cobas® liat CT/NG nucleic acid test

K240217 · Roche Molecular Systems, Inc. · QEP · Jan 17, 2025 · Microbiology

Device Facts

• Record ID: K240217
• Device Name: cobas® liat CT/NG nucleic acid test
• Applicant: Roche Molecular Systems, Inc.
• Product Code: QEP · Microbiology
• Decision Date: Jan 17, 2025
• Decision: SESE
• Submission Type: Dual Track
• Regulation: 21 CFR 866.3393
• Device Class: Class 2

Intended Use

The cobas liat CT/NG nucleic acid test is an automated, qualitative in vitro nucleic acid diagnostic test that utilizes realtime polymerase chain reaction (PCR) for the direction of Chlamydia (CT) and Neisseria gonorthoeae (NG) nucleic acid in male urine and vaginal swabs, all in cobas PCR Media (Roche Molecular Systems, Inc.). This test is intended as an aid in the diagnosis of urogenital infections in both symptomatic individuals.

Device Story

Automated, qualitative in vitro diagnostic test using real-time PCR to detect Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid. Input: male urine or vaginal swabs in cobas PCR Media. Operation: cobas liat analyzer integrates sample purification, nucleic acid amplification, and detection. Targets: Cryptic plasmid/23S rRNA (CT) and pivNG/NGR9 (NG). Includes Internal Control (IC) for processing and inhibition monitoring. Used in clinical settings; operated by clinicians or laboratory staff. Output: qualitative detection result. Assists healthcare providers in diagnosing urogenital infections, enabling timely clinical decision-making and patient management.

Clinical Evidence

Prospective multi-site clinical study (N=4780) compared cobas liat CT/NG results to a Composite Comparator Algorithm (CCA) or Patient Infected Status (PIS) using FDA-cleared NAATs. For CT: Male urine sensitivity 97.3%, specificity 99.9%; Vaginal swabs PPA 98.2%, NPA 99.8%. For NG: Male urine sensitivity 100%, specificity 99.9%; Vaginal swabs PPA 95.2%, NPA 99.8%. Study included 48 operators across 13 sites. Invalid rate was 0.1% after retesting.

Technological Characteristics

Multiplex real-time PCR assay. Targets: CT (cryptic plasmid, 23S rRNA) and NG (pivNG, NGR9). Uses cobas liat analyzer with automated sample processing, amplification, and detection. Closed, flexible assay tube with pre-packaged reagents and breakable seals. Internal process control (armored RNA) monitors extraction, amplification, and inhibition. Connectivity: USB, network, LIS, or DMS export. Software-controlled via Assay Specific Package (LASP).

Indications for Use

Indicated for symptomatic and asymptomatic individuals requiring diagnosis of urogenital infections caused by Chlamydia trachomatis or Neisseria gonorrhoeae. Specimens include male urine and vaginal swabs (clinician or self-collected) in cobas PCR Media.

Regulatory Classification

Identification

A device to detect nucleic acids from non-viral microorganism(s) causing sexually transmitted infections and associated resistance marker(s) is an in vitro diagnostic device intended for the detection and identification of nucleic acids from non-viral microorganism(s) and their associated resistance markers in clinical specimens collected from patients suspected of sexually transmitted infections. The device is intended to aid in the diagnosis of non-viral sexually transmitted infections in conjunction with other clinical and laboratory data. These devices do not provide confirmation of antibiotic susceptibility since mechanisms of resistance may exist that are not detected by the device.

Special Controls

A device to detect nucleic acids from non-viral microorganism(s) causing sexually transmitted infections and associated resistance marker(s) must comply with the following special controls: (1) The intended use for the 21 CFR 809.10 labeling must include a detailed description of targets the device detects, the results provided to the user, the clinical indications appropriate for test use, and the specific population(s) for which the device is intended. (2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device: alternatively, the sample collection device must be cleared in a premarket submission as a part of this device. (3) The 21 CFR 809.10(b) labeling must include: (i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens; (ii) Detailed discussion of the performance characteristics of the device for all claimed specimen types based on analytical studies, including, but not limited to. Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, with-in lab precision, and reproducibility, as appropriate; (iii) Detailed descriptions of the test procedure, the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing. (iv) Limiting statements indicating that: (A)a negative test result does not preclude the possibility of infection; (B) the test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician; (C) reliable results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe procedures in any one of these steps can lead to incorrect results; and (D)if appropriate (e.g., recommended by CDC, by current well-accepted clinical guidelines, or by published peer reviewed research), that the clinical performance is inferior in a specific clinical subpopulation or for a specific claimed specimen type. (v) If the device is intended to detect antimicrobial resistance markers, limiting statements, as appropriate, indicating that: (A)negative results for claimed resistance markers do not indicate susceptibility of detected microorganisms, as resistance markers not measured by the assay or other potential mechanisms of antibiotic resistance may be present; (B) detection of resistance markers cannot be definitively linked to specific microorganisms and the source of a detected resistance marker may be an organism not detected by the assay, including colonizing flora; (C) detection of antibiotic resistance markers may not correlate with phenotypic gene expression; and (D) therapeutic failure or success cannot be determined based on the assay results, since nucleic acid may persist following appropriate antimicrobial therapy. (4) Design verification and validation must include: (i) Detailed device description documentation, including, but not limited to, methodology from obtaining sample to result, design of primer/probe sequences, rationale for target sequence selection, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported result). (ii) Detailed documentation of analytical studies including but not limited to, Limit of Detection, inclusivity, cross-reactivity, microbial interference, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, with-in lab precision, and reproducibility, as appropriate. (iii) Detailed documentation and performance results from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, when determined to be appropriate by FDA, additional characterized clinical 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 FDA accepted comparator methods. Documentation from the clinical studies must include the clinical study protocol (including a predefined statistical analysis plan) study report, testing results, and results of all statistical analyses. (iv) A detailed description of the impact of any software, including, but not limited to, software applications and hardware-based devices that incorporate software, on the device's functions.

