a surrogate for tracheal aspirate matrix. Samples prepared in ARM matrix were shown to generate equivalent results to samples prepared in natural tracheal aspirate matrix (See Section L.2.b below).

For the LoD study, samples were inoculated with multiple organisms (i.e., multispiked). To mitigate potential for competitive inhibition, sample compositions were designed to ensure that only one or two analytes were amplified in each multiplex PCR chamber.

LoD estimates were initially determined by testing small numbers of replicates at multiple different organism concentrations. The LoD for each analyte was then confirmed by testing 20 replicates at the estimated LoD concentration. The final LoD is defined as the lowest concentration (reported as CFU/mL, IFU/mL, or CCU/mL) of sample for which ≥95% of sample replicates generate positive results. Tables 4 and 5 below summarize the confirmed LoDs for each LRT panel microorganism and antibiotic resistance marker.

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| LRT Panel Microorganism | ID of Tested Reference
Strain | LoD Concentration
[CFU/mL, or other as indicated in
footnote] |
|-------------------------------------|----------------------------------------------|---------------------------------------------------------------------|
| Acinetobacter spp. | ATCC 19606 ( A.
baumannii ) | 1x105 |
| Chlamydia pneumoniae (in IFU/mL) a | ATCC VR-2282 | 1.5x104 |
| Citrobacter freundii | ATCC 8090 | 2x105 |
| Escherichia coli | ATCC 11775 | 7.5x104 |
| Enterobacter cloacae complex | ATCC 13047 ( E. cloacae ) | 5x106 |
| Haemophilus influenzae | ATCC 33391 | 2x106 |
| Klebsiella oxytoca | ATCC 13182 | 2x105 |
| Klebsiella pneumoniae Variant 1 | ATCC 13883 | 4.5x105 |
| Klebsiella pneumoniae Variant 2 | ATCC 700603 | 5x104 |
| Klebsiella variicola | ATCC BAA-830 | 1x105 |
| Legionella pneumophila | ATCC 33152 | 2x106 |
| Moraxella catarrhalis | ATCC 25238 | 8x105 |
| Morganella morganii | ATCC 25830 | 5x105 |
| Mycoplasma pneumoniae (in CCU/mL) b | ATCC 29085 | 1x105 |
| Proteus mirabilis | ATCC 29906 | 1x105 |
| Proteus vulgaris | ATCC 29905 | 6x105 |
| Pseudomonas aeruginosa | ATCC 10145 | 5x104 |
| Serratia marcescens | ATCC 13880 | 1x105 |
| Staphylococcus aureus | ATCC 12600 | 5x106 |
| Stenotrophomonas maltophilia | ATCC 13637 | 3x104 |
| Streptococcus pneumoniae | ATCC 49619 | 5x105 |

Table 4: Limit of detection for LRT panel microorganisms

a IFU: inclusion-forming units

b CCU: color-changing units

LRT Panel Antibiotic Resistance Marker	ID of Tested Reference Strain	LoD Concentration [CFU/mL]
ctx-M	NRZ-00751 ( K. pneumoniae )	1x105
kpc	NRZ-00281 ( E. coli )	5x105
mecA	ATCC 33591 ( S. aureus )	2x106
ndm	JMI 50067 ( E. coli )	5x104
oxa-23	NCTC 13301 ( A. baumannii )	2x107
oxa-24	NCTC 13302 ( A. baumannii )	5x105
oxa-48	NCTC 13442 ( K. pneumoniae )	2x106
oxa-58	NCTC 13305 ( A. baumannii )	7.5x105
tema	NCTC 13351 ( E. coli )	6x104
vim	DSM-24600 ( P. aeruginosa )	5x104

Table 5: Limit of detection for I RT panel antibiotic resistance markers

a Although the LRT Application reports tem only with H. influenzae, LoD was determined with an E. coli strain positive for tem. Note that inclusivity testing was successfully performed with two tem positive H. influenzae strains.

e. Analytical Reactivity (Inclusivity)

The inclusivity study of the LRT Application was assessed using samples inoculated at low positive concentrations with well-characterized bacterial isolates. Most strains were evaluated in samples prepared at Enterobacter asburiae | ATCC 35953 | 1.5x107 | 3x | 2/2 |
| Escherichia coli | ATCC 11775 | 1x105 | 1.3x | 2/2 |
| Escherichia coli | ATCC 25922 | 1x105 | 1.3x | 2/2 |
| Escherichia coli | ATCC 35218 | 1x105 | 1.3x | 4/4 |
| Escherichia coli | ATCC BAA-2523 | 1x105 | 1.3x | 2/2 |
| Escherichia coli | NCTC 13351 | 5x104 | 0.7x | 2/2 |
| Escherichia coli | NCTC 13476 | 1x105 | 1.3x | 2/2 |
| Escherichia coli | JMI 50067 | 6x104 | 0.8x | 2/2 |
| Escherichia coli | NRZ-00176 | 1x105 | 1.3x | 2/2 |
| Escherichia coli | NRZ-00222 | 6x104 | 0.8x | 2/2 |
| Escherichia coli | NRZ-00281 | 1.8x105 | 2.4x | 2/2 |
| Haemophilus influenzae (serotype a) | ATCC 9006 | 2x106 | 1x | 2/2 |
| Haemophilus influenzae (serotype c) | ATCC 9007 | 6x106 | 3x | 2/2 |
| Haemophilus influenzae (serotype b) | ATCC 10211 | 6x106 | 3x | 2/2 |
| Haemophilus influenzae (serotype b) | ATCC 49247 | 6x106 | 3x | 2/2 |
| Haemophilus influenzae
(non-typeable/non-capsulated) | ATCC 33391 | 6x106 | 3x | 2/2 |
| Haemophilus influenzae (serotype b) | ATCC 49766 | 6x106 | 3x | 2/2 |
| Haemophilus influenzae (serotype b) | NCTC 8468 | 2x106 | 1x | 2/2 |
| Klebsiella oxytoca | ATCC 13182 | 8x104 | 0.4x | 6/6 |
| Klebsiella oxytoca | ATCC 43863 | 8x104 | 0.4x | 2/2 |
| Klebsiella oxytoca | ATCC 8724 | 1.6x105 | 0.8x | 2/2 |
| Klebsiella oxytoca | ATCC 49131 | 8x104 | 0.4x | 2/2 |
| Klebsiella oxytoca | NCIMB 12819 | 4x105 | 2x | 2/2 |
| Klebsiella oxytoca | NRZ-22060 | 8x104 | 0.4x | 2/2 |
| Klebsiella pneumoniae | ATCC 13883 | 4x105 | 0.9x | 4/4 |
| Klebsiella pneumoniae | NCTC 13439 | 1x106 | 2.2x | 2/2 |
| Klebsiella pneumoniae | NCTC 13440 | 6x105 | 1.3x | 2/2 |
| Klebsiella pneumoniae | NCTC 13442 | 4x105 | 0.9x | 2/2 |
| Klebsiella pneumoniae | NCTC 13443 | 4x105 | 0.9x | 2/2 |
| Klebsiella pneumoniae | Micromyx 4653 | 8x105 | 1.8x | 4/4 |
| Klebsiella pneumoniae | Micromyx 4676 | 1x106 | 2.2x | 2/2 |
| Klebsiella pneumoniae | JMI 49831 | 6x105 | 1.3x | 2/2 |
| Klebsiella pneumoniae | JMI 49767 | 9x105 | 2x | 2/2 |
| Klebsiella pneumoniae | NRZ-00002 | 4x105 | 0.9x | 2/2 |
| Klebsiella pneumoniae | NRZ-00103 | 1.5x106 | 3.3x | 2/2 |
| Klebsiella pneumoniae | NRZ-00223 | 9x105 | 2x | 4/4 |
| Klebsiella pneumoniae | NRZ-00249 | 6x105 | 1.3x | 2/2 |
| Klebsiella pneumoniae | NRZ-00472 | 9x105 | 2x | 2/2 |
| Klebsiella pneumoniae | NRZ-00751 | 9x105 | 2x | 2/2 |
| Klebsiella pneumoniae variant II ( K. quasipneumoniae ) | ATCC 700603 | 6x105 | 1.3x | 2/2 |
| Strain | Strain ID | Test Conc.
[CFU/mL] | LoD
factora | # Positive/

Tests |

| Klebsiella variicola | ATCC BAA-830 | 3x105 | 3x | 2/2 |
| Klebsiella variicola | clinical strain 1 | 3.9x105 | 3.9x | 2/2 |
| Klebsiella variicola | clinical strain 2 | 2.6x105 | 2.6x | 2/2 |
| Klebsiella variicola | clinical strain 3 | 1.5x105 | 1.5x | 2/2 |
| Klebsiella variicola | clinical strain 4 | 2.4x105 | 2.4x | 3/3 |
| Klebsiella variicola | clinical strain 5 | 1.5x105 | 1.5x | 2/2 |
| Legionella pneumophila (serotype 1) | ATCC 33152 | 8x105 | 0.4x | 2/2 |
| Legionella pneumophila (serotype 2) | ATCC 33154 | 6x106 | 3x | 2/2 |
| Legionella pneumophila (serotype 3) | ATCC 33155 | 4x105 | 0.2x | 2/2 |
| Legionella pneumophila (serotype 6) | ATCC 33215 | 2x106 | 1x | 2/2 |
| Legionella pneumophila (serotype 8) | ATCC 35096 | 4x105 | 0.2x | 2/2 |
| Legionella pneumophila (serotype 10) | ATCC 43283 | 4x105 | 0.2x | 2/2 |
| Legionella pneumophila | UCLA L1 | 4x105 | 0.2x | 2/2 |
| Legionella pneumophila | UCLA L5 | 4x105 | 0.2x | 2/2 |
| Legionella pneumophila | UCLA L6 | 4x105 | 0.2x | 2/2 |
| Moraxella catarrhalis | ATCC 25238 | 2x106 | 2.5x | 0/2 |
| Moraxella catarrhalis | ATCC 43617 | 4x105 | 0.5x | 2/2 |
| Moraxella catarrhalis | ATCC 8176 | 4x105 | 0.5x | 2/2 |
| Moraxella catarrhalis | ATCC 25240 | 4x105 | 0.5x | 2/2 |
| Moraxella catarrhalis | ATCC 23246 | 4x105 | 0.5x | 2/2 |
| Moraxella catarrhalis | ATCC 49143 | 2x106 | 2.5x | 2/2 |
| Morganella morganii | ATCC 8019 | 1x105 | 0.2x | 2/2 |
| Morganella morganii | ATCC 25829 | 5x105 | 1x | 2/2 |
| Morganella morganii | ATCC 25830 | 1x105 | 0.2x | 2/2 |
| Morganella morganii spp. sibonii | ATCC 49948 | 1.5x106 | 3x | 2/2 |
| Mycoplasma pneumoniae | ATCC 29085 | 3x105 CCU/mLc | 3x | 2/2 |
| Mycoplasma pneumoniae | ATCC 29343 | 3x105 CCU/mLc | 3x | 2/2 |
| Mycoplasma pneumoniae | ATCC 15492 | 3x106 CFU/mL | 3x | 2/2 |
| Mycoplasma pneumoniae | ATCC 15531 | 3x106copies/mL | 3x | 2/2 |
| Proteus mirabilis | ATCC 12453 | 4x104 | 0.4x | 2/2 |
| Proteus mirabilis | ATCC 14153 | 3x105 | 3x | 2/2 |
| Proteus mirabilis | ATCC 25933 | 4x105 | 4x | 0/2 |
| Proteus mirabilis | ATCC 25933 | 5x105 | 5x d | 2/2 |
| Proteus mirabilis | ATCC 29906 | 3x105 | 3x | 4/4 |
| Proteus vulgaris | ATCC 6380 | 2.4x106 | 4x | 0/2 |
| Proteus vulgaris | ATCC 6380 | 3x106 | 5x d | 2/2 |
| Proteus vulgaris | ATCC 8427 | 4x104 | 0.1x | 2/2 |
| Proteus vulgaris | ATCC 29905 | 1.8x106 | 3x | 4/4 |
| Proteus hauseri | ATCC 700826 | 5x104e | 0.1x | 2/2 |
| Proteus penneri | ATCC 33519 | 5x104e | 0.1x | 2/2 |
| Pseudomonas aeruginosa | ATCC 10145 | 1x105 | 2x | 2/2 |
| Strain | Strain ID | Test Conc.
[CFU/mL] | LoD
factora | # Positive/

Tests |

| Pseudomonas aeruginosa | ATCC 27853 | 1x105 | 2x | 2/2 |
| Pseudomonas aeruginosa | DSM-24600 | 5x104 | 1x | 4/4 |
| Pseudomonas aeruginosa | NCTC 13437 | 2x104 | 0.4x | 4/4 |
| Pseudomonas aeruginosa | Micromyx 2562 | 1x105 | 2x | 4/4 |
| Pseudomonas aeruginosa | NRZ-00196 | 2x104 | 0.4x | 2/2 |
| Pseudomonas aeruginosa | NRZ-03961 | 5x104 | 1x | 2/2 |
| Pseudomonas aeruginosa | UCLA P20 | 4x104 | 0.8x | 2/2 |
| Serratia marcescens | ATCC 8100 | 3x105 | 3x | 2/2 |
| Serratia marcescens | ATCC 13880 | 3x105 | 3x | 2/2 |
| Serratia marcescens | ATCC 14756 | 3x105 | 3x | 2/2 |
| Serratia marcescens | ATCC 15365 | 3x105 | 3x | 2/2 |
| Serratia marcescens | ATCC 27117 f | 2x105 | 2x | 1/2 |
| | | 3x105 | 3x | 1/2 |
| | | 5x105 | 5x | 1/2 |
| | | 1x106 | 10x | 0/2 |
| Serratia marcescens | ATCC 43861 | 2x105 | 2x | 2/2 |
| Serratia marcescens ssp. sakuensis | DSM-17174 | 3x105 | 3x | 2/2 |
| Staphylococcus aureus | IDEXX VB962455 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | IDEXX VB9981353 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | IDEXX VB969039 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | ATCC BAA-2312 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | NCTC 12493 | 6x106 | 1.2x | 2/2 |
| Staphylococcus aureus | ATCC 33591 | 6x106 | 1.2x | 2/2 |
| Staphylococcus aureus | DSM-17091 | 6x106 | 1.2x | 2/2 |
| Staphylococcus aureus | ATCC 12600 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | ATCC 29213 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | ATCC 43300 | 1.5x107 | 3x | 2/2 |
| Staphylococcus aureus | RKI 07-03165 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | RKI 01-00694 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | RKI 09-00187 | 8x106 | 1.6x | 2/2 |
| Staphylococcus aureus | RKI 08-02492 | 8x106 | 1.6x | 2/2 |
| Stenotrophomonas maltophilia | ATCC 13636 | 5x104 | 1.7x | 2/2 |
| | | 2x104 | 0.7x | 2/2 |
| | | 3x104 | 1x | 1/2 |
| Stenotrophomonas maltophilia | ATCC 13637 g | 5x104 | 1.7x | 5/6 |
| | | 1x105 | 3.3x | 1/2 |
| | | 2x105 | 6.7x | 1/2 |
| Stenotrophomonas maltophilia | ATCC 17666 | 2x104 | 0.7x | 2/2 |
| Stenotrophomonas maltophilia | ATCC 49130 | 2x104 | 0.7x | 2/2 |
| Stenotrophomonas maltophilia | DSM-50173 g
[ATCC 17444] | 2x104 | 0.7x | 1/4 |
| | | 3x104 | 1x | 0/1 |
| | | 5x104 | 1.7x | 3/4 |
| | | 1x105
2x105 | 3.3x
6.7x | 0/2
1/2 |
| Strain | Strain ID | Test Conc.
[CFU/mL] | LoD
factora | # Positive/