*Classification.* Class II (special controls). The special controls for this device are:(1) The intended use for the labeling required under § 809.10 of this chapter must include a detailed description of targets the device detects, the results provided to the user, the clinical indications appropriate for test use, and the specific population(s) for which the device is intended. (2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device. (3) The labeling required under § 809.10(b) of this chapter must include: (i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens; (ii) Detailed discussion of the performance characteristics of the device for all claimed specimen types based on analytical studies, including Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, within lab precision, and reproducibility, as appropriate; (iii) Detailed descriptions of the test procedure, the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing; (iv) Limiting statements indicating that: (A) A negative test result does not preclude the possibility of infection; (B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician; (C) Reliable results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and (D) If appropriate ( *e.g.,* recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer reviewed research), that the clinical performance is inferior in a specific clinical subpopulation or for a specific claimed specimen type; and(v) If the device is intended to detect antimicrobial resistance markers, limiting statements, as appropriate, indicating that: (A) Negative results for claimed resistance markers do not indicate susceptibility of detected microorganisms, as resistance markers not measured by the assay or other potential mechanisms of antibiotic resistance may be present; (B) Detection of resistance markers cannot be definitively linked to specific microorganisms and the source of a detected resistance marker may be an organism not detected by the assay, including colonizing flora; (C) Detection of antibiotic resistance markers may not correlate with phenotypic gene expression; and (D) Therapeutic failure or success cannot be determined based on the assay results, since nucleic acid may persist following appropriate antimicrobial therapy. (4) Design verification and validation must include: (i) Detailed device description documentation, including methodology from obtaining sample to result, design of primer/probe sequences, rationale for target sequence selection, and computational path from collected raw data to reported result ( *e.g.,* how collected raw signals are converted into a reported result).(ii) Detailed documentation of analytical studies, including, Limit of Detection, inclusivity, cross-reactivity, microbial interference, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, within lab precision, and reproducibility, as appropriate. (iii) Detailed documentation and performance results from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, when determined to be appropriate by FDA, additional characterized clinical 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 FDA accepted comparator methods. Documentation from the clinical studies must include the clinical study protocol (including a predefined statistical analysis plan) study report, testing results, and results of all statistical analyses. (iv) A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's functions.

Predicate Devices

• cobas CT/NG for use on cobas 6800/8800 systems (K173887)

Submission Summary (Full Text)

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January 17, 2025

Image /page/0/Picture/1 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left, there is a seal with an abstract design. To the right of the seal, there is a blue square with the letters "FDA" in white. Next to the blue square, the words "U.S. FOOD & DRUG" are written in blue, with the word "ADMINISTRATION" written in a smaller font size below.

Roche Molecular Systems, Inc. Deborah Leu Regulatory Affairs Project Manager 4300 Hacienda Drive Pleasanton, California 94588

## Re: K240217

Trade/Device Name: cobas liat CT/NG nucleic acid test Regulation Number: 21 CFR 866.3393 Regulation Name: Device To Detect Nucleic Acids From Non-Viral Microorganism(S) Causing Sexually Transmitted Infections And Associated Resistance Marker(S) Regulatory Class: Class II Product Code: QEP, LSL, MKZ Dated: January 25, 2024 Received: January 26, 2024

Dear Deborah Leu:

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

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

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Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30. Design controls; 21 CFR 820.90. Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

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

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatory

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assistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

# Himani Bisht -S

Himani Bisht, Ph.D. Assistant Director Viral Respiratory and HPV Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) K240217

Device Name cobas liat CT/NG nucleic acid test

### Indications for Use (Describe)

The cobas liat CT/NG nucleic acid test is an automated, qualitative in vitro nucleic acid diagnostic test that utilizes realtime polymerase chain reaction (PCR) for the direction of Chlamydia (CT) and Neisseria gonorthoeae (NG) nucleic acid in male urine and vaginal swabs, all in cobas PCR Media (Roche Molecular Systems, Inc.).

This test is intended as an aid in the diagnosis of urogenital infections in both symptomatic individuals.

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

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# cobas® liat CT/NG nucleic acid test 510(k) Summary

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

| Submitter Name | Roche Molecular Systems, Inc. |
|----------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Address | 4300 Hacienda Drive,<br>Pleasanton, CA 94588-2722 |
| Contact | Deborah Leu<br>Phone: [phone]<br>Email: [e-mail] |
| Date Prepared | January 15, 2025 |
| Proprietary Name | cobas® liat CT/NG nucleic acid test |
| Common Name | cobas® liat CT/NG |
| Classification Name | Nucleic Acid Detection System For Non-Viral Microorganism(S) Causing<br>Sexually Transmitted Infections<br>DNA probe, Nucleic Acid Amplification, Chlamydia<br>Neisseria spp. direct serological test reagents |
| Product Codes | QEP<br>MKZ<br>LSL |
| Predicate Devices | cobas® 6800/8800 CT/NG |
| Establishment Registration | Roche Molecular Systems, Inc. (2243471) |

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#### DEVICE DESCRIPTION 1.

The test is performed on the cobas® liat analyzer which automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in biological samples using real-time PCR assays. The assay targets both the Cryptic plasmid and 23S rRNA of Chlamydia trachomatis and the pivNG and NGR9 of Neisseria gonorrhoeae. An Internal Control (IC) is also included. The IC is present to control for adequate processing of the target bacteria through steps of sample purification, nucleic acid amplification, and to monitor the presence of inhibitors in the PCR processes.

#### 2. INDICATIONS FOR USE

The cobas® liat CT/NG nucleic acid test is an automated, qualitative in vitro nucleic acid diagnostic test that utilizes real-time polymerase chain reaction (PCR) for the direct detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) nucleic acid in male urine and vaginal swabs, all in cobas® PCR Media (Roche Molecular Systems, Inc.).

This test is intended as an aid in the diagnosis of urogenital infections in both symptomatic and asymptomatic individuals.

#### TECHNOLOGICAL CHARACTERISTICS 3.

The primary technological characteristics and intended use of the RMS cobas® liat CT/NG nucleic acid test are substantially equivalent to other legally marketed nucleic acid amplification tests intended for the qualitative detection of CT and NG.

As indicated in Table 1, the RMS cobas® liat CT/NG nucleic acid test is substantially equivalent to significant characteristics of the identified predicate device, the currently cleared cobas® 6800/8800 CT/NG (K173887) for use on cobas® 6800/8800 Systems.

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| | Submitted Device:<br>cobas® liat CT/NG nucleic acid test | Predicate Device:<br>cobas® 6800/8800 CT/NG for use on cobas®<br>6800/8800 Systems. |
|------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Regulation Name | 866.3393<br>866.3120<br>866.3390 | 866.3390<br>866.3120<br>862.2570 |
| Product Code | QEP<br>MKZ<br>LSL | LSL<br>MKZ<br>OOI |
| Intended Use | The cobas® liat CT/NG nucleic acid test<br>is an automated, qualitative in vitro nucleic<br>acid diagnostic test that utilizes real-time<br>polymerase chain reaction (PCR) for the<br>direct detection of Chlamydia trachomatis<br>(CT) and Neisseria gonorrhoeae (NG)<br>nucleic acid in male urine and vaginal<br>swabs, all in cobas® PCR Media (Roche<br>Molecular Systems, Inc.).<br>This test is intended as an aid in the<br>diagnosis of urogenital infections in both<br>symptomatic and asymptomatic<br>individuals. | The cobas® CT/NG on the cobas® 6800/8800<br>system is an automated, qualitative in vitro<br>nucleic acid diagnostic test, that utilizes real-time<br>polymerase chain reaction (PCR), for the direct<br>detection of Chlamydia trachomatis (CT) and/or<br>Neisseria gonorrhoeae (NG) DNA in male and<br>female urine, clinician-instructed self-collected<br>vaginal swab specimens (collected in a clinical<br>setting), clinician-collected vaginal swab<br>specimens, and endocervical swab specimens,<br>all collected in cobas® PCR Media (Roche<br>Molecular Systems, Inc.), and cervical specimens<br>collected in PreservCyt® solution. This test is<br>intended as an aid in the diagnosis of chlamydial<br>and gonococcal disease in both symptomatic and<br>asymptomatic individuals. |
| Sample Type | Male and female urine, vaginal swabs | Male and female urine,<br>Self-collected/clinician-collected vaginal swab<br>specimens in cobas® PCR Media,<br>Endocervical swab specimens in cobas® PCR<br>Media,<br>Cervical specimens in PreservCyt® solution. |
| Analyte Targets | Chlamydia trachomatis (CT)<br>Neisseria gonorrhoeae (NG) | Chlamydia trachomatis (CT),<br>Neisseria gonorrhoeae (NG) |
| Ancillary Collection<br>Kits | cobas® PCR Urine Sample Kit<br>cobas® PCR Media Uni Swab Sample Kit | cobas® PCR Media Dual Swab Sample Kit<br>cobas® PCR Media Uni Swab Sample Kit<br>cobas® PCR Urine Sample Kit |
| Sample Preparation | Automated | Same |
| Amplification<br>Technology | Real-time PCR | Same |
| Detection<br>Chemistry | Assay using different reporter dyes for<br>target and control | Paired reporter and quencher fluorescence<br>labeled probes (TaqMan Technology) using<br>fluorescence resonance energy transfer (FRET) |
| Controls Used | Sample processing control (IC) Positive<br>and negative control | Same |
| Results Analysis | PCR Cycle threshold analysis | Same |

#### Comparison of the cobas® liat CT/NG nucleic acid test and the Predicate Device Table 1:

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#### NON-CLINICAL PERFORMANCE EVALUATION 4.

#### 4.1. Analytical sensitivity (Limit of Detection)

Analytical sensitivity was determined by analyzing a dilution series of two representative strains/serovars of Chlamydia trachomatis (CT, Serovar D and I) and Neisseria gonorrhoeae (NG, Strains 2948 and 891). The CT and NG cultures were diluted in pooled negative urine (UR) or pooled negative vaginal swab (VS) clinical specimens to 7 concentration levels. All levels were tested with at least 20 replicates per concentration tested across 3 unique lots of reagents. LoD for each specimen type is shown in Table 2 and Table 3 for CT and NG respectively as the target concentration which can be detected in ≥ 95% of the replicates for all lots.

Table 2: CT concentration levels with at least 95% observed hit rate for all lots tested

| Specimen Types | CT<br>Serovar D LoD<br>(EB/mL) | CT<br>Serovar D Mean Ct<br>Value | CT<br>Serovar I LoD<br>(EB/mL) | CT<br>Serovar I Mean Ct<br>Value |
|-------------------------------------|--------------------------------|----------------------------------|--------------------------------|----------------------------------|
| Urine in cobas® PCR<br>Media | 0.085 | 36.2 | 0.784 | 36.0 |
| Vaginal Swab in<br>cobas® PCR Media | 0.170 | 35.3 | 0.784 | 35.7 |

EB = Elementary Bodies

| Table 3: NG concentration levels with at least 95% observed hit rate for all lots tested |
|------------------------------------------------------------------------------------------|
| |

| Specimen Types | NG<br>Strain 2948 LoD<br>(CFU/mL) | NG<br>Strain 2948 Mean Ct<br>Value | NG<br>Strain 891 LoD<br>(CFU/mL) | NG<br>Strain 891 Mean Ct<br>Value |
|-------------------------------------|-----------------------------------|------------------------------------|----------------------------------|-----------------------------------|
| Urine in cobas® PCR<br>Media | 0.250 | 34.7 | 0.200 | 34.5 |
| Vaginal Swab in<br>cobas® PCR Media | 0.500 | 34.2 | 0.200 | 34.5 |