Tests |

| Stenotrophomonas maltophilia | DSM-21874 g
[NCIMB 9528] | 2x104
3x104
5x104
1x105
2x105 | 0.7x
1x
1.7x
3.3x
6.7x | 1/4
0/2
2/4
0/2
0/2 |
| | Streptococcus pneumoniae | ATCC 33400 | 1.5x106
2.5x106 | 3x
5x |
| Streptococcus pneumoniae
(serotype 19F) | ATCC 49619 | 1.5x106 | 3x | 2/2 |
| Streptococcus pneumoniae
(serotype 3) | ATCC 6303 | 1.5x106
2.5x106 | 3x
5x | 1/2
2/2 |
| Streptococcus pneumoniae
(serotype 5) | ATCC 6305 | 2x105 | 0.4x | 2/2 |
| Streptococcus pneumoniae | ATCC 49150 | 1.5x106 | 3x | 2/2 |
| Streptococcus pneumoniae
(serotype 1) | ATCC 6301 | 2x105
1x106 | 0.4x
2x | 1/2
2/2 |
| Streptococcus pneumoniae | ATCC 10015 | 2x105 | 0.4x | 2/2 |
| Streptococcus pneumoniae
(serotype 2) | ATCC 27336 | 1.5x106 | 3x | 2/2 |
| Streptococcus pneumoniae
(serotype 9V) | DSM-11865 | 2x105 | 0.4x | 2/2 |
| Streptococcus pneumoniae
(serotype 23F) | DSM-11866 | 1.5x106 | 3x | 2/2 |
| Streptococcus pneumoniae
(serotype 6B) | DSM-11867 | 1.5x106 | 3x | 2/2 |

Table 6: Microorganism targets, Inclusivity strains

15

16

17

18

Analyte LoDs were established using "artificial respiratory medium" (ARM) as sample matrix surrogate for aspirates. Inclusivity testing was performed by contrived sample matrix. Test concentrations are given as multiples of claimed ARM LoDs. PBS and ARM LoDs are comparable for most analytes PBS and ARM LoDs differ by a factor between 0.3-3.

b Concentration of C. pneumoniae strains was determined as IFU (inclusion-forming units) / mL.

For Mycoplasma pneumoniae inclusivity testing different source materials were used: strain cultures determined in CCU (color changing units) / mL or CFU / mL, or a genomic DNA extract (in copies / mL); test concentrations are referenced against an LoD of 1x103 CCUmL that correlates approximately to 1x106 CFU / mL or 1x106 copies / mL.

Sequencing of one P. mirabilis and one P. vulgaris strain with reduced performance revealed single mismatches for internal probes.

C Test concentrations for P. hauseri and P. penneri are shown as LoD multiples as determined for P. vulgaris.

One strain did not perform consistently at concentrations close to the LoD; sequencing attempts have failed.

8 Strain ATCC 13637 (type strain) was used to establish LoD. During inclusivity testing, single failures at concentrations above LoD were observed, although no probe mismatches were present. Inclusivity testing was repeated for this strain at 1x ARM LoD and 2/2 positive replicates were obtained.

Strain DSM-50173 showed reduced sensitivity and sequencing revealed multiple mismatches for one internal probe.

Strain DSM-21874 showed reduced sensitivity, however, sequencing did not reveal any mismatches to primers or probes. Testing was repeated for this strain at 1x LoD (0/2), 1.7x (0/2) and 3.3x LoD (2/2).

To supplement inclusivity testing for each LRT panel microorganism target, in-silico analysis was performed to assess Unyvero LRT primer and probe sequences for the predicted detection of microorganism strains with applicable sequences available in the GenBank database (search performed January 2018).

Results from the in-silico analyses identified strain entries that are predicted to be

19

detected when the organism is present at LoD concentrations (match of relevant primer and hybridization probe sequences), predicted to be detected with reduced sensitivity (typically, single relevant primer or probe sequence mismatches; detection likely at higher than LoD concentrations only) or predicted to be not detected at clinically relevant concentrations (multiple relevant primer mismatches in primer and probe sequences).

Table 7 lists microorganisms that were evaluated in inclusivity testing that are also predicted to be detected at LoD concentrations based on in-silico analysis. Table 8 lists microorganisms evaluated in inclusivity testing for which one or more strain entries are predicted to be detected with reduced sensitivity.

Additional microorganisms were evaluated by in-silico analysis only. For these microorganisms, Table 9 lists those that are predicted to be detected when present at LoD concentrations and Table 10 lists microorganisms that are predicted to be detected with reduced sensitivity for one or more strain entries.

The following language was included in the device labeling along with results from the in-silico analyses performed:

. In-silico analysis results were provided as supplementary data. The results are not intended to be a surrogate for wet testing and do not assure that specific strains will be detected.
. The performance of the Unyvero LRT Application has not been established for those microorganism species that were evaluated by in-silico analysis only.

Microorganism	Microorganism
Acinetobacter baumannii	Mycoplasma pneumoniae
Acinetobacter calcoaceticus	Proteus mirabilis
Acinetobacter lwoffii	Proteus vulgaris
Acinetobacter haemolyticus	Proteus hauseric
Chlamydia pneumoniae	Proteus penneric
Enterobacter cloacaea	Pseudomonas aeruginosa
Escherichia coli	Serratia marcescens
Klebsiella pneumoniaeb	Staphylococcus aureus
Klebsiella variicola	Streptococcus pneumoniaed

Table 7: Microorganisms with reference strains detected at or near LoD concentrations in inclusivity wet testing that are predicted to be detected at LoD based on in-silico analysis for all strain entries

ª Including E. cloacae ssp. dissolvens

b Including K. pneumoniae variant 2 (K. quasipneumoniae)

6 No Genbank entries available, BLAST search was performed using the whole genome shotgun (wgs) database d In-silico analysis includes serotype 7F

20

Table 8: Microorganisms with reference strains detected at or near LoD concentrations in the Inclusivity study and are predicted to be detected with reduced sensitivity for one or more strain entries

| Microorganism | # entries predicted to be detected
at LoD | # entries predicted to be detected with
reduced sensitivity |
|------------------------------|----------------------------------------------|----------------------------------------------------------------|
| Citrobacter freundii | 11 | 3 |
| Enterobacter asburiae | - | 9 |
| Enterobacter hormaechei | 11a | 1 |
| Klebsiella oxytoca | 8 | 3 |
| Legionella pneumophila | 31 | 36b |
| Moraxella catarrhalis | 11 | 4 |
| Morganella morganii | 1 | 5 |
| Stenotrophomonas maltophilia | 17 | 2 |

a In-silico analysis includes subspecies. oharae and steigerwaltii

8 Inclusivity wet testing included three strains predicted with reduced sensitivity by in-silico analysis (ATCC 33152, ATCC 33154); all strains were detected at concentrations at LoD.

Table 9: Microorganisms predicted to be detected at LoD based on in-silico analysis only

Microorganism
Acinetobacter nosocomialis	Acinetobacter oleivorans
Acinetobacter pittii	Acinetobacter schindleri
Acinetobacter junii	Enterobacter kobei
Acinetobacter parvus	Enterobacter ludwigii
Acinetobacter lactucae	Enterobacter xiangfangensis

Table 10: Microorganisms predicted to be detected with reduced sensitivity for one or more strain entries based on in-silico analysis only

| Microorganism | # entries predicted to be detected at
LoD | # entries predicted to be detected with
reduced
sensitivity |
|--------------------------|----------------------------------------------|-------------------------------------------------------------------|
| Acinetobacter ursingii | - | 9 a,b |
| Acinetobacter soli | - | 1 |
| Acinetobacter guillouiae | - | 1 |

a No Genbank entries available, BLAST search was performed using the whole genome shotgun (wgs) database

b Tests with reference strain DSM-16037 were negative at 107 CFU/mL

Based on the results from the Inclusivity study and in-silico analyses for microorganism targets, the following limitation is included in the Unyvero LRT labeling:

. Based on in-silico analyses, some strains may either not be detected or be detected with reduced sensitivity due to variations in targeted sequences for

21

the following microorganisms: C freundii, K. oxytoca, L. pneumophilia, M. catarrhalis, M. morganii. Based on wet testing for Proteus spp. and S. marcescens and based on in-silico/wet testing for M. catarrhalis and S. maltophilia some strains may be detected with reduced sensitivity due to variations in targeted sequences.

Inclusivity/Resistance Marker Targets:

Inclusivity testing for resistance marker targets included testing of samples prepared with well-characterized strains determined to carry resistance markers targeted by the Unyvero LRT Application. Samples were prepared at near LoD concentrations ( To supplement inclusivity testing for specific resistance marker variants and subgroups, in-silico analysis was performed with Unyvero LRT primer and probe sequences compared to sequences available in the GenBank database. Variants predicted to be detected at LoD (match of relevant primer and probe sequences), variants predicted to be detected with reduced sensitivity (typically, single relevant

24

mismatches of primer or probe sequences; detection likely at higher than LoD concentrations only), and variants predicted to be not detected at clinically relevant concentrations (multiple relevant mismatches in primer and probe sequences) are listed in Table 12.

The following statements are included in the assay labeling:

In-silico analysis results were provided as supplementary data. Results are not . intended to be a surrogate for wet testing and do not assure that specific resistance marker variants will be detected by the assay.
. The performance of the Unyvero LRT Application has not been established for those resistance marker variants that were evaluated by in-silico analysis only.

| Resistance Marker:
Subgroup | Variants predicted at LoD | Variants predicted at
reduced sensitivity | Variants not predicted |
|-------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| ctx-M:
ctx-M1 subgroup | 1, 3, 10, 11, 15, 22, 23, 28-
30, 32-34, 36, 37, 42, 52-
55, 57, 58, 61, 66, 69, 71,
72, 79, 80, 83, 88, 101, 103,
108, 109, 114, 116, 117,
132, 136, 138, 139, 142,
144, 150, 155-158, 162-
164, 166, 167, 170, 172,
173, 175-177, 179-184,
186, 188, 189, 190 | 12, 60, 62, 64, 68, 82, 96,
107, 133, 169 | - |
| ctx-M:
ctx-M2
ctx-M8
ctx-M9
ctx-M25
ctx-M45
subgroups | - | - | 2, 4-9, 13, 14, 16-21, 24-27,
31, 35, 38-41, 43-51, 56,
59, 63, 65, 67, 73-78, 81,
84-87, 89-95, 97-100, 102,
104-106, 110-113, 115,
121-126, 129-131, 134,
137, 141, 147, 148, 152,
159-161, 165, 168, 171,
174, 185, 191 |
| kpc | 1-32 | - | - |
| ndm | 1, 3-19, 21 | 2 | - |
| oxa:
oxa-23 | 23, 27, 49, 73, 134, 146,
165-171, 225, 239, 366,
398, 422, 423, 435, 440,
469, 481, 482, 483, 565 | 103, 133 | - |
| oxa:
oxa-24 | 24-26, 33, 40, 72, 139, 160,
207, 437 | - | - |
| 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, 566, 567 | 204, 547 | 54, 436, 535 |
| oxa:
oxa-58 | 58, 96, 97, 164, 397, 467 | 512 | 420 |

Table 12: In-silico predicted detection, resistance marker variants

25

| Resistance Marker:
Subgroup | Variants predicted at LoD | Variants predicted at
reduced sensitivity | Variants not predicted |
|--------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|------------------------|
| tem | 1-4, 6, 8-12, 15-17, 19, 20-
22, 24, 26, 28-30, 32-36,
40, 43, 45, 47-49, 52-55,
57, 60, 63, 67, 68, 70-72,
76-88, 90-96, 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 | 97, 98, 99, 151, 163 | 178 |
| vim | 1-4, 6, 8-12, 14, 15-20, 23,
24, 26-29, 30, 31, 33-37,
39-46, 48, 50-52, 54, 55 | 5, 25, 38, 49 | 7, 13, 47 |

Although the Unyvero LRT Application can detect multiple tem variants as shown in Table 13 above, positive or negative tem results are reported by the assay only if H. influenzae is concurrently detected in the specimen. It is noted that the tem-1 variant is carried by H. influenzae while other gram negative rods species can carry tem-1 or other tem variants. If the source of positive tem result is not H. influenzae and H. influenzae is not detected, the tem results will not be reported by the LRT software (i.e., it will be masked). However, if the source of a positive tem result is another microorganism and H. influenzae is also detected in the specimen, the tem result will be reported.

The following limitation is included in the Unyvero LRT Application labeling:

Because the tem gene is ubiquitous in members of the Enterobacteriaceae. . positive LRT results for tem may be due to the presence of Enterobacteriaceae in the specimen.
In summary, based on the results from wet-testing and in-silico analyses for targeted resistance markers, the following limitation is included in the Unyvero LRT labeling:
. Wet testing was not performed for all known resistance marker types and/or subtypes. Based on in-silico analyses and inclusivity wet testing, some antibiotic resistance marker variants may either not be detected or detected with reduced sensitivity due to variations in targeted sequences for ctx-M1 subgroup, ndm. oxa-23. oxa-48. oxa-58. vim.
The potential for false negative results for targeted resistance markers is further mitigated by inclusion of the following limitation in the Unyvero LRT Application labeling and report:
Antimicrobial resistance may occur via multiple mechanisms other than the . resistance markers detected by the Unyvero LRT assay. Negative results for LRT resistance markers do not indicate antimicrobial susceptibility of

26

detected organisms.