CFU = Colony Forming Units

#### 4.2. Inclusivity

Inclusivity was performed for an additional 15 CT serovars and 43 NG strains using one lot of reagents. Testing was performed using CT and NG cultures that were spiked into pools of negative clinical specimens. Three replicates per dilution level were tested for each subtype per

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specimen type. The lowest level at which all three replicates tested as positive are reported in Table 4 and Table 5 for CT and NG respectively.

| Serovar Type or Variant | Urine Specimens (EB/mL) | Vaginal Swab Specimens (EB/mL) |
|-------------------------|-------------------------|--------------------------------|
| A | 0.1 | 0.2 |
| B | 0.4 | 0.2 |
| Ba | 0.4 | 1 |
| C | 0.7 | 0.7 |
| E | 2 | 36 |
| F | 0.4 | 0.04 |
| G | 0.4 | 0.4 |
| H | 0.4 | 3 |
| J | 0.1 | 0.2 |
| K | 0.1 | 0.04 |
| LGV Type 1 | 0.1 | 0.04 |
| LGV Type 2 | 1600 | 200 |
| LGV Type 3 | 0.1 | 0.7 |
| nvCT | 0.1 | 0.7 |
| Finnish-nvCT | 1:100 of Patient Sample | 1:100 of Patient Sample |

Table 4: Inclusivity testing for CT serovars

#### Inclusivity testing for NG strains Table 5:

| Strain ID | Urine Specimens<br>(CFU/mL) | Vaginal Swab Specimens (CFU/mL) |
|------------------------------------------------------------------------------|-----------------------------|---------------------------------|
| ATCC 27633 | 0.2 | 0.5 |
| ATCC 49226 | 1 | 0.006 |
| ATCC 700825 | 0.01 | 0.001 |
| Clinical Isolate SS169 | 0.06 | 0.02 |
| NBL 1606 | 0.3 | 0.08 |
| NBL 1952 | 0.2 | 0.1 |
| NBL 2012 | 0.2 | 0.3 |
| NRL 1977 | 0.02 | 0.02 |
| NRL 8042 - Belgium | 0.02 | 0.02 |
| NRL 13477 | 0.09 | 0.1 |
| NRL 13819 | 0.006 | 0.004 |
| NRL 33155 - Atlanta | 0.09 | 0.001 |
| NRL 33641 | 0.01 | 0.07 |
| Strain ID | Urine Specimens<br>(CFU/mL) | Vaginal Swab Specimens (CFU/mL) |
| NRL 35495 | 0.01 | 0.07 |
| NRL DAN 09612 | 0.02 | 0.03 |
| NRL DN 7896 - DENMARK | 0.9 | 0.3 |
| NRL DN 7901 - DENMARK | 0.02 | 0.02 |
| NRL DOM 362 - Dominican Republic | 0.09 | 0.09 |
| NRL DOM 1271 - Dominican Republic | 0.4 | 0.1 |
| NRL KPO 1148 - KENYA (KPO) | 0.2 | 0.07 |
| NRL KPO 1161 - KENYA (KPO) | 0.02 | 0.02 |
| NRL Peru 33 | 0.07 | 0.07 |
| NRL Peru 83 | 0.02 | 0.02 |
| NRL PITT 94-4833 - PITTSBURGH (PITT) | 0.02 | 0.02 |
| NRL PITT 94-8561 - PITTSBURGH (PITT) | 0.09 | 0.1 |
| NRL PP 132 - PHILLIPINES | 0.09 | 0.1 |
| NRL SEN 97 P-292 - SENEGAL (SEN) | 0.006 | 0.02 |
| NRL SEN 97 P-301 - SENEGAL (SEN) | 0.006 | 0.07 |
| Roche Diagnostics K.K.,Japan RDN001-<br>00193 | 0.02 | 0.03 |
| Roche Diagnostics, Australia 04D125:<br>Darwin Northern Territory, Australia | 0.09 | 0.1 |
| Roche Diagnostics, Australia 04D127:<br>Darwin Northern Territory, Australia | 0.09 | 0.1 |
| Roche Diagnostics, Australia 04D129:<br>Darwin Northern Territory, Australia | 0.09 | 0.1 |
| Roche Diagnostics, Australia 04D130:<br>Darwin Northern Territory, Australia | 0.4 | 0.1 |
| Roche Diagnostics, Australia 04D132:<br>Darwin Northern Territory, Australia | 0.09 | 0.09 |
| Roche Diagnostics, Australia 05D003:<br>Darwin Northern Territory, Australia | 0.02 | 0.03 |
| Roche Diagnostics, Australia 05D004:<br>Darwin Northern Territory, Australia | 0.006 | 0.004 |
| Roche Diagnostics, Australia 4551 -<br>Western Australia | 0.02 | 0.02 |
| Statens Serum Institut 223/06 | 0.006 | 0.006 |
| Statens Serum Institut 1498/46 | 0.02 | 0.02 |
| Statens Serum Institut 2170/46 | 0.02 | 0.02 |
| Statens Serum Institut 2222/46 | 0.4 | 0.09 |
| Statens Serum Institut 6973/45 | 0.09 | 0.09 |
| UCSF58 | 0.06 | 0.07 |

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

{10}------------------------------------------------

#### 4.3. Analytical specificity/cross reactivity

A panel of 181 strains of bacteria, fungi and viruses, including those commonly found in patient specimens, as well as 52 representative strains of non-gonorrhoeae Neisseria species and other phylogenetically unrelated organisms, were tested to assess analytical specificity. The organisms listed in Table 6 were spiked at concentrations of ≥ 1 x 106 units/mL* for bacteria or fungi and ≥ 1 x 105 units/mL for viruses into pools of negative vaginal swab specimens collected in cobas® PCR Media and negative urine specimens stabilized in cobas® PCR Media. Testing was performed with each potential interfering organism in the absence of, as well as mixed with. CT and NG cultures at ~3x LoD. Results indicated that 180 of the non-target organisms tested did not generate any false positive or false negative results due to cross-reactivity or interference. One strain of Neisseria lactamica (CCUG 26479), at concentrations greater than 1 x 104 CFU/mL, interfered with detection of NG at ~3x LoD. At 1 x 104 CFU/mL, this N. lactamica strain did not interfere with detection of NG at ~3x LoD, nor did 8 additional strains of N. lactamica when tested at concentrations ≥ 1 x 10° CFU/mL.

*Four bacteria could only be tested at a concentration below 1 x 106 units/mL and above 7 x 104 units/mL due to low stock titers.

| Acholeplasma laidlawii | Eikenella corrodens | Mobiluncus curtisii | Peptostreptococcus<br>anaerobius |
|-----------------------------------------------------------|--------------------------------------------------------------------------|-------------------------------------------------------|---------------------------------------------------------|
| Acholeplasma oculi1,3 | Enterobacter aerogenes<br>(Klebsiella aerogenes) | Moraxella catarrhalis | Plesiomonas shigelloides |
| Acinetobacter calcoaceticus | Enterobacter cloacae | Moraxella lacunata | Prevotella bivia |
| Acinetobacter lwoffii | Enterococcus avium | Moraxella osloensis | Cutibacterium acnes |
| Actinomyces israelii1,3 | Enterococcus faecalis (2<br>strains) | Morganella morganii | Proteus mirabilis |
| Actinomyces pyogenes<br>(Trueperella pyogenes) | Enterococcus faecium (2<br>strains) | Mycobacterium smegmatis | Proteus vulgaris |
| Aerococcus viridans | Erwinia herbicola<br>(Pantoea agglomerans) | Mycoplasma faucium1,3 | Providencia stuartii |
| Aeromonas hydrophila | Erysipelothrix rhusiopathiae | Mycoplasma fermentans | Pseudomonas aeruginosa |
| Alcaligenes faecalis | Escherichia coli | Mycoplasma hominis | Pseudomonas fluorescens |
| Atopobium vaginae<br>(Fannyhessea vaginae) | Flavobacterium<br>meningosepticum<br>(Elizabethkingia<br>meningoseptica) | Mycoplasma orale | Pseudomonas putida |
| Bacillus subtilis | Fusobacterium nucleatum | Mycoplasma penetrans | Rahnella aquatilis |
| Bacteroides fragilis | Gardnerella vaginalis | Mycoplasma pirum | Rhizobium radiobacter<br>(Agrobacterium<br>tumefaciens) |
| Bacteroides ureolyticus<br>(Campylobacter<br>ureolyticus) | Gemella haemolysans | Mycoplasma pneumoniae | Rhodospirillum rubrum |
| Bifidobacterium<br>adolescentis | Giardia Intestinalis | Mycoplasma primatum | Saccharomyces cerevisiae |
| Bifidobacterium breve | Haemophilus ducreyi | Mycoplasma salivarium | Salmonella minnesota |
| Blautia producta | Haemophilus influenzae | Mycoplasma<br>spermatophilum | Salmonella typhimurium |
| Brevibacterium linens | Herpes simplex virus I | Neisseria cinerea (4<br>strains) | Serratia marcescens |
| Campylobacter jejuni | Herpes simplex virus II | Neisseria denitrificans<br>(Bergeriella denitrifican) | Staphylococcus aureus |
| Candida albicans (2 strains) | HIV-1 | Neisseria elongata (3<br>strains) | Staphylococcus epidermidis |
| Candida glabrata<br>(Nakaseomyces glabratus) | Human papilloma virus 16<br>(CaSki cells) | Neisseria flava | Staphylococcus<br>saprophyticus |
| Candida parapsilosis | Kingella denitrificans | Neisseria flavescens (2<br>strains) | Streptococcus agalactiae |
| Candida tropicalis | Kingella kingae | Neisseria lactamica (9<br>strains)2 | Streptococcus bovis |
| Chlamydia pneumoniae | Klebsiella oxytoca | Neisseria macacae | Streptococcus mitis |
| Chlamydia psittaci | Klebsiella pneumoniae | Neisseria meningitidis<br>Serogroup A | Streptococcus mutans |
| Chromobacterium<br>violaceum | Lactobacillus acidophilus | Neisseria meningitidis<br>Serogroup B | Streptococcus pneumoniae |
| Citrobacter braakii | Lactobacillus brevis<br>(Levilactobacillus brevis) | Neisseria meningitidis<br>Serogroup C<br>(4 strains) | Streptococcus pyogenes |
| Citrobacter freundii | Lactobacillus crispatus | Neisseria meningitidis<br>Serogroup D | Streptococcus salivarius |
| Clostridium difficile<br>(Clostridioides difficile) | Lactobacillus jensenii | Neisseria meningitidis<br>Serogroup W135 | Streptococcus sanguinis |
| Clostridium perfringens | Lactobacillus lactis | Neisseria meningitidis<br>Serogroup Y | Streptomyces griseinus |
| Corynebacterium<br>genitalium | Lactobacillus vaginalis<br>(Limosilactobacillus<br>vaginalis) | Neisseria mucosa (3<br>strains) | Trichomonas tenax |
| Corynebacterium xerosis | Legionella pneumophila (2<br>strains) | Neisseria perflava | Ureaplasma parvum |
| Cryptococcus neoformans | Leptotrichia buccalis | Neisseria polysaccharea | Ureaplasma urealyticum1,3 |
| Cytomegalovirus | Leuconostoc<br>mesenteroides | Neisseria sicca (3 strains) | Veillonella parvula |
| Deinococcus radiodurans | Leuconostoc<br>paramesenteroides<br>(Weissella<br>paramesenteroides) | Neisseria subflava (14<br>strains) | Vibrio parahaemolyticus |
| Derxia gummosa | Listeria monocytogenes | Paracoccus denitrificans | Yersinia enterocolitica |
| Dientamoeba fragilis | Micrococcus luteus | Pentatrichomonas hominis | - |

Microorganisms tested for analytical specificity/cross reactivity Table 6:

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

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

1 Organism was tested at a concentration of < 1.0e+6 units/mL and > 7.0e+4 units/mL.

2 One strain of organism was tested at a concentration of < 1.0e+6 units/mL and > 1.0e+4 units/mL. 3Tested at highest concentration possible per stock concentration.