Cross-Reactivity/Exclusivity: f.

A study was conducted to evaluate the potential for cross-reactivity (exclusivity) of Unyvero LRT Application targets with closely related microorganisms as well as commensal microorganisms that are commonly present in the respiratory tract. Study samples were prepared with microorganisms at high concentrations (~107 CFU/mL) and testing was performed in duplicate.

In addition to testing of exclusivity samples, in-silico (BLAST) analysis was used to evaluate the potential for cross-reactivity for any microorganism strain entries with applicable sequences available in the Gen-Bank database.

The following cross-reactivity with Unyvero LRT targets was either observed in the evaluation of test samples or predicted based on in-silico analysis.

. Cross-reactivity with the Citrobacter freundii target is predicted with Citrobacter braakii and Kluyvera georgiana.
. Cross-reactivity with the Enterobacter cloacae complex target is predicted with Enterobacter soli, Enterobacter mori, and Enterobacter nickellidurans.
Cross-reactivity with the E. coli target is predicted with Shigella dysenteriae, . Shigella boydii, Shigella flexneri, Shigella sonnei, Escherichia albertii and Escherichia fergusonii.
. Cross-reactivity with the Haemophilus influenzae target was demonstrated with Haemophilus haemolyticus and Haemophilus parainfluenzae. Note that wet- testing of one Haemophilus parainfluenzae strain generated expected negative results.
. Cross-reactivity with the Klebsiella oxytoca target is predicted with Klebsiella michagenensis.
Cross-reactivity with the Staphylococcus aureus target is predicted with . Staphylococcus argenteus and Staphylococcus simiae.
. Cross-reactivity with the Stenotrophomonas maltophilia target is predicted with Stenotrophomonas nitritireducens, Stenotrophomonas daejeonensis, Stenotrophomonas acidaminiphila, Stenotrophomonas koreensis and Stenotrophomonas rhizophila. All Stenotrophomonas species predicted to be cross-reactive are not associated with human respiratory infection.

Based on in-silico analysis, no cross-reactivity is predicted for the following LRT panel targets: Acinetobacter spp., C. pneumoniae, K. pneumoniae, K. variicola, L. pneumophila, M. catarrhalis, M. morganii, M. pneumoniae, Proteus spp., P. aeruginosa, S. marcescens, ctx-M, kpc, ndm, oxa-23, oxa-24, oxa-48, oxa-58, tem, vim, and mecA.

The following limitation regarding cross-reactivity of the Unyvero LRT Application is include in device labeling:

27

. Based on in-silico analysis and exclusivity wet testing, the following LRT panel microorganism assays are expected to cross-react with closely related clinically relevant species: Citrobacter freundii (cross-reactive to C. braakii, Khyvera georgiana;), Escherichia coli (cross-reactive to E. albertii, E. fergusonii and Shigella spp. (S. dysenteriae, S. sonnei, S. flexneri, S. boydii)), Haemophilus influenzae (cross-reactive to H. haemolyticus and H. parainfluenzae), Klebsiella oxytoca (cross-reactive to K. michiganensis).
Results of in-silico analysis and wet-testing for near-neighbor microorganism strains is shown in Table 13.

No cross reactivity was observed in testing of the commensal microorganism strains listed in Table 14.

| Close Neighbor Strain | Cross-Reactivity Prediction
(in-silico analysis) | Wet Testing
Result
at 107
CFU/mL,
Strain ID | Cross-Reactions
observed in Clinical
Study
(N = number of
specimens) |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------|
| Citrobacter freundii | Detection predicted at higher
than LoD concentrations b | - | - |
| Citrobacter braakii | Detection predicted at higher
than LoD concentrations | - | - |
| Kluyvera georgiana | Detection predicted at higher
than LoD concentrations | - | - |
| Citrobacter koseri | Detection not predicted | negative
ATCC 27156 | - |
| Enterobacter cloacae complex | - | - | - |
| Enterobacter soli a | Detection predicted at LoD | - | - |
| Enterobacter mori a | Detection predicted at LoD | - | - |
| Enterobacter nickellidurans a | Detection predicted at LoD | - | - |
| Escherichia coli | Detection predicted at LoD | - | - |
| Shigella dysenteriae a | Detection predicted at LoD | - | - |
| Shigella boydii a | Detection predicted at LoD | - | - |
| Shigella flexneri a | Detection predicted at LoD | - | - |
| Shigella sonnei a | Detection predicted at LoD | - | - |
| Escherichia albertii | Detection predicted at LoD | - | - |
| Escherichia fergusonii | Detection predicted at LoD | - | - |
| Haemophilus influenzae | - | - | - |
| Haemophilus haemolyticus | Detection predicted at higher
than LoD concentrations | positive
ATCC 33390 | 2 |
| Haemophilus parahaemolyticus | Detection not predicted | negative
ATCC 10014 | - |
| Haemophilus parainfluenzae | Detection not predicted | negative
ATCC 33392 | 1 |
| Aggregatibacter actino-
mycetemcomitans | Detection not predicted | negative
ATCC 33384 | - |
| Close Neighbor Strain | Cross-Reactivity Prediction
(in-silico analysis) | Wet Testing
Result
at 107
CFU/mL,
Strain ID | Cross-Reactions
observed in Clinical
Study
(N = number of
specimens) |
| Aggregatibacter aphrophilus | Detection not predicted | negative
ATCC 19415 | - |
| Klebsiella oxytoca | | - | - |
| Klebsiella michiganensis | Detection predicted at LoD/
Detection predicted at higher
than LoD concentrations c | - | - |
| Staphylococcus aureus | | - | - |
| Staphylococcus argenteus a | Detection predicted at LoD | - | - |
| Staphylococcus simiae a | Detection predicted at LoD | - | - |
| CNS:
S. epidermidis
S. capitis
S. lugdunensis
S. haemolyticus
S. saprophyticus | Detection not predicted | negative
ATCC 51625
ATCC 27840
ATCC 43809
ATCC 29970
ATCC 15305 | - |
| Stenotrophomonas maltophilia | | | |
| Stenotrophomonas spp.,
(environmental/soil microorganisms):a
S. nitritireducens
S. daejeonensis
S. acidaminiphila
S. koreensis
S. rhizophila | Detection predicted at LoD | - | - |
| Xanthomonas spp. a | Detection predicted at higher
than LoD concentrations- | - | - |
| Pseudoxanthomonas spp. a | Detection predicted at higher
than LoD concentrations- | - | - |
| Streptococcus pneumoniae | | | |
| other Streptococcus sp.:
S. agalactiae
S. anginosus
S. dysgalactiae
S. gordonii
S. intermedius
S. mitis
S. mutans
S. oralis
S. parasanguinis
S. pseudopneumoniae
S. pyogenes
S. salivarius
S. sanguinis
S. vestibularis | Detection not predicted | negative
ATCC 13813
ATCC 33397
ATCC 43078
ATCC 10558
ATCC 27335
ATCC 49456
ATCC 25175
ATCC 35037
ATCC 15912
ATCC BAA-
960
ATCC 12344
ATCC 7073
ATCC 10556 | - |

Table 13: Exclusivity testing: In-silico prediction, wet-testing of exclusivity samples, and cross-reactivity observed in the clinical study

28

ª No clinical relevance for respiratory infections

b Strains are predicted to be detected at higher than LoD concentrations due to primer and probe mismatches

E. coli ),
NRZ-00223 (9), Micromyx 4653 (10),
Micromyx 4676 ( K. pneumoniae ) (10) | 3 x 106 (6x) | 25/25 | 100.0
(86.7 - 100.0) | | |
| | total | 48/49 | 98.0
(89.3 - 99.6) | 245/245 | 100.0
(98.5 - 100.0) |
| ndm
JMI 50067 (6) ( E. coli ), NCTC 13443 (6),
JMI 49831 (6) ( K. pneumoniae ), JMI 49755
(5) ( A. baumannii ), JMI 46239 (6) ( E. cloacae ) | 1 x 105 (2x) | 13/14 | 92.9
(68.5 - 98.7) | | |
| | 5 x 105 (10x) | 15/15 | 100.0
(79.6 - 100.0) | | |
| | total | 28/29 | 96.6
(82.8 - 99.4) | 237/263 e | 90.1
(85.9 - 93.2) |
| oxa-23
Micromyx 4410 (6), Micromyx 6148 (6),
Micromyx 6149 (4), Micromyx 6153 (6),
UCLA A5 (6) ( A. baumannii ) (6 each) | 1 x 107 (0,5x) | 13/14 | 92.9
(68.5 - 98.7) | | |
| | 2 x 107 (1x) | 14/14 | 100.0
(78.5 - 100.0) | | |
| | total | 27/28 | 96.4
(82.3 - 99.4) | 139/140 | 99.3
(96.1 - 99.9) |
| oxa-24
NCTC 13302, NRZ-00449, UCLA A4, two
clinical isolates ( A. baumannii ) (10 each) | 6 x 104 (0,1x) | 25/25 | 100.0
(86.7 - 100.0) | | |
| | 3 x 105 (0,6x) | 25/25 | 100.0
(86.7 - 100.0) | | |
| | total | 50/50 | 100.0
(92.9 - 100.0) | 90/90 | 100.0
(95.9 - 100.0) |
| oxa-48
NRZ-00176 (19), ATCC BAA-2523 (10) ( E. coli ), NCTC 13442 (10), NRZ-00002 (10)
( K. pneumoniae ) | 4 x 106 (2x) | 24/24 | 100.0
(86.2 - 100.0) | | |
| | 1 x 107 (5x) | 24/25 | 96.0
(80.5 - 99.3) | | |
| | total | 48/49 | 98.0
(89.3 - 99.6) | 205/205 | 100.0
(98.2 - 100.0) |
| oxa-58
NCTC 13305 (18), NRZ-00518 (12)
( A. baumannii ) | 4 x 105 (0,5x) | 15/15 | 100.0
(79.6 - 100.0) | | |
| | 1 x 106 (1.3x) | 15/15 | 100.0
(79.6 - 100.0) | | |
| | total | 30/30 | 100.0
(88.7 - 100.0) | 138/138 | 100.0
(97.3 - 100.0) |
| vim
NRZ-00452 (10) ( C. freundii ), NRZ-00239
(20) ( E. cloacae ),DSM-24600 (19)
( P. aeruginosa ) | 1 x 105 (2x) | 22/24 | 91.7
(74.2 - 97.7) | | |
| | 3 x 105 (6x) | 25/25 | 100.0
(86.7 - 100.0) | | |
| | total | 47/49 | 95.9
(86.3 - 98.9) | 234/234 | 100.0
(98.4 - 100.0) |

49

a total of 50 tests (for C. freundii, L. pneumophila, M. pneumoniae, cx-M, kpc, oxa-24, oxa-48. vin) or 30 tests (for K. oxytoca, K. variicola, M. morganii, ndm, oxa-23, oxa-58) or C. pneumoniae) was performed. Missing results were due to invalid test results.

50

0 Numbers in parentheses indicate number of tests performed for an individual strain

C IFU: inclusion-forming units

4 CCU: color-changing units, concentration used for ATCC 15492: 1 x 106 and 3 x 10° CFU/mL (1 CCU/mL was estimated to be equivalent to 10 CFU/mL)

° Eight positive ctx-M results and 24 positive ndm results were linked to contamination in test materials

Clinical Performance, Resistance Marker Targets

Performance characteristics for LRT Application antibiotic resistance marker targets were evaluated in the prospective study (603 aspirate specimens) and supplemented with results from contrived specimens (results shown in Table 29 above).

To assess the performance of the Unyvero LRT Application for detection of each resistance marker target, positive and negative percent agreement was calculated as compared to results of validated multiplex PCR assays followed by bi-directional sequencing.

It is noted that antibiotic resistance marker results are only reported by the LRT Application if at least one corresponding host microorganism is simultaneously detected. If an applicable microorganism is not detected in the specimen, positive resistance marker results are masked on the results screen (i.e., the result is masked and the report indicates N/A regardless whether the resistance marker is detected or not detected). Evaluation of assay performance for detection of resistance markers by the LRT Application was performed both with and without application of masking/reporting rules.

Table 30 includes performance of the LRT assay for detection of resistance marker targets as observed in the prospective study and compared to PCR/bi-directional sequencing. Analysis includes all positive resistance marker results (i.e., without software masking).

51

Table 30: Prospective Study, Resistance marker performance as compared to molecular comparator assays (multiplex PCR followed by bi-directional sequencing), without application of software masking

| | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|--------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 15/16 | 93.8
(71.7 - 98.9) | 584/587 | 99.5
(98.5 - 99.8) |
| kpc | 7/7 | 100.0
(64.6 - 100.0) | 596/596 | 100.0
(99.4 - 100.0) |
| ndm | 0/0 | na | 603/603 | 100.0
(99.4 - 100.0) |
| oxa-23 | 6/7 | 85.7
(48.7 - 97.4) | 594/596 | 99.7
(98.8 - 99.9) |
| oxa-24 | 2/3 | 66.7
(20.8 - 93.9) | 600/600 | 100.0
(99.4 - 100.0) |
| oxa-48 | 0/0 | na | 602/603 | 99.8
(99.1 - 100.0) |
| oxa-58 | 0/0 | na | 603/603 | 100.0
(99.4 - 100.0) |
| tem | 54/54 | 100.0
(93.4 - 100.0) | 537/549 | 97.8
(96.2 - 98.7) |
| vim | 2/2 | 100.0
(34.2 - 100.0) | 601/601 | 100.0
(99.4 - 100.0) |
| mecA | 108/124 | 87.1
(80.1 - 91.9) | 453/479 | 94.6
(92.2 - 96.3) |

Table 31 shows LRT performance in the prospective study for each targeted resistance marker based on comparison to molecular comparator assays (PCR/bi-directional sequencing) for only those specimens in which an applicable LRT microorganism target was detected by the LRT assay (i.e., results shown are after application of software masking). Based on the reporting rules for the Unyvero LRT assay, performance for ctx-M, blaxec, blands and blaym includes specimens that were positive by the Unyvero LRT Application for a targeted microorganism of the Enterobacteriaceae, Acinetobacter spp. and/or P. geruginosa. For oxa-48. performance includes only those specimens that were positive for one or more of the Enterobacteriaceae targets. For oxa-23, oxa-24 and oxa-58. the performance includes only those specimens that were positive for Acinetobacter spp. For blaTEM, performance includes only those specimens that were positive for H. influenzae. For mecA, performance includes only those specimens that were positive for S. aureus.

52

Table 31: Prospective Study, Resistance marker performance as compared to molecular comparator assays (multiplex PCR followed by bi-directional sequencing), with application of masking.

| | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) | Not reported
(masked) |
|--------|----------------------------|-------------------------|----------------------------|-------------------------|--------------------------|
| ctx-M | 15/16 | 93.8
(71.7 - 98.9) | 177/179 | 98.9
(96.0 - 99.7) | 408 |
| kpc | 6/6 | 100.0
(61.0 - 100.0) | 189/189 | 100.0
(98.0 - 100.0) | 408 |
| ndm | 0/0 | na | 195/195 | 100.0
(98.1 - 100.0) | 408 |
| oxa-23 | 6/7 | 85.7
(48.7 - 97.4) | 18/20 | 90.0
(69.9 - 97.2) | 576 |
| oxa-24 | 2/2 | 100.0
(34.2 - 100.0) | 25/25 | 100.0
(86.7 - 100.0) | 576 |
| oxa-48 | 0/0 | na | 138/139 | 99.3
(96.0 - 99.9) | 464 |
| oxa-58 | 0/0 | na | 27/27 | 100.0
(87.5 - 100.0) | 576 |
| tem | 8/8 | 100.0
(67.6 - 100.0) | 16/16 | 100.0
(80.6 - 100.0) | 579 |
| vim | 2/2 | 100.0
(34.2 - 100.0) | 193/193 | 100.0
(98.0 - 100.0) | 408 |
| mecA | 54/59 | 91.5
(81.6 - 96.3) | 53/61 | 86.9
(76.2 - 93.2) | 483 |

Tables 32-41 include performance for resistance marker targets for each applicable microorganism detected by the LRT assay. Performance for each resistance marker target is evaluated as compared to PCR/bi-directional sequencing. Each table includes only the subset of specimens that are positive by LRT for the specified microorganism target; therefore, the results shown are after application of software masking.

53

Table 32: Prospective Study, Resistance marker performance as compared molecular comparator assays, stratified for LRT positive samples for Acinetobacter spp. (N=27). Note that detection of each resistance marker cannot be definitively linked to Acinetobacter spp.

| Acinetobacter
spp. | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|-----------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 5/5 | 100.0
(56.6 - 100.0) | 21/22 | 95.5
(78.2 - 99.2) |
| kpc | 2/2 | 100.0
(34.2 - 100.0) | 25/25 | 100.0
(86.7 - 100.0) |
| ndm | 0/0 | na | 27/27 | 100.0
(87.5 - 100.0) |
| oxa-23 | 6/7 | 85.7
(48.7 - 97.4) | 18/20 | 90.0
(69.9 - 97.2) |
| oxa-24 | 2/2 | 100.0
(34.2 - 100.0) | 25/25 | 100.0
(86.7 - 100.0) |
| oxa-58 | 0/0 | na | 27/27 | 100.0
(87.5 - 100.0) |
| vim | 1/1 | 100.0
(20.7 - 100.0) | 26/26 | 100.0
(87.1 - 100.0) |

Table 33: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for Citrobacter freundii (N=3)

| Citrobacter
freundii | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|-------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 0/0 | na | 3/3 | 100.0
(43.9 - 100.0) |
| kpc | 0/0 | na | 3/3 | 100.0
(43.9 - 100.0) |
| ndm | 0/0 | na | 3/3 | 100.0
(43.9 - 100.0) |
| oxa-48 | 0/0 | na | 3/3 | 100.0
(43.9 - 100.0) |
| vim | 0/0 | na | 3/3 | 100.0
(43.9 - 100.0) |

54

Table 34: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for Enterobacter cloacae complex (N=20). Note that detection of each resistance marker cannot be definitively linked to E. cloacae complex.

| Enterobacter
cloacae
complex | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|------------------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 0/0 | na | 20/20 | 100.0
(83.9 - 100.0) |
| kpc | 1/1 | 100.0
(20.7 - 100.0) | 19/19 | 100.0
(83.2 - 100.0) |
| ndm | 0/0 | na | 20/20 | 100.0
(83.9 - 100.0) |
| oxa-48 | 0/0 | na | 20/20 | 100.0
(83.9 - 100.0) |
| vim | 0/0 | na | 20/20 | 100.0
(83.9 - 100.0) |

Table 35: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for K. oxytoca (N=15). Note that detection of each resistance marker cannot be definitively linked to K. oxytoca.

| Klebsiella
oxytoca | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|-----------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 0/0 | na | 14/15 | 93.3
(70.2 - 98.8) |
| kpc | 0/0 | na | 15/15 | 100.0
(79.6 - 100.0) |
| ndm | 0/0 | na | 15/15 | 100.0
(79.6 - 100.0) |
| oxa-48 | 0/0 | na | 15/15 | 100.0
(79.6 - 100.0) |
| vim | 0/0 | na | 15/15 | 100.0
(79.6 - 100.0) |

55

Table 36: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for K. pneumoniae (N=38). Note that detection of each resistance marker cannot be definitively linked to K. pneumoniae.

| Klebsiella
pneumoniae | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|--------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 8/8 | 100.0
(67.6 - 100.0) | 29/30 | 96.7
(83.3 - 99.4) |
| kpc | 2/2 | 100.0
(34.2 - 100.0) | 36/36 | 100.0
(90.4 - 100.0) |
| ndm | 0/0 | na | 38/38 | 100.0
(90.8 - 100.0) |
| oxa-48 | 0/0 | na | 38/38 | 100.0
(90.8 - 100.0) |
| vim | 0/0 | na | 38/38 | 100.0
(90.8 - 100.0) |

Table 37: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for K. variicola (N=4). Note that detection of each resistance marker cannot be definitively linked to K. variicola.

| Klebsiella
variicola | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|-------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 0/0 | na | 4/4 | 100.0
(51.0 - 100.0) |
| kpc | 0/0 | na | 4/4 | 100.0
(51.0 - 100.0) |
| ndm | 0/0 | na | 4/4 | 100.0
(51.0 - 100.0) |
| oxa-48 | 0/0 | na | 4/4 | 100.0
(51.0 - 100.0) |
| vim | 0/0 | na | 4/4 | 100.0
(51.0 - 100.0) |

56

Table 38: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for Morganella morganii (N=10). Note that detection of each resistance marker cannot be definitively linked to Morganella morganii.

| Morganella
morganii | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 2/2 | 100.0
(34.2 - 100.0) | 8/8 | 100.0
(76.6- 100.0) |
| kpc | 2/2 | 100.0
(34.2 - 100.0) | 8/8 | 100.0
(67.6 - 100.0) |
| ndm | 0/0 | na | 10/10 | 100.0
(72.3 - 100.0) |
| oxa-48 | 0/0 | na | 9/10 | 90.0
(59.6 - 98.2) |
| vim | 1/1 | 100.0
(20.7 - 100.0) | 9/9 | 100.0
(70.1 - 100.0) |

Table 39: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for Proteus spp. (N=29). Note that detection of each resistance marker cannot be definitively linked to Proteus spp.

| Proteus
spp. | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|-----------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 3/3 | 100.0
(43.9 - 100.0) | 26/26 | 100.0
(87.1 - 100.0) |
| kpc | 1/1 | 100.0
(20.7 - 100.0) | 28/28 | 100.0
(87.9 - 100.0) |
| ndm | 0/0 | na | 29/29 | 100.0
(88.3 - 100.0) |
| oxa-48 | 0/0 | na | 29/29 | 100.0
(88.3 - 100.0) |
| vim | 1/1 | 100.0
(20.7 - 100.0) | 28/28 | 100.0
(87.9 - 100.0) |

57

Table 40: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for Pseudomonas aeruginosa (N=84). Note that detection of each resistance marker cannot be definitively linked to Pseudomonas aeruginosa.

| Pseudomonas
aeruginosa | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|---------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 9/9 | 100.0
(70.1 - 100.0) | 75/75 | 100.0
(95.1 - 100.0) |
| kpc | 3/3 | 100.0
(43.9 - 100.0) | 81/81 | 100.0
(95.5 - 100.0) |
| ndm | 0/0 | na | 84/84 | 100.0
(95.6 - 100.0) |
| oxa-48 | 0/0 | na | 83/84 | 98.8
(93.6 - 99.8) |
| vim | 2/2 | 100.0
(34.2 - 100.0) | 82/82 | 100.0
(95.5 - 100.0) |

Table 41: Prospective Study, Resistance marker performance as compared to molecular comparator assays, stratified for LRT positive samples for Serratia marcescens (N=25). Note that detection of each resistance marker cannot be definitively linked Serratia marcescens.

| Serratia
marcescens | Positivity
TP / (TP+FN) | PPA
[%]
(95 % CI) | Negativity
TN / (TN+FP) | NPA
[%]
(95 % CI) |
|------------------------|----------------------------|-------------------------|----------------------------|-------------------------|
| ctx-M | 1/1 | 100.0
(20.7 - 100.0) | 24/24 | 100.0
(86.2 - 100.0) |
| kpc | 4/4 | 100.0
(51.0 - 100.0) | 21/21 | 100.0
(84.5 - 100.0) |
| ndm | 0/0 | na | 25/25 | 100.0
(86.7 - 100.0) |
| oxa-48 | 0/0 | na | 25/25 | 100.0
(86.7 - 100.0) |
| vim | 0/0 | na | 25/25 | 100.0
(86.7 - 100.0) |

58

For the prospective study, additional analyses of clinical performance for detection of resistance marker targets was conducted in combination with results from microorganism detection. For each marker, two different 3x3 tables were generated; one 3x3 table with Comparator 'A', reflecting culture as the reference method for LRT microorganism targets and one 3x3 table with Comparator 'B', reflecting the composite comparator method (culture plus PCR/bi-directional sequencing) for LRT microorganism targets. For the resistance markers, the comparator method was PCR/bi-directional sequencing for all analyses.

Agreement rates were determined for the following resistance marker/microorganism combinations following the Unyvero LRT reporting rules:

tem: H. influenzae
ctx-M, kpc, vim: All Enterobacteriaceae targets, Acinetobacter spp., and P. . aeruginosa combined
oxa-48: all Enterobacteriaceae targets combined .
oxa-23. oxa-24: Acinetobacter spp. .
mecA: S. aureus .

Numbers of available culture isolates, results of linkage analysis (confirmation that the host microorganism strain is the source of the antibiotic resistance marker determined by PCR/bi-directional sequencing of culture isolates), and the number of multi-detection samples (more than one host microorganism detected by the composite comparator (comparator A) or SoC/culture (comparator B) are indicated as footnotes to each of the agreement tables. Agreement tables are not presented for ndm and oxa-58 as there were no positive results for these analytes by either the LRT or comparator assays.

Results are shown in Tables 42 through 49, with data presented in two tables for each resistance marker (one with Comparator A and one with Comparator B).

59

| H. influenzaec
tem | | Composite Comparator Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|-------------------------------------|------------|--------------------------------------------------------------------------------------------------|------------|-------------|-------|
| | Org+/Res+a | Org+/Res+b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+ | 6 | 0 | 2 | 8 |
| | Org+/Res- | 0 | 9 | 6 | 15 |
| | Org- | 1 | 1 | 577 | 579 |
| total | | 7 | 10 | 585 | 602 |
| Haemophilus influenzae/tem | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 85.7 | 6/7 | 48.7 - 97.4 | |
| Agreement (Org+/Res-) | | 90.0 | 9/10 | 59.6 - 98.2 | |
| Agreement (Org-) | | 98.6 | 577/585 | 97.3 - 99.3 | |

Table 42: Agreement rates for tem between LRT and comparator methods A and B, (single) host microorganism: H. influenzae

4 isolate linkage: 3 culture positive cases, 2 isolates [2 of 2 confirmed]

b multi-detection specimens: not applicable (tem reported only with H. influenzae)

& note that other LRT panel microorganisms (Enterobacter spp., P. aeruginosa) could be the source of tem

B

| H. influenzaec
tem | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|-------------------------------------|------------|-------------------------------------------------------------------------------------|-----------|------------|--------------|
| | | Org+/Res+b | Org+/Res- | Org- | total |
| | Org+/Res+a | 3 | 0 | 5 | 8 |
| Unyvero
Result | Org+/Res- | 0 | 5 | 11 | 16 |
| | Org- | 0 | 0 | 579 | 579 |
| | total | 3 | 5 | 595 | 603 |
| | | Haemophilus influenzae/tem | rate [%] | positivity | 95 % CI |
| | | Agreement (Org+/Res+) | 100 | 3/3 | 43.9 - 100.0 |
| | | Agreement (Org+/Res-) | 100 | 5/5 | 56.6 - 100.0 |
| | | Agreement (Org-) | 97.3 | 579/595 | 95.7 - 98.3 |

ª Isolate linkage: 2 isolates [2 of 2 confirmed]

b Multi-detection specimens: not applicable (tem reported only with H. influenzae)

6 Note that other LRT panel microorganisms (Enterobacter spp., P. aeruginosa could be the source of tem

60

| Corr. Host Microorganism
ctx-M | | Composite Comparator Result for Host Microorganism -
PCR/Seq for Antibiotic Resistance Marker | | | |
|-----------------------------------|------------|--------------------------------------------------------------------------------------------------|------------|-------------|-------|
| | | Org+/Res+b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+a | 15 | 1 | 1 | 17 |
| | Org+/Res- | 1 | 156 | 20 | 177 |
| | Org- | 0 | 16 | 392 | 408 |
| total | | 16 | 173 | 413 | 602 |
| Corr. host microorganism /ctx-M | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 93.8 | 15/16 | 71.7 - 98.9 | |
| Agreement (Org+/Res-) | | 90.2 | 156/173 | 84.8 - 93.8 | |
| Agreement (Org-) | | 94.9 | 392/413 | 92.4 - 96.7 | |