#### Interference 4.4.

The effects of over-the-counter or prescription products that may be present in urine or vaginal swab clinical specimens were evaluated at the concentration listed in Table 7. Testing was executed using pooled clinical specimens spiked with potential interferents at levels expected from normal patient usage. Interferents were tested in CT/NG negative specimen pools as well as in positive specimen pools spiked with CT/NG at ~3x LoD for each specimen type using one lot of reagents. Five replicates each of CT/NG negative sample and CT/NG positive sample (for each of two culture subtypes per microorganism) were tested with each exogenous substance in each specimen type, except for Azo Urinary Pain Relief, which was tested in urine only.

Of the products tested, no interference was observed in 15 substances when tested at concentrations of 1.5 mg/mL. Azo Urinary Pain Relief and carbomer-containing Replens™ Long-Lasting Vaginal Moisturizer resulted in false negative results in at least one replicate when tested at higher concentrations. Azo Urinary Pain Relief and Replens™ Long-Lasting Vaginal Moisturizer at concentrations greater than 0.5 mg/mL, respectively, may interfere with the assay performance. Levels of substances tolerated by the assay for all specimen types are shown in Table 7.

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

| Product Name | Urine (mg/mL) | Vaginal Swabs (mg/mL) |
|------------------------------------------------|---------------|-----------------------|
| Azo Urinary Pain Relief (urine only) | 0.5* | - |
| Clindamycin Phosphate Vaginal Cream | 1.5 | 1.5 |
| Equate tioconazole 1 Day | 1.5 | 1.5 |
| Equate Vagicaine Anti-Inch Cream | 1.5 | 1.5 |
| Estradiol Vaginal Cream | 1.5 | 1.5 |
| 7 Day vaginal cream | 1.5 | 1.5 |
| K-Y® UltraGel | 1.5 | 1.5 |
| Metronidazole Vaginal Gel | 1.5 | 1.5 |
| Monistat Miconazole Nitrate Vaginal Cream (2%) | 1.5 | 1.5 |
| Monistat® Instant Itch Relief Cream | 1.5 | 1.5 |
| Norforms Deodorant Suppositories | 1.5 | 1.5 |
| Premarin Vaginal Cream | 1.5 | 1.5 |
| Replens™ Long-Lasting Vaginal Moisturizer | 1.0* | 1.5 |
| Summer's Eve Ultra Freshening Spray | 1.5 | 1.5 |
| VCF - Vaginal Contraceptive Gel | 1.5 | 1.5 |
| Yeast Gard Gel Treatment | 1.5 | 1.5 |
| RepHresh™ Vaginal Gel | 1.5 | 1.5 |

Table 7: List of products tested for interference

*Note: Concentrations above this level may cause interference in clinical samples.

Endogenous substances that may be present in urine or vaginal swab clinical specimens were evaluated at the concentration listed in Table 8. Testing was executed using pooled clinical specimens spiked with potential endogenous interferents at levels expected in a typical clinical sample. Endogenous substances were tested in CT/NG negative specimen pools as well as in positive specimen pools spiked with CT/NG at ~3x LoD for each relevant specimen type using one lot of reagents. Five replicates each of CT/NG negative sample and CT/NG positive sample (for each of two culture subtypes per microorganism) were tested with each endogenous substance in each relevant sample type.

For all endogenous substances tested, no interference was observed. Levels of endogenous substances tolerated by the assay for each specimen types are shown in Table 8.

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

| Endogenous Substance | Urine | Vaginal Swab |
|---------------------------------|--------------------------|--------------------------|
| Human cells (PBMCs) cells/mL | 1.0E+06 | 1.0E+06 |
| Mucus | 1 swab dipped into mucus | 1 swab dipped into mucus |
| Whole blood (v/v) | 10% | 10% |
| Semen (v/v) (vaginal swab only) | - | 1.5% |
| Albumin (w/v) (urine only) | 5% | - |
| Bilirubin (w/v) (urine only) | 1% (w/v) | - |
| Glucose (w/v) (urine only) | 1% (w/v) | - |
| Acidic pH (urine only) | pH 4 | - |

#### Summary of endogenous substance concentrations that do not show Table 8: interforonco

#### Competitive inhibition 4.5.

Alkaline pH (urine only)

To assess competitive inhibition between CT and NG, a total of six different combinations of low concentration of target (~2x LoD) were mixed with high concentrations of the other targets in both urine and vaginal swab clinical specimen matrices. Each combination was tested in replicates of 10 using one lot of reagents.

pH 9

Testing results indicated that when one or two target microorganisms were present at high concentrations, no interference was observed for microorganisms that were present at low concentrations (~2x LoD), when tested in both urine and vaginal swab clinical specimen matrices.

#### REPRODUCIBILITY STUDIES 5.

A reproducibility study was performed across different sites, lots, days, operators, instruments for cobas® liat CT/NG panels prepared from vaginal swabs and urine in cobas® PCR Media. Testing was performed at three external sites with a minimum of 3 cobas® liat analyzers per site. Operators at the CLIA-waived sites that met the definition of intended use operators were considered for this study. Selected operators were provided with the assay's IFU, Quick Reference Instructions, and the cobas® liat system User Guide. Operators were asked to read the materials before beginning any study testing. No assay or instrument training was provided to the operators.