Table 43: Agreement rates for ctx-M between LRT and comparator methods A and B, host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

a At least one isolate available for 10 specimens [linkage confirmed for 5 of 10 specimens]

b Multi-detection specimens (two or more corresponding LRT host microorganisms): 9 of 16

B

| Corr. Host Microorganism
ctx-M | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|-----------------------------------|-------------|-------------------------------------------------------------------------------------|------------|------------|-------------|
| | | Org+/Res+ b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+ a | 11 | 0 | 6 | 17 |
| | Org+/Res- | 1 | 128 | 49 | 178 |
| | Org- | 0 | 10 | 398 | 408 |
| total | | 12 | 138 | 453 | 603 |
| Corr. host microorganism /ctx-M | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | | 91.7 | 11/12 | 64.6 - 98.5 |
| Agreement (Org+/Res-) | | | 92.8 | 128/138 | 87.2 - 96.0 |
| Agreement (Org-) | | | 87.9 | 398/453 | 84.5 - 90.6 |

a At least one isolate available for 10 specimens [linkage confirmed for 5 of 10 specimens]

b Multi-detection specimens (two or more corresponding LRT host microorganisms): 6 of 12

61

| A | | Corr. Host Microorganism
kpc | Composite Comparator Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|---|-----------------------|----------------------------------------|--------------------------------------------------------------------------------------------------|------------|--------------|-------|
| | | | Org+/Res+ b | Org+/Res- | Org- | total |
| | | Org+/Res+ a | 6 | 0 | 0 | 6 |
| | Unyvero
Result | Org+/Res- | 0 | 167 | 21 | 188 |
| | | Org- | 0 | 16 | 392 | 408 |
| | | total | 6 | 183 | 413 | 602 |
| | | Corr. host microorganism / kpc | rate [%] | positivity | 95 % CI | |
| | Agreement (Org+/Res+) | | 100.0 | 6/6 | 61.0 - 100.0 | |
| | Agreement (Org+/Res-) | | 91.3 | 167/183 | 86.3 - 94.5 | |
| | Agreement (Org-) | | 94.9 | 392/413 | 92.4 - 96.7 | |

Table 44: Agreement rates for kpc between LRT and comparator methods A and B, host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

a Sample linkage: at least one isolate available for 5 specimens [linkage confirmed for 4 of 5 specimens] b Multi-detection specimens (two or more corresponding LRT host microorganisms): 4 of 6

B

| Corr. Host Microorganism
kpc | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|---------------------------------|-------------|-------------------------------------------------------------------------------------|-----------|------------|--------------|
| | | Org+/Res+ b | Org+/Res- | Org- | total |
| | Org+/Res+ a | 6 | 0 | 0 | 6 |
| Unyvero | Org+/Res- | 0 | 134 | 55 | 189 |
| Result | Org- | 0 | 10 | 398 | 408 |
| | total | 6 | 144 | 453 | 603 |
| Corr. host microorganism /kpc | | | rate [%] | positivity | 95 % CI |
| Agreement (Org+/Res+) | | | 100 | 6/6 | 61.0 - 100.0 |
| Agreement (Org+/Res-) | | | 93.1 | 134/144 | 87.7 - 96.2 |
| Agreement (Org-) | | | 87.9 | 398/453 | 84.5 - 90.6 |

a Sample linkage: at least one isolate available for 5 specimens [linkage confirmed for 4 of 5 specimens] b Multi-detection specimens (two or more corresponding LRT host microorganisms): 2 of 6

62

| Corr. Host Microorganism
vim | | Composite Comparator Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|----------------------------------------|------------|--------------------------------------------------------------------------------------------------|-----------|------------|--------------|
| | | Org+/Res+b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+a | 2 | 0 | 0 | 2 |
| | Org+/Res- | 0 | 171 | 21 | 192 |
| | Org- | 0 | 16 | 392 | 408 |
| | total | 2 | 187 | 413 | 602 |
| | | Corr. host microorganism/ vim | rate [%] | positivity | 95 % CI |
| | | Agreement (Org+/Res+) | 100.0 | 2/2 | 34.2 - 100.0 |
| | | Agreement (Org+/Res-) | 91.4 | 171/187 | 86.6 - 94.7 |
| | | Agreement (Org-) | 94.9 | 392/413 | 92.4 - 96.7 |

Table 45: Agreement rates for vim between LRT and comparator methods A and B, host microorganisms: Enterobacteriaceae, Acinetobacter spp., P. aeruginosa.

a Sample linkage: at least 1 isolate available for 2 specimens [linkage confirmed for 2 of 2 specimens] b Multi-detection specimens (two or more corresponding LRT host microorganisms): 1 of 2

B

| Corr. Host Microorganism
vim | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|---------------------------------|------------|-------------------------------------------------------------------------------------|------------|--------------|-------|
| | | Org+/Res+b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+a | 2 | 0 | 0 | 2 |
| | Org+/Res- | 0 | 138 | 55 | 193 |
| | Org- | 0 | 10 | 398 | 408 |
| total | | 2 | 148 | 453 | 603 |
| Corr. host microorganism/vim | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 100.0 | 2/2 | 34.2 - 100.0 | |
| Agreement (Org+/Res-) | | 93.2 | 138/148 | 88.0 - 96.3 | |
| Agreement (Org-) | | 87.9 | 398/453 | 84.5 - 90.6 | |

a Sample linkage: at least 1 isolate available for 2 specimens [linkage confirmed for 2 of 2 specimens] b Multi-detection specimens (two or more corresponding LRT host microorganisms): 1 of 2

63

Table 46: Agreement rates for oxa-48 between LRT and comparator methods A and
B, host microorganisms: Enterobacteriaceae.

| A | Corr. Host Microorganism
oxa-48 | | Composite Comparator Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|---|------------------------------------|------------|--------------------------------------------------------------------------------------------------|----------|------------|-------------|
| | | Org+/Res+b | Org+/Res- | Org- | total | |
| | Unyvero
Result | Org+/Res+a | 0 | 0 | 1 | 1 |
| | | Org+/Res- | 0 | 119 | 19 | 138 |
| | | Org- | 0 | 10 | 453 | 463 |
| | total | | 0 | 129 | 473 | 602 |
| | Corr. host microorganism/oxa-48 | | | rate [%] | positivity | 95 % CI |
| | Agreement (Org+/Res+) | | | na | 0/0 | na |
| | Agreement (Org+/Res-) | | | 92.2 | 119/129 | 86.3 - 95.7 |
| | Agreement (Org-) | | | 95.8 | 453/473 | 93.6 - 97.2 |

ª Sample linkage: N/A, no culture positive specimens, no isolates

b Multi-detection specimens (two or more corresponding host microorganisms): none

B

| Corr. Host Microorganism
oxa-48 | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|------------------------------------|-------------|-------------------------------------------------------------------------------------|------------|-------------|-------|
| | | Org+/Res+ b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+ a | 0 | 0 | 1 | 1 |
| | Org+/Res- | 0 | 80 | 59 | 139 |
| | Org- | 0 | 8 | 455 | 463 |
| | total | 0 | 88 | 515 | 603 |
| Corr. host microorganism/oxa-48 | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | na | 0/0 | na | |
| Agreement (Org+/Res-) | | 90.9 | 80/88 | 83.1 - 95.3 | |
| Agreement (Org-) | | 88.3 | 455/515 | 85.3 - 90.8 | |

^a Sample linkage: N/A, no culture positive specimens, no isolates

ª Sample linkage: N/A, no culture positive specimens, no isolates
b Multi-detection specimens (two or more corresponding host microorganisms): none

64

| A | Acinetobacter spp.
oxa-23 | | Composite Comparator Result for Host Microorganism
PCR/Seq for Antibiotic Resistance Marker | | |
|---------------------------|------------------------------|------------|------------------------------------------------------------------------------------------------|-------------|-------|
| | | Org+/Res+b | Org+/Res- | Org- | total |
| | Org+/Res+a | 6 | 2 | 0 | 8 |
| Unyvero
Result | Org+/Res- | 1 | 13 | 5 | 19 |
| | Org- | 0 | 1 | 575 | 576 |
| | total | 7 | 16 | 580 | 603 |
| Acinetobacter spp./oxa-23 | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 85.7 | 6/7 | 48.7 - 97.4 | |
| Agreement (Org+/Res-) | | 81.3 | 13/16 | 57.0 - 93.4 | |
| Agreement (Org-) | | 99.1 | 575/580 | 98.0 - 99.6 | |

Table 47: Agreement rates for oxa-23 between LRT and comparator methods A and B, (single) host microorganism: Acinetobacter spp.

a Isolate linkage: 2 culture positive specimens, 2 isolates [2 of 2 confirmed]

b Multi-detection specimens: not applicable (oxa-23 reported only with Acinetobacter spp.)

| Acinetobacter spp.
oxa-23 | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|------------------------------|-------------|-------------------------------------------------------------------------------------|------------|--------------|-------|
| | | Org+/Res+ b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+ a | 2 | 0 | 6 | 8 |
| | Org+/Res- | 1 | 7 | 11 | 19 |
| | Org- | 0 | 0 | 576 | 576 |
| | total | 3 | 7 | 593 | 603 |
| Acinetobacter spp./oxa-23 | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 66.7 | 2/3 | 20.8 - 93.9 | |
| Agreement (Org+/Res-) | | 100.0 | 7/7 | 64.6 - 100.0 | |
| Agreement (Org-) | | 97.1 | 576/593 | 95.5 - 98.2 | |

4 Isolate linkage: 2 isolates [2 of 2 confirmed]

B

b Multi-detection specimens: not applicable (oxa-23 reported only with Acinetobacter spp.)

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| | Acinetobacter spp. | Composite Comparator Result for Host Microorganism –
PCR/Seq for Antibiotic Resistance Marker | | | |
|---------------------------|-----------------------|--------------------------------------------------------------------------------------------------|-----------|------------|--------------|
| | oxa-24 | Org+/Res+ b | Org+/Res- | Org- | total |
| | Org+/Res+ a | 2 | 0 | 0 | 2 |
| Unyvero | Org+/Res- | 0 | 20 | 5 | 25 |
| Result | Org- | 0 | 1 | 575 | 576 |
| | total | 2 | 21 | 580 | 603 |
| Acinetobacter spp./oxa-24 | | | rate [%] | positivity | 95 % CI |
| | Agreement (Org+/Res+) | | 100.0 | 2/2 | 34.2 - 100.0 |
| | | Agreement (Org+/Res-) | 95.2 | 20/21 | 77.3 - 99.2 |
| | | Agreement (Org-) | 99.1 | 575/580 | 98.0 - 99.6 |

Table 48: Agreement rates for oxa-24 between LRT and comparator methods A and B. host microorganisms: Enterobacteriaceae. Acinetobacter spp., P. geruginosa.

alisolate linkage: 1 culture positive specimen, 1 isolate [1 of 1 confirmed]

b Multi-detection specimens: not applicable (oxa-24 reported only with Acinetobacter spp.)

B

| Acinetobacter spp.
oxa-24 | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|------------------------------|------------|-------------------------------------------------------------------------------------|------------|--------------|-------|
| | | Org+/Res+b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+a | 1 | 0 | 1 | 2 |
| | Org+/Res- | 0 | 9 | 16 | 25 |
| | Org- | 0 | 0 | 576 | 576 |
| | total | 1 | 9 | 593 | 603 |
| Acinetobacter spp./oxa-24 | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 100.0 | 1/1 | 20.7 - 100.0 | |
| Agreement (Org+/Res-) | | 100.0 | 9/9 | 70.1 - 100.0 | |
| Agreement (Org-) | | 97.1 | 576/593 | 95.5 - 98.2 | |

ª Isolate linkage: 1 isolate [1 of 1 confirmed]

b Multi-detection specimens: not applicable (oxa-24 reported only with Acinetobacter spp.)

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| | S. aureus
mecA | Composite Comparator Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|-------------------|---------------------------------|--------------------------------------------------------------------------------------------------|-----------|------------|-------------|
| | | Org+/Res+ b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+ a | 53 | 7 | 2 | 62 |
| | Org+/Res- | 5 | 44 | 9 | 58 |
| | Org- | 4 | 6 | 473 | 483 |
| | total | 62 | 57 | 484 | 603 |
| | S. aureus/mecA | | rate [%] | positivity | 95 % CI |
| | Agreement (Org+/Res+) | | 85.5 | 53/62 | 74.7 - 92.2 |
| | Agreement (Org+/Res-) | | 77.2 | 44/57 | 64.8 - 86.2 |
| | Agreement (Org-) | | 97.7 | 473/484 | 96.0 - 98.7 |

Table 49. Agreement rates for mecA between LRT and comparator methods A and B, (single) host microorganism: S. aureus.

a Isolate linkage: 48 culture positive specimens, 36 isolates [29 of 36 confirmed] b Multi-detection specimens: not applicable (mecA only reported with S. aureus)

| S. aureus
mecA | | Culture Result for Host Microorganism +
PCR/Seq for Antibiotic Resistance Marker | | | |
|---------------------------------|-------------|-------------------------------------------------------------------------------------|------------|-------------|-------|
| | | Org+/Res+ b | Org+/Res- | Org- | total |
| Unyvero
Result | Org+/Res+ a | 44 | 4 | 14 | 62 |
| | Org+/Res- | 4 | 29 | 25 | 58 |
| | Org- | 0 | 2 | 481 | 483 |
| | total | 48 | 35 | 520 | 603 |
| S. aureus/mecA | | rate [%] | positivity | 95 % CI | |
| Agreement (Org+/Res+) | | 91.7 | 44/48 | 80.4 - 96.7 | |
| Agreement (Org+/Res-) | | 82.9 | 29/35 | 67.3 - 91.9 | |
| Agreement (Org-) | | 92.5 | 481/520 | 89.9 - 94.5 | |

4 Isolate linkage: 36 isolates [29 of 36 confirmed]

B

b Multi-detection specimens: not applicable (mecA reported only with S. aureus)

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For the prospective study, clinical performance of the Unyvero LRT Application for detection of mecA was also compared to phenotypic antimicrobial susceptibility (AST) testing results for all S. aureus isolates recovered from the reference culture. Culture results were the reference method S. aureus detection and cefoxitin and/or oxacillin AST results were used as the phenotypic reference method for mecA. Results are shown in Table 50 below.

| S. aureus
mecA | | Culture Result for S. aureus and Cefoxitin/Oxacillin AST
results | | | |
|---------------------------------|-----------------------|----------------------------------------------------------------------------|--------------------|------------|-----------|
| | | SA+/Res+
(MRSA) | SA+/Res-
(MSSA) | SA- | total |
| Unyvero
Result | SA+/mecA+ | 40 | 7 | 14 | 61 |
| | SA+/mecA-- | 1 | 31 | 25 | 57 |
| | SA- | 2 | 0 | 481 | 483 |
| | total | 43 | 38 | 520 | 601 |
| | S. aureus/mecA | | rate [%] | positivity | 95 % CI |
| Agreement (SA+/Res+) | | | 93.0 | 40/43 | 81.4-97.6 |
| Agreement (SA+/Res-) | | | 81.6 | 31/38 | 66.6-90.8 |
| Agreement (SA-) | | | 92.5 | 481/520 | 89.9-94.5 |

Table 50: Agreement rates for S. aureus mecA between LRT and phenotypes of corresponding S. aureus strains.

A known limitation for detection of LRT resistance marker targets directly from tracheal aspirate specimens is that detection of markers cannot be definitively linked to corresponding detected microorganisms. For example, on-panel microorganisms present in the specimen that are not detected by LRT or off-panel microorganisms could serve as the source of a detected marker. In addition, tracheal aspirate specimens commonly contain multiple microorganisms, of which more than one microorganism could be the source of a detected resistance marker.

For the prospective and archived U.S. specimen cohorts, an analysis of genotypic 'linkage' as well as phenotypic agreement was performed to evaluate the relationship between detection of resistance markers by the LRT Application directly from tracheal aspirate specimens, the presence of the marker in corresponding culture isolates (based on PCR/bi-directional sequencing), and agreement with phenotypic AST results of reference culture isolates. Included in the analysis were specimens that were positive for targeted resistance markers by the LRT assay and were also 'true positive' for applicable LRT microorganism targets (i.e., positive by LRT and positive by culture for applicable microorganism targets). The analysis also included only those specimens for which isolates were available for confirmatory genotypic testing. For 'true positive' specimens, positive results for antibiotic resistance marker targets were compared to individual available isolates from SoC/culture positive specimens for:

68

1. Genotypic Linkage: Genotypic linkage of positive LRT antibiotic resistance marker results to sequencing of cultured isolates (presence of the antibiotic resistance marker in the genome of one (or more) host microorganism strains isolated from a specific specimen, determined by PCR/bi-directional sequencing). Note that only a subset of isolates was available for this analysis. Confirmed genotypic linkage is defined as a positive LRT result for a resistance marker where a corresponding culture isolate is also positive by sequencing).
1. Phenotypic Analysis: Agreement of positive LRT resistance marker results to associated phenotypic antimicrobial susceptibility testing (AST) results. AST results were collected for the antimicrobials listed in Table 51. Analysis of AST agreement with positive LRT results is presented in Tables 53-59 only for the subset of specimens to include those LRT positive specimens demonstrating genotypic linkage of the detected resistance marker.

For evaluation of phenotypic agreement of positive LRT resistance marker results. AST results for applicable antibiotics were collected and evaluated for all applicable reference culture isolates (Table 51). AST results were reported as MIC values or zone diameters (for Kirby-Bauer tests). AST results were determined using breakpoints listed in CLSI guidance M100S (Performance Standards for Antimicrobial Susceptibility Testing. 26th Edition 2016). "Intermediate" AST results were regarded as "resistant" and any strain was regarded as "resistant" if at least one of the corresponding drug AST results were "intermediate" or "resistant". Any strain was regarded as "susceptible" if AST results were susceptible for all applicable tested antibiotics.

69

| Antibiotic resistance

marker	Associated resistance	AST assay
tem	Penicillins	Ampicillin, Cefinase
ctx-M	3rd Generation Cephalosporins and
Cefepime	Enterobacteriaceae:
Cefotaxime, Ceftazidime, Ceftriaxone
Acinetobacter spp.:
Cefotaxime, Ceftazidime, Ceftriaxone,
Cefepime
P. aeruginosa :
Ceftazidime, Cefepime
kpc, ndm	Carbapenems	Enterobacteriaceae:
Meropenem, Ertapenem, Imipenem
Acinetobacter spp./
P. aeruginosa :
Meropenem, Imipenem
oxa-48		Enterobacteriaceae: Meropenem, Ertapenem,
Imipenem
oxa-23, oxa-24, and
oxa-58		Acinetobacter spp.:
Meropenem, Imipenem
mecA	Oxacillin	S. aureus :
Oxacillin, Cefoxitin

Table 51: AST assays used for evaluation of antibiotic resistance markers detected by LRT

Evaluation of genotypic linkage and phenotypic agreement for resistance markers detected by the LRT Application was performed on a per specimen basis. For many specimens, more than one isolate with the potential to carry a detected resistance marker was recovered from the reference culture.

For specimens that were positive for LRT antibiotic resistance markers, the overall number and percent of specimens with confirmed genotypic linkage as observed in the archived (N=185) and prospective (N=603) studies combined is shown in Table 52. Results are not presented for ndm and oxa-58 (no positive LRT results) and for oxa-48 (one positive LRT result, no isolate available).

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| Resistance Marker
Detected | LRT Microorganism
Target | Number of
specimens with
demonstrated
linkage/Number of
True positive LRT
Microorganism
Results | Percentage of
Specimens with
Confirmed
Linkage |
|-------------------------------|--------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------|
| tem | H. influenzae | 1/2 | 50% |
| ctx-M | Enterobacteriaceae, P.
aeruginosa, and/or
Acinetobacter spp. | 9/16 | 56.3% |
| kpc | Enterobacteriaceae, P.
aeruginosa, and/or
Acinetobacter spp. | 4/5 | 80% |
| vim | Enterobacteriaceae, P.
aeruginosa, and/or
Acinetobacter spp. | 2/3 | 66.7% |
| oxa-23 | Acinetobacter spp. | 4/4 | 100% |
| oxa-24 | Acinetobacter spp. | 1/1 | 100% |
| mecA | S. aureus | 37/47 | 78.7% |

Table 52: Performance. Linkage Analysis

Further details of the genotypic linkage and phenotypic agreement analyses are shown in Tables 53-59 with 'true positive' specimens presented in individual table rows. An additional column is added to each table listing specimens with positive resistance markers results and corresponding microorganism targets detected by LRT but not recovered by culture. It is noted that these microorganisms may be the source of the detected resistance marker; however, due to the lack of isolates, linkage and agreement with AST results cannot be evaluated.

Analysis of phenotypic agreement for the subset of specimens with positive LRT Application resistance marker results showed 100% agreement (i.e., resistance) for all positive specimens with culture isolates demonstrating genotypic linkage (i.e., corresponding culture isolate is positive for the resistance marker).

The following microorganism abbreviations are used in Tables 53-59: Acinetobacter spp. (Aci), Enterobacter cloacae complex (Ecl), Escherichia coli (Eco), Haemophilus influenzae (Hae), Klebsiella oxytoca (Kox), Klebsiella pneumoniae (Kpn), Morganella morganii (Mor), Proteus spp. (Pro), Pseudomonas aeruginosa (Pse), Serratia marcescens (Ser), Staphylococcus aureus (Sau).

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Table 53: Linkage analysis for LRT positive specimens for tem that are true positive for H. influenzae and have available isolates. Agreement of AST results for specimens with confirmed linkage

| True Positive Host
Microorganisms a | # Specimens | | | Isolate Linkage | | | Agreement of AST
result for linked
microorganisms
[%] b
(95 % CI) |
|----------------------------------------|-------------|-------|--------|-----------------|-------|-----------------------------|-------------------------------------------------------------------------------|
| | all | arch. | prosp. | not
conf. | conf. | Linkage
[%]
(95 % CI) | |
| tem | 2 d | 0 | 2 | 1 | 1 | 1/2
50.0
(9.4-90.6) | 1/1
100.0
(20.7-100.0) |
| Hae* | 1 | 0 | 1 | 0 | 1 | | R |
| Hae, [Eco*] c | 1 | 0 | 1 | 1 | 0 | | - |

a True positive: SoC positive H. influenzae detected by LRT, *: indicates microorganisms for a certain specimen with confirmed presence of tem ("linkage") by PCR/bi-directional sequencing, "[Org.]": indicates other true positive microorganisms (Enterobacteriaceae, Acinetobacter spp., P. aeruginosa) that could potentially be the source of tem.

b R: resistant to Penicillin, S: susceptible to Penicillin, na: no AST data available.

C Note that tem was linked to an E. coli isolate isolated from this specimen and the H. influenzae isolate also isolated from this specimen was susceptible to cefinase.

d In total, four H. influenzae true positive specimens (one archived, three prospective) were

reported positive for tem by LRT; H. influenzae isolates were available for two cases.

Table 54: Linkage analysis for LRT positive specimens for ctx-M that are true positive for applicable LRT microorganisms and have available isolates. Agreement of AST results for specimens with confirmed linkage

| True Positive Host
Microorganismsa | # Specimens | | | Isolate Linkage | | Linkage
[%]
(95 % CI) | Agreement of
AST result for
linked
microorganismsb
[%]
(95 % CI) | Specimens that are
LRT positive/culture
negative for
additional applicable
microorganismsc |
|---------------------------------------|-------------|-------|--------|-----------------|-------|-----------------------------|---------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------|
| | all | arch. | prosp. | not
conf. | conf. | | | |
| ctx-M | 16d | 6 | 10 | 7 | 9 | 9/16
56.3
(33.2-76.9) | 7/7
100.0
(64.6-100.0) | |
| Eco*, Pse | 2 | 0 | 2 | 0 | 2 | | R (1), na (1) | 0 of 2 |
| Kpn* | 2 | 1 | 1 | 0 | 2 | | R (1), na (1) | 1 of 2 |
| Eco* | 1 | 0 | 1 | 0 | 1 | | R | 0 of 1 |
| Kpn*, Pse | 1 | 1 | 0 | 0 | 1 | | R | 1 of 1 |
| (Kpn), Pse, Pro* | 1 | 1 | 0 | 0 | 1 | | R | 0 of 1 |
| Kpn, Pro* | 1 | 1 | 0 | 0 | 1 | | R | 1 of 1 |
| Pse, Pro* | 1 | 0 | 1 | 0 | 1 | | R | 1 of 1 |
| Pse | 4 | 1 | 3 | 4 | 0 | | - | 4 of 4 |
| (Kpn), Pse | 1 | 0 | 1 | 1 | 0 | | - | 0 of 1 |
| Mor, Pse, Ser | 1 | 1 | 0 | 1 | 0 | | - | 1 of 1 |
| (Pse), (Pro), Ser | 1 | 0 | 1 | 1 | 0 | | - | 1 of 1 |

4 True positive: SoC positive microorganisms detected by LRT, *: indicates microorganisms for a certain specimen with confirmed presence of cts-M ("linkage") by PCR bi-directional sequencing, "(Org.)": indicates microorganisms for which no isolate was collected and linkage analysis could not be performed.

b R: resistant to Third Generation Cephalosporins, na: no AST data

72

Additional LRT positive culture negative results for applicable targeted microorganisms that could potentially be the source of detected antibiotic resistance markers.

In total, 23 specimens were reported positive for ex-M by LRT (12 archived, 11 prospective); isolates were available for 16 cases.

Table 55: Linkage analysis for LRT positive specimens for kpc that are true positive for applicable LRT microorganisms and have available isolates. Agreement of AST results for specimens with confirmed linkage

| True Positive Host
Microorganisms a | # Specimens | | | Isolate Linkage | | Linkage
[%]
(95 % CI) | Agreement of
AST result for
linked
microorganisms
[%] b
(95 % CI) | Specimens that are
LRT
positive/culture
negative for
additional
applicable
microorganisms c |
|----------------------------------------|-------------|-------|--------|-----------------|-------|-----------------------------|----------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------|
| | all | arch. | prosp. | not
conf. | conf. | | | |
| kpc | 5 d | 0 | 5 | 1 | 4 | 4/5
80.0
(37.6-96.4) | 3/3
100.0
(43.9-100.0) | |
| Ecl* | 1 | 0 | 1 | 0 | 1 | | na | 0 of 1 |
| Kpn* | 1 | 0 | 1 | 0 | 1 | | R | 0 of 1 |
| Mor* | 1 | 0 | 1 | 0 | 1 | | R | 1 of 1 |
| Pse* | 1 | 0 | 1 | 0 | 1 | | R | 1 of 1 |
| (Pse), (Pro), Ser | 1 | 0 | 1 | 1 | 0 | | - | 1 of 1 |

a True positive: SoC positive microorganisms detected by LRT, *: indicates microorganisms for a certain specimen with confirmed presence of kpc ("linkage") by PCR bi-directional sequencing, "(Org.)": indicates microorganisms for which no isolate was collected and linkage analysis could not be performed.

b R: resistant to Carbapenems, na: no AST data available

C Additional LRT positive/culture negative for applicable targeted microorganisms that could potentially be the source of detected antibiotic resistance markers.

a In total, nine specimens were reported positive for kpc by LRT (three archived, six prospective); isolates were available for five cases.

Table 56: Linkage analysis for LRT positive specimens for vim that are true positive for applicable LRT microorganisms and have available isolates. Agreement of AST results for specimens with confirmed linkage

True Positive Host Microorganismsa	# Specimens			Isolate Linkage		Linkage [%] (95 % CI)	Agreement of AST result for linked microorganismsb [%] (95 % CI)	Specimens that are LRT positive/culture negative for additional applicable microorganismsc
vim	3d	1	2	1	2	2/3
66.7 %
(20.8-93.9)	2/2
100.0
(34.2-100.0)
Pse*	1	0	1	0	1		R	0 of 1
Pse*, (Pro)	1	0	1	0	1		R	1 of 1
Kpn, (Pro)	1	1	0	1	0		-	1 of 1

a True positive: SoC positive microorganisms detected by LRT, *: indicates microorganisms for a certain specimen with confirmed

73

presence of vim ("linkage") by PCR/bi-directional sequencing, "(Org.)": indicates microorganisms for which no isolate was collected and linkage analysis could not be performed.

b R: resistant to Carbapenems, na: no AST data available.

6 Additional LRT positive culture negative results for applicable targeted microorganisms that could potentially be source of detected antibiotic resistance markers.