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

Two operators at each site each tested 1 panel per specimen type per day (1 complete panel consists of 3 panel members each tested in triplicate) for a total of 15 days. All replicates for each panel member were always tested on the same analyzer. Each panel, per specimen type, consisted of a negative panel member (negative for all 3 analytes), a low positive panel member, and a moderate positive panel member with each positive panel member being co-formulated with all 3 analytes. For each panel member, approximately 270 results were produced.

The Reproducibility Study was executed with a total of 1618 tests consisting of 811 tests for the vaginal specimen type and 807 tests for the urine specimen type.

Table 9 and Table 10 show the site-to-site reproducibility study results for cobas® liat CT/NG by sample type and panel member concentration, respectively for CT and NG.

| Specimen<br>Type | Panel Member<br>Concentration | Site 1* | Site 2* | Site 3* | Overall* |
|------------------|-------------------------------|-------------------------------------|-------------------------------------|-------------------------------------|---------------------------------------|
| Vaginal | 1-2x LoD | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(89/89)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(269/269)<br>(98.6% - 100.0%) |
| Vaginal | 3-5x LoD | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(270/270)<br>(98.6% - 100.0%) |
| Vaginal | Negative | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(83/83)<br>(95.6 - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(263/263)<br>(98.6% - 100.0%) |
| Urine | 1-2x LoD | 87.8%<br>(79/90)<br>(79.4%- 93.0%) | 93.3%<br>(83/89)<br>(86.1% - 96.9%) | 91.1%<br>(82/90)<br>(83.4% - 95.4%) | 90.7%<br>(244/269)<br>(86.6%- 93.6%) |
| Urine | 3-5x LoD | 95.6%<br>(86/90)<br>(89.1% - 98.3%) | 98.9%<br>(88/89)<br>(93.9% - 99.8%) | 94.4%<br>(85/90)<br>(87.6% - 97.6%) | 96.3%<br>(259/269)<br>(93.3%- 98.0%) |
| Urine | Negative | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(80/80)<br>(95.6% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>260/260<br>(98.5% - 100.0%) |

Summary CT of site-to-site reproducibility results with cobas® liat CT/NG Table 9:

Note: LoD : limit of detection

*Percent Agreement with Expected Results (n/N) (95% Confidence Interval)

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

| Specimen<br>Type | Panel<br>Member<br>Conc. | Site 1* | Site 2* | Site 3* | Overall* |
|------------------|--------------------------|-------------------------------------|-------------------------------------|-------------------------------------|---------------------------------------|
| Vaginal | 1-2x LoD | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(89/89)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(269/269)<br>(98.6% - 100.0%) |
| Vaginal | 3-5x LoD | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(270/270)<br>(98.6% - 100.0%) |
| Vaginal | Negative | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(83/83)<br>(95.6 - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(263/263)<br>(98.6% - 100.0%) |
| Urine | 1-2x LoD | 100%<br>(90/90)<br>(95.9% - 100.0%) | 98.9%<br>(88/89)<br>(93.9% - 99.8%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 99.6%<br>(268/269)<br>(97.9% - 99.9%) |
| Urine | 3-5x LoD | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(89/89)<br>(95.9% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(269/269)<br>(98.6% - 100.0%) |
| Urine | Negative | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(80/80)<br>(95.6% - 100.0%) | 100%<br>(90/90)<br>(95.9% - 100.0%) | 100%<br>(260/260)<br>(98.5% - 100.0%) |

Table 10: Summary NG of site-to-site reproducibility results with cobas® liat CT/NG

Note: LoD : limit of detection

*Percent Agreement with Expected Results (n/N) (95% Confidence Interval)

Table 11 and Table 12 present the total SD, and total percent CV (%) for Cycle Threshold Values from the Reproducibility Study for each specimen panel type run in cobas® liat CT/NG, respectively for CT and NG.

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

Table 11: CT - Overall mean estimate, standard deviations, and coefficients of variation (%) for cycle threshold values by sample type and expected concentration for cobas® liat CT/NG by sample type and positive panel member concentration

| | | Bet-<br>ween<br>Site | Bet-<br>ween<br>Site | Bet-<br>ween<br>Lot | Bet-<br>ween<br>Lot | Bet-<br>ween<br>Day | Bet-<br>ween<br>Day | Bet-ween<br>Operator/<br>Run | Bet-ween<br>Operator/<br>Run | Within-<br>Run | Within-<br>Run | Total | | | |
|----------------|---------------------------------------|----------------------|----------------------|---------------------|---------------------|---------------------|---------------------|------------------------------|------------------------------|----------------|----------------|-------|------|------|------|
| Sample<br>Type | Panel<br>Member<br>Concen-<br>tration | n/Na | Mean<br>Ct | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | | |
| Vagin<br>al | 1x-2x<br>LoD | 269/269 | 33.4 | 0.00 | 0.00 | 0.53 | 1.60 | 0.22 | 0.67 | 0.00 | 0.00 | 0.84 | 2.52 | 1.02 | 3.06 |
| Vagin<br>al | 3x-5x<br>LoD | 270/270 | 32.1 | 0.21 | 0.64 | 0.58 | 1.82 | 0.30 | 0.93 | 0.00 | 0.00 | 1.00 | 3.13 | 1.22 | 3.79 |
| Urine | 1x-2x<br>LoD | 244/269 | 34.8 | 0.15 | 0.44 | 0.84 | 2.41 | 0.31 | 0.88 | 0.00 | 0.00 | 0.91 | 2.61 | 1.28 | 3.69 |
| Urine | 3x-5x<br>LoD | 259/269 | 34.0 | 0.15 | 0.45 | 0.70 | 2.07 | 0.23 | 0.68 | 0.00 | 0.00 | 0.98 | 2.89 | 1.24 | 3.65 |

Ct: cycle threshold; CV%: percent coefficient of variation; LoD: Limit of Detection; SD: standard deviation. ªn is the number of tests in agreement with expected results. N is the total number of valid tests for the panel member.