In total, three specimens (one archived, two prospective) were reported positive for vim by LRT, isolates were available for all cases.

Table 57: Linkage analysis for LRT positive specimens for oxa-23 that are true positive for Acinetobacter spp. and have available isolates. Agreement of AST results for specimens with confirmed linkage

| True Positive Host
Microorganisms a | # Specimens | | | Isolate Linkage | | Agreement of
AST result for
linked
microorganisms | |
|----------------------------------------|-------------|-------|--------|-----------------|-------|------------------------------------------------------------|------------------------------|
| | all | arch. | prosp. | not
conf. | conf. | Linkage
[%]
(95 % CI) | [%] b
(95 % CI) |
| oxa-23 | 4c | 2 | 2 | 0 | 4 | 4/4
100.0
(51.0-100.0) | 3/3
100.0
(43.9-100.0) |
| Aci* | 4 | 2 | 2 | 0 | 4 | | R (3), na (1) |

a True positive: SoC positive Acinetobacter spp. detected by LRT, *: indicates microorganisms for a certain specimen with confirmed presence of oxa-23 ("linkage") by PCR/bi-directional sequencing.

b R: resistant to Carbapenems, S: susceptible to Carbapenems, na: no AST data available

C In total, eight specimens were reported positive for oxa-23 by LRT (six archived, two prospective); isolates were available for four cases.

Table 58: Linkage analysis for LRT positive specimens for exa-24 that are true positive for Acinetobacter spp. and have available isolates. Agreement of AST results for specimens with confirmed linkage

| True Positive Host
Microorganisms a | # Specimens | | | Isolate Linkage | | Linkage
[%]
95 % CI | Agreement of AST
result for linked
microorganisms
[%] b
(95 % CI) |
|----------------------------------------|-------------|-------|--------|-----------------|-------|------------------------------|-------------------------------------------------------------------------------|
| | all | arch. | prosp. | not
conf. | conf. | | |
| oxa-24 | 1 c | 0 | 1 | 0 | 1 | 1/1
100.0
(20.7-100.0) | 1/1
100.0
(20.7-100.0) |
| Aci* | 1 | 0 | 1 | 0 | 1 | | R |

a True positive: SoC positive Acinetobacter spp. detected by LRT, *: indicates microorganisms for a certain specimen with confirmed presence of oxa-24 ("linkage") by PCR/bi-directional sequencing.

b R: resistant to Carbapenems, S: susceptible to Carbapenems, na: no AST data available.

In total, one prospective specimen with available isolate was reported positive for oxa-24 by LRT.

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Table 59: Linkage analysis for LRT positive specimens for mecA that are true positive for S. aureus and have available isolates. Agreement of AST results for specimens with confirmed linkage.

| True Positive Host
Microorganisms a | # Specimens | | | Isolate Linkage | | Linkage
[%]
(95 % CI) | Agreement of AST
result for linked
microorganisms
[%] b
(95 % CI) |
|----------------------------------------|-------------|-------|--------|-----------------|-------|------------------------------|-------------------------------------------------------------------------------|
| | all | arch. | prosp. | not
conf. | conf. | | |
| mecA | 47 c | 11 | 36 | 10 | 37 | 37/47
78.7
(65.1-88.0) | 36/36
100.0
(90.4-100.0) |
| Sau* | 37 | 8 | 29 | 0 | 37 | | R (36), na (1) |
| Sau | 10 | 3 | 7 | 10 | 0 | | - |

4 True positive: SoC positive S. aureus detected by LRT, *. indicates microorganisms for a certain specimen with confirmed presence of mecA ("linkage") by PCR/bi-directional sequencing.

b R: resistant to oxacillin. S: susceptible to oxacillin. na: no AST data available.

6 In total, 81 specimens were reported positive for mecA by LRT (33 archived, 48 prospective); isolates were available for 47 cases.

Summary of Clinical Study:

The clinical study design for evaluation of the Unyvero LRT assay did not include evaluation of patient outcome and therefore the potential impact of test results on patient care is unknown. The assay labeling and assay report include multiple limitations and warnings for the laboratory and clinician, most importantly stating that results from this assay must be used in conjunction with results from culture.

Microorganism Targets ('typical' bacteria): Clinical performance of the Unvvero LRT Application for detection of 'typical' microorganisms in tracheal aspirate specimens was evaluated by comparing LRT results to traditional semi-quantitative culture as well as to a composite comparator consisting of culture plus a molecular comparator (PCR followed by bidirectional sequencing). For most microorganism targets, assay sensitivity and specificity compared to culture were greater than 90% and 95% respectively.

The analysis and interpretation of tracheal aspirate cultures can be somewhat subjective, with the judgement of the laboratory technologist playing a critical role in determining the final culture results. Current recommendations for reporting semi-quantitative results for tracheal aspirate cultures are based on the relative quantities of all potential pathogens as well as the amount of normal respiratory flora that grow on culture plates.

LRT false positive results when compared to culture are not unexpected as the Unyvero LRT assay does not distinguish between viable and non-viable organisms, does not quantify the amount of DNA present and reports detected microbial DNA without providing information on the presence or amount of normal respiratory flora.

Although the LRT assay generated a significant number of false positive results compared to culture, most of these results were confirmed to be positive by PCR and sequencing. demonstrating that the targeted bacterial DNA was present in the specimen and the Unyvero

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LRT assay correctly detected the presence of microbial DNA targets. For many false positive results, culture results were reported as mixed flora or normal respiratory flora and many specimens were positive by the LRT assay for two or more targeted microorganisms as shown in tables 25 above.

False positive results for 'typical' bacterial analytes can be mitigated by limitations provided in the assay labeling and test report informing the laboratory and clinician that detected microorganisms may be from colonizing flora and may not be the causative agent of disease. Most importantly, false positive and false negative Unvyero results can be mitigated by the requirement that concomitant culture is performed for all tracheal aspirate specimens tested with the Unyvero LRT Application.

Microorganism targets ('atypical' bacteria): Performance for the 'atypical' analytes was demonstrated primarily with contrived specimens with Unyvero LRT results showing acceptable agreement to expected results. The following limitation is included in the package insert:

. A negative result for the 'atypical' microorganisms (C. pneumoniae, L. pneumophila, and M. pneumoniae) does not exclude the presence of this microorganism in the patient specimen. A positive result should be evaluated in the overall context of the patient's clinical condition and other laboratory results being part of the standard-of-care routine.
Resistance marker targets: The Unyvero LRT assay demonstrated acceptable performance for detection of targeted resistance markers when compared to validated PCR and sequencing assays. The assay masking rules applied by the assay software allow reporting of positive resistance marker results only for specimens with concurrently detected microorganisms that have the potential to carry the detected marker. However, despite these rules, detected resistance markers cannot always be definitively be linked to the concurrently detected microorganism as shown in Table 52 above. False positive results for resistance markers can be mitigated by the requirement for concomitant culture and subsequent AST testing. False negative results for resistance markers are mitigated by the following limitation included in the assay report:
. Antimicrobial resistance may occur via multiple mechanisms other than the resistance markers detected by the Unyvero LRT assay. Negative results for LRT resistance markers do not indicate antimicrobial susceptibility of detected organisms.
Note: Greater than (b) a additional prospectively collected specimens were evaluated in the pivotal clinical study for the Unyvero LRT Application and results from this testing were submitted in the De Novo application. Results for this specimen cohort were reviewed and considered as additional supportive evidence in the assessment of Unyvero LRT Application performance.

4. Clinical cut-off:

Not applicable

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5. Expected values/Reference range:

Of the 603 evaluable tracheal aspirate specimens in the prospective study, the LRT Application reported 312 specimens with at least one positive LRT panel microorganism, including 125 specimens with multi-detections for two or more microorganisms. Reference culture testing reported 236 specimens with at least one LRT panel microorganism, including 62 specimens with two or more LRT panel microorganisms reported. Expected values (number of positive results for each microorganism reported by the Unyvero LRT Application) are presented in Table 60 for all prospectively tested specimens as well as for specimens positive by LRT for multiple microorganisms. Table 61 includes the numbers of positive resistance marker results reported by the LRT assay in the prospective study.

| | Expected Values for all
Specimens
(N= 603) | | Expected Values for Multi-
detection Specimens
(N= 125) | |
|------------------------------|--------------------------------------------------|------|---------------------------------------------------------------|------|
| | # specimens | [%] | # specimens | [%] |
| Acinetobacter spp. | 27 | 4.5 | 20 | 16.0 |
| Chlamydia pneumoniae | 0 | 0.0 | 0 | 0.0 |
| Citrobacter freundii | 3 | 0.5 | 2 | 1.6 |
| Enterobacter cloacae complex | 20 | 3.3 | 11 | 8.8 |
| Escherichia coli | 45 | 7.5 | 32 | 25.6 |
| Haemophilus influenzae | 24 | 4.0 | 15 | 12.0 |
| Klebsiella oxytoca | 15 | 2.5 | 11 | 8.8 |
| Klebsiella pneumoniae | 38 | 6.3 | 25 | 20.0 |
| Klebsiella variicola | 4 | 0.7 | 4 | 3.2 |
| Legionella pneumophila | 2 | 0.3 | 1 | 0.8 |
| Moraxella catarrhalis | 13 | 2.2 | 9 | 7.2 |
| Morganella morganii | 10 | 1.7 | 10 | 8.0 |
| Mycoplasma pneumoniae | 3 | 0.5 | 2 | 1.6 |
| Proteus spp. | 29 | 4.8 | 25 | 20.0 |
| Pseudomonas aeruginosa | 84 | 13.9 | 54 | 43.2 |
| Serratia marcescens | 25 | 4.1 | 20 | 16.0 |
| Staphylococcus aureus | 120 | 19.9 | 57 | 45.6 |
| Stenotrophomonas maltophilia | 52 | 8.6 | 39 | 31.2 |
| Streptococcus pneumoniae | 13 | 2.2 | 3 | 2.4 |

Table 60: Expected values of LRT panel microorganism targets, prospective study

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| | Expected Values
(N= 603) | |
|--------|-----------------------------|------|
| | # specimens | [%] |
| ctx-M | 17 | 2.8 |
| kpc | 6 | 1.0 |
| ndm | 0 | 0.0 |
| oxa-23 | 8 | 1.3 |
| oxa-24 | 2 | 0.3 |
| oxa-48 | 1 | 0.2 |
| oxa-58 | 0 | 0.0 |
| tem | 8 | 1.3 |
| vim | 2 | 0.3 |
| mecA | 62 | 10.3 |

Table 61: Expected values of LRT panel antibiotic resistance markers as determined by the LRT Application for the prospective aspirate study

Table 62 includes the numbers of positive results for resistance markers and concurrently detected microorganism targets as observed in the prospective clinical study. Results are stratified by the number of resistance marker targets detected.

	Table 62: Expected Values of resistance markers and microorganism targets,
prospective study

| Microorganism | Resistance
Marker | # Specimens |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|-------------|
| No resistance marker reported | | 513 |
| negative (no microorganisms detected) | - | 291 |
| Pseudomonas aeruginosa | - | 30 |
| Staphylococcus aureus | - | 29 |
| Stenotrophomonas maltophilia | - | 13 |
| Escherichia coli | - | 11 |
| Streptococcus pneumoniae | - | 10 |
| Klebsiella pneumoniae | - | 9 |
| Pseudomonas aeruginosa, Stenotrophomonas
maltophilia | - | 9 |
| Enterobacter cloacae complex | - | 8 |
| Haemophilus influenzae | - | 7 |
| Acinetobacter spp. | - | 6 |
| Serratia marcescens | - | 5 |
| Moraxella catarrhalis | - | 4 |
| Proteus spp. | - | 4 |
| Enterobacter cloacae complex, Escherichia coli | - | 3 |
| Klebsiella oxytoca | - | 3 |
| Moraxella catarrhalis, Staphylococcus aureus | - | 3 |
| Pseudomonas aeruginosa, Staphylococcus aureus | - | 3 |
| Staphylococcus aureus, Stenotrophomonas
maltophilia | - | 3 |
| Enterobacter cloacae complex, Klebsiella oxytoca,
Stenotrophomonas maltophilia | - | 2 |
| Escherichia coli, Klebsiella pneumoniae | - | 2 |
| Microorganism | Resistance Marker | # Specimens |
| Escherichia coli, Klebsiella pneumoniae, Proteus spp. | - | 2 |
| Escherichia coli, Pseudomonas aeruginosa | - | 2 |
| Escherichia coli, Staphylococcus aureus | - | 2 |
| Haemophilus influenzae, Proteus spp., Staphylococcus aureus | - | 2 |
| Haemophilus influenzae, Staphylococcus aureus | - | 2 |
| Klebsiella oxytoca, Stenotrophomonas maltophilia | - | 2 |
| Klebsiella pneumoniae, Stenotrophomonas maltophilia | - | 2 |
| Proteus spp., Pseudomonas aeruginosa | - | 2 |
| Proteus spp., Staphylococcus aureus | - | 2 |
| Acinetobacter spp., Enterobacter cloacae complex, Pseudomonas aeruginosa | - | 1 |
| Acinetobacter spp., Klebsiella pneumoniae, Saureus, Stenotrophomonas maltophilia | - | 1 |
| Acinetobacter spp., Moraxella catarrhalis, Morganella morganii, Proteus spp., Pseudomonas aeruginosa, Serrratia marcescens, Stenotrophomonas maltophilia, Streptococcus pneumoniae | - | 1 |
| Acinetobacter spp., Proteus spp., Pseudomonas aeruginosa, Serrratia marcescens, Stenotrophomonas maltophilia | - | 1 |
| Acinetobacter spp., Serrratia marcescens, Stenotrophomonas maltophilia | - | 1 |
| Citrobacter freundii | - | 1 |
| Citrobacter freundii, Klebsiella oxytoca | - | 1 |
| Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Morganella morganii | - | 1 |
| Escherichia coli, Haemophilus influenzae, Staphylococcus aureus | - | 1 |
| Escherichia coli, Moraxella catarrhalis, Pseudomonas aeruginosa | - | 1 |
| Escherichia coli, Proteus spp., Pseudomonas aeruginosa, Serratia marcescens | - | 1 |
| Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens | - | 1 |
| Escherichia coli, Stenotrophomonas maltophilia | - | 1 |
| Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa | - | 1 |
| Enterobacter cloacae complex, Klebsiella variicola, Moraxella catarrhalis | - | 1 |
| Enterobacter cloacae complex, Proteus spp. | - | 1 |
| Enterobacter cloacae complex, Staphylococcus aureus | - | 1 |
| Haemophilus influenzae, Klebsiella pneumoniae | - | 1 |
| Haemophilus influenzae, Moraxella catarrhalis | - | 1 |
| Haemophilus influenzae, Pseudomonas aeruginosa, Serratia marcescens | - | 1 |
| Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa, Serratia marcescens | - | 1 |
| Klebsiella oxytoca, Pseudomonas aeruginosa | - | 1 |
| Klebsiella oxytoca, Pseudomonas aeruginosa, Stenotrophomonas maltophilia | - | 1 |
| Microorganism | Resistance
Marker | # Specimens |
| Klebsiella pneumoniae, Klebsiella variicola | - | 1 |
| Klebsiella pneumoniae, Morganella morganii, Staphylococcus aureus | - | 1 |
| Klebsiella pneumoniae, Pseudomonas aeruginosa | - | 1 |
| Klebsiella pneumoniae, Serratia marcescens, Staphylococcus aureus | - | 1 |
| Klebsiella variicola, Stenotrophomonas maltophilia | - | 1 |
| Legionella pneumophila | - | 1 |
| Legionella pneumophila, Staphylococcus aureus | - | 1 |
| Moraxella catarrhalis, Proteus spp., Pseudomonas aeruginosa, Serratia marcescens | - | 1 |
| Mycoplasma pneumoniae | - | 1 |
| Mycoplasma pneumoniae, Streptococcus pneumoniae | - | 1 |
| Proteus spp., Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia | - | 1 |
| Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus | - | 1 |
| Pseudomonas aeruginosa, Serratia marcescens | - | 1 |
| Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia | - | 1 |
| Serratia marcescens, Staphylococcus aureus | - | 1 |
| Serratia marcescens, Stenotrophomonas maltophilia | - | 1 |
| One resistance marker reported | | 78 |
| Staphylococcus aureus | mecA | 34 |
| Klebsiella pneumoniae, Staphylococcus aureus | mecA | 3 |
| Acinetobacter spp., Staphylococcus aureus | mecA | 2 |
| Escherichia coli, Staphylococcus aureus | mecA | 2 |
| Acinetobacter spp., Morganella morganii, Proteus spp., Staphylococcus aureus | mecA | 1 |
| Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumoniae, Staphylococcus aureus | mecA | 1 |
| Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus | mecA | 1 |
| Escherichia coli, Serratia marcescens, Staphylococcus aureus, Stenotrophomonas maltophilia | mecA | 1 |
| Enterobacter cloacae complex, Escherichia coli, Staphylococcus aureus | mecA | 1 |
| Klebsiella variicola, Staphylococcus aureus | mecA | 1 |
| Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus | mecA | 1 |
| Morganella morganii, Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus | mecA | 1 |
| Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus | mecA | 1 |
| Proteus spp., Staphylococcus aureus | mecA | 1 |
| Pseudomonas aeruginosa, Staphylococcus aureus | mecA | 1 |
| Serratia marcescens, Staphylococcus aureus | mecA | 1 |
| Staphylococcus aureus, Stenotrophomonas maltophilia | mecA | 1 |
| Klebsiella pneumoniae | ctx-M | 3 |
| Escherichia coli | ctx-M | 2 |
| Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae | ctx-M | 1 |
| Microorganism | Resistance
Marker | # Specimens |
| Acinetobacter spp., Klebsiella pneumoniae | ctx-M | 1 |
| Escherichia coli, Pseudomonas aeruginosa | ctx-M | 1 |
| Escherichia coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia | ctx-M | 1 |
| Klebsiella oxytoca | ctx-M | 1 |
| Enterobacter cloacae complex | kpc | 1 |
| Klebsiella pneumoniae | kpc | 1 |
| Morganella morganii, Serratia marcescens | kpc | 1 |
| Acinetobacter spp., Proteus spp., Pseudomonas aeruginosa, Stenotrophomonas maltophilia | oxa-23 | 1 |
| Acinetobacter spp., Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia | oxa-23 | 1 |
| Acinetobacter spp. | oxa-24 | 1 |
| Morganella morganii, Pseudomonas aeruginosa | oxa-48 | 1 |
| Haemophilus influenzae | tem | 2 |
| Haemophilus influenzae, Streptococcus pneumoniae | tem | 1 |
| Haemophilus influenzae, Mycoplasma pneumoniae | tem | 1 |
| Escherichia coli, Haemophilus influenzae | tem | 1 |
| Haemophilus influenzae, Staphylococcus aureus | tem | 1 |
| Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia | vim | 1 |
| Two resistance markers reported | | |
| Escherichia coli, Staphylococcus aureus | ctx-M, mecA | 1 |
| Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus | ctx-M, mecA | 1 |
| Klebsiella pneumoniae, Morganella morganii, Proteus spp., Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia | ctx-M, kpc | 1 |
| Acinetobacter spp., Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia | ctx-M, oxa-24 | 1 |
| Acinetobacter spp., Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia | kpc, oxa-23 | 1 |
| Acinetobacter spp., Proteus spp., Staphylococcus aureus | mecA, oxa-23 | 1 |
| Acinetobacter spp., Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus | mecA, oxa-23 | 1 |
| Haemophilus influenzae, Staphylococcus aureus | mecA, tem | 1 |
| Three resistance markers reported | | |
| Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus | ctx-M, mecA, oxa-23 | 1 |
| Acinetobacter spp., Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia | ctx-M, mecA, tem | 1 |
| Acinetobacter spp., Morganella morganii, Proteus spp., Pseudomonas aeruginosa | ctx-M, oxa-23, vim | 1 |
| Acinetobacter spp., Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Stenotrophomonas maltophilia | kpc, mecA, oxa-23 | 1 |

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79

80

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M. Instrument Name:

Unyvero System

N. System Descriptions:

1. Modes of Operation:
  Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

Yes __________________________________________________________________________________________________________________________________________________________________________

1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes ______________ or No _____________________________________________________________________________________________________________________________________________________

Level of Concern Moderate

Software Description

A detailed description of the Unyvero System software was provided and included the function of each Unyvero System software component. A summary of the features of the System component software is shown in Table 63.

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| Component of Unyvero

System	Description and Function of System Component
Unyvero Lysator	The Lysator software is responsible for specimen lysis and
communicates with the Cockpit software. The Lysator software manages
internal mechanical/electrical function of the Lysator instrument.
Unyvero Analyzer	The Analyzer software controls the integrated mechanical, electronic and
optical elements to execute a test run within the Unyvero LRT Cartridge.
The Analyzer software is responsible for managing run analysis
workflow, generation of test results and communicating with the
Unyvero Cockpit software. The Analyzer software monitors internal
mechanical/electrical functions of the Analyzer instrument.
Unyvero Cockpit	The Unyvero Cockpit software provides the main user interface for the
overall Unyvero System. The Cockpit software is responsible for
managing communication between all Unyvero System components
(Cockpit, Lysator, Analyzer), management of analysis workflow, and
presentation and storage of test results.
Entire Unyvero System	Software for each component is involved in management of
communication between components.

Table 63: Unyvero System

Device Hazard Analysis:

A risk analysis and corresponding risk management plan was provided for the Unyvero LRT Application (i.e., assay) and Unyvero System. The risk analysis included potential risks to the patient. Risks identified for the patient were mitigated to Low/Moderate risk levels with assay and instrument controls, use of barcodes for process control, verification and validation procedures and detailed instructions for use. The risk analysis also included potential risks to the operator. Risks identified for the operator were mitigated to Low/Moderate risk levels through labeling, verification and validation, manufacturing quality control measures, and EMC testing. The Device Hazard Analysis for the Unyvero LRT Application and Unyvero System was acceptable.

Architecture Design Chart: A detailed structure of the software used in the Unyvero System was provided.

Software Requirements Specification (SRS): SRS documentation was provided describing requirements and specifications for each of the software components of the Unyvero System was described.

Traceability Analysis: Documentation of traceability matrix that links all product requirements, functional specifications, and verification and validation testing for the complete Unyvero system was provided.

Software Development Environment Description: A description of Unyvero software development environment was provided and was acceptable.

Verification and Validation Testing:

The sponsor provided adequate documentation of verification and validation (V & V) testing covering all software/instrument components of the Unyvero System. V & V

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testing of Unyvero System software was successfully completed at the individual component and system integration levels. The normal operation and user interface of all Unyvero software components were also tested and verified.

Revision Level History:

The firm provided a software revision level history that detailed the updates to the system software corresponding to each version.

Unresolved Anomalies: All major residual risks and unresolved anomalies were properly mitigated. Any remaining anomalies did not present major concerns for safety and efficacy for either the user or the patient.

EMC Testing: The Lysator, Analyzer, and Cockpit components of the Unyvero System were subjected to EMC testing. Testing was conducted according to acceptable standards and no EMC issues were observed.

3. Specimen Identification:

Specimen Identification information can be manually entered or automatically entered using the integrated barcode reader.

4. Specimen Sampling and Handling:

Before starting the test, the user scans the clinical identification (barcode) from the primary specimen container using the built-in barcode reader of the Unvvero Cockpit or the information may be entered manually on the cockpit on-screen keyboard.

The specimen is initially vortexed and then manually pipetted into the Unyvero Sample Tube. If the specimen is viscous, a Unyvero T1 Sample Transfer Tool may be used to facilitate pipetting the specimen into the Sample Tube.

After the specimen is placed in the Sample Tube, the user then places the Unvvero Cap on the Sample Tube. scans the Sample Tube barcode and places it in the Unyvero Lysator. After processing on the Lysator is finished, the user places the Sample Tube and thawed Mastermix into the Unyvero LRT Cartridge, scans the Cartridge barcode and places it into the indicated position in the Unyvero Analyzer as per Unyvero software instructions. The Unyvero software then instructs the user to start the test which is fully automated until completion.

5. Calibration:

Calibration is not required by the user.

1. Quality Control:

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See section L1(c) for information on internal and external controls.

O. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

Not applicable

P. Proposed Labeling: The labeling is sufficient and satisfies the requirements of 21 CFR parts 801 and 809 as well as the Special Controls for this type of device.

Q. Identified Risks to Health and Mitigation Measures:

Identified Risks	Mitigation Measures
Incorrect identification or lack of
identification of a pathogenic microorganism
by the device can lead to improper patient
management	General Controls and Special Controls (1),
(2), (3) and (4)
Failure to correctly interpret test results	General Controls and Special Controls (1),
(2)(iii), (2)(iv), (2)(v), (2)(vi), (2)(vii),
(2)(viii), and (3)
Failure to correctly operate the instrument	General Controls and Special Controls (1),
(2)(i), (4)(ii), (4)(iii) and (4)(iv)

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R. Benefit/Risk Analysis:

Summary
Summary of
the Benefit(s)	The Unyvero LRT Application is the first multiplex PCR assay to detect and identify nucleic acids from bacteria and antimicrobial resistance markers directly from tracheal aspirate specimens. The Unyvero LRT Application can provide bacterial identification results and antimicrobial resistance marker results in approximately 4.5 hours compared to traditional bacterial culture for which final bacterial identification and antimicrobial susceptibility testing can take several days. The performance of the Unyvero LRT Application demonstrated acceptable performance for detection of assay targets in clinical specimens. Although sensitivity/PPA did not exceed 95% for many analytes for the primary efficacy endpoint and specificity/NPA demonstrated a relatively high rate of false positive results, clinical performance is mitigated by use of the assay in conjunction with traditional culture and interpretation by healthcare providers. Detection of resistance markers by the Unyvero LRT assay correlated with phenotypic antimicrobial resistance in cultured isolates that carried each antimicrobial resistance marker. Positive antimicrobial resistance marker results from the LRT assay may identify patients for which broad empiric therapy may be necessary.
Summary of
the Risk(s)	False positive results and false negative results are the primary risks associated with use of the Unyvero LRT Application. A false positive result may lead to unnecessary antimicrobial therapy, and potential adverse drug reactions, such as allergic reactions, C. difficile colitis and/or increased antimicrobial resistance. A false negative result may result in a delay of effective antimicrobial therapy, with subsequent worsening of infection and associated increase in morbidity or mortality. Misinterpretation of antimicrobial resistance genes could result in unnecessary broad-spectrum antimicrobial therapy or a delay to effective therapy, which could lead to potential adverse drug reactions or increased morbidity or mortality.
Summary of
Other
Factors	None.

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| Conclusions
Do the
probable
benefits
outweigh the
probable
risks? | The probable benefits of the Unyvero LRT assay outweigh the potential risks in light of
the listed special controls and applicable general controls. The Unyvero LRT assay is the
first multiplex PCR diagnostic device to detect and identify bacterial nucleic acids and
antimicrobial resistance genes directly from tracheal specimens and is likely to benefit
patients by more rapidly diagnosing tracheitis or ventilator-associated pneumonia and
identifying highly resistance bacterial infections. The clinical performance observed in
comparison to traditional bacterial culture and/or validated PCR assays indicated that the
Unyvero LRT assay could provide potential benefits to patients by rapid and accurate
diagnosis of lower respiratory tract infections. The proposed special controls will ensure
that errors will be uncommon, and potential errors are further mitigated by current
laboratory practices, which include standard of care bacterial culture, other diagnostics,
and product labeling. |

-------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

S. Patient Perspectives

This submission did not include specific information on patient perspectives for this device.

T. Conclusion:

The information provided in this de novo submission to classify this device into class II under regulation 21 CFR 866.3985. FDA believes that the stated special controls, and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	QBH
Device Type:	Device to detect and identify microorganisms and associated resistance
marker nucleic acids directly in respiratory specimens
Class:	II (special controls)
Regulation:	21 CFR 866.3985

(a) Identification. A device to detect and identify microorganisms and associated resistance marker nucleic acids directly in 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:

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(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, instruments, 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.
- If applicable, a limiting statement that detected microorganisms may not be the (vi) 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.
The 21 CFR 809.10(b) labeling and any test report generated by the device must (3) include a limiting statement that negative results for resistance markers do not indicate susceptibility of detected microorganisms.

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(4) Design verification and validation must include:
- Performance characteristics from clinical studies that include prospective (i) (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.
- A detailed device description including the following: (ii)
  - (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.
- As part of the risk management activities, an appropriate end user device training (iv) program must be offered as an effort to mitigate the risk of failure from user error.