Table 12: NG - Overall mean estimate, standard deviations, and coefficients of variation (%) for cycle threshold values by sample type and expected concentration for cobas® liat CT/NG by sample type and positive panel member concentration

| | | | Between Site | | Between Lot | | Between Day | | Between Operator/ Run | | Within Run | | Total | | |
|-------------|----------------------------|---------|--------------|------|-------------|------|-------------|------|-----------------------|------|------------|------|-------|------|------|
| Sample Type | Panel Member Concentration | n/Na | Mean Ct | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| Vaginal | 1x-2x LoD | 269/269 | 32.2 | 0.11 | 0.34 | 0.59 | 1.83 | 0.29 | 0.89 | 0.14 | 0.42 | 0.59 | 1.83 | 0.90 | 2.79 |
| Vaginal | 3x-5x LoD | 270/270 | 30.9 | 0.10 | 0.33 | 0.15 | 0.50 | 0.18 | 0.57 | 0.00 | 0.00 | 0.41 | 1.33 | 0.48 | 1.56 |
| Urine | 1x-2x LoD | 268/269 | 32.9 | 0.16 | 0.47 | 0.70 | 2.12 | 0.26 | 0.78 | 0.46 | 1.41 | 0.74 | 2.25 | 1.16 | 3.51 |
| Urine | 3x-5x LoD | 269/269 | 31.4 | 0.07 | 0.23 | 0.25 | 0.80 | 0.16 | 0.51 | 0.00 | 0.00 | 0.56 | 1.79 | 0.64 | 2.04 |

Ct: cycle threshold; CV%: percent coefficient of variation; LoD: Limit of Detection; SD: standard deviation.

ªn is the number of tests in agreement with expected results. N is the total number of valid tests for the panel member.

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

In the Reproducibility Study, the PPA for CT in urine panel members was less than the expected 95%. Therefore, a supplemental Precision Study was performed at one site across different lots, days, operators and instruments for cobas® liat CT/NG for the detection of CT in urine from urine panels prepared at negative, 1x-2x and 3x-5xLoD concentration levels. There were six total untrained operators and the level of instructional material were the same for this supplemental Precision study. Each operator tested 1 panel per day for 5 non-consecutive days for each lot (1 complete panel consisted of 3 panel members). This supplemental Precision Study was executed with a total of 810 evaluable tests on urine panel members.

Table 13 shows the supplemental between operator Precision Study for cobas® liat CT/NG by panel member concentration for CT in urine.

| Panel Member<br>Concentration | Operator | n/N a | Agreement with Expected<br>Results (%) |
|-------------------------------|----------|-------|----------------------------------------|
| 1-2x LoD | 1 | 44/45 | 97.8% |
| 1-2x LoD | 2 | 44/44 | 100.0% |
| 1-2x LoD | 3 | 45/45 | 100.0% |
| 1-2x LoD | 4 | 44/44 | 100.0 % |
| 1-2x LoD | 5 | 45/45 | 100.0% |
| 1-2x LoD | 6 | 45/45 | 100.0% |
| 3-5x LoD | 1 | 45/45 | 100.0% |
| 3-5x LoD | 2 | 45/45 | 100.0% |
| 3-5x LoD | 3 | 45/45 | 100.0% |
| 3-5x LoD | 4 | 45/45 | 100.0% |
| 3-5x LoD | 5 | 45/45 | 100.0% |
| 3-5x LoD | 6 | 44/44 | 100.0% |
| Negative | 1 | 43/44 | 97.7% |
| Negative | 2 | 45/45 | 100.0% |
| Negative | 3 | 45/45 | 100.0% |
| Negative | 4 | 45/45 | 100.0% |
| Negative | 5 | 45/45 | 100.0% |
| Negative | 6 | 44/44 | 100.0% |

Table 13: Summary of CT Precision/Repeatability study results

a n is the number of tests with expected results. N is the total number of valid tests.

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

Table 14 shows the supplemental Reproducibility Study for cobas® liat CT/NG standard deviation (SD) and coefficient of variation (CV) of Cycle Threshold Values for each factor as well as the total SD and total CV (%) for each positive panel member.

## Table 14: CT - Overall mean estimate, standard deviations, and coefficients of variation (%) for cycle threshold values and expected concentration for cobas® liat CT/NG by positive panel member concentration in urine

| - | | | Between<br>Instrument | Between<br>Instrument | Between<br>Lot | Between<br>Lot | Between<br>Day | Between<br>Day | Between<br>Operator<br>/Run | Between<br>Operator<br>/Run | Within-<br>Run | Within-<br>Run | Total | Total |
|----------------------------------|-------------|------------|-----------------------|-----------------------|----------------|----------------|----------------|----------------|-----------------------------|-----------------------------|----------------|----------------|-------|-------|
| Panel<br>Member<br>Concentration | n/Na | Mean<br>Ct | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1x-2xLOD | 267/<br>268 | 35.3 | 0.00 | 0.00 | 0.03 | 0.08 | 0.00 | 0.00 | 0.00 | 0.00 | 0.85 | 2.41 | 0.85 | 2.41 |
| 3x-5xLOD | 269/<br>269 | 33.7 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.46 | 1.35 | 1.07 | 3.19 | 1.17 | 3.47 |

Note: Ct = cycle threshold, CT=Chlamydia trachomatis, CV(%) = percent coefficient of variation, LoD = Limit of Detection, NG=Neisseria gonorrhoeae, SD =standard deviation.

ªn is the number of tests in agreement with expected results. N is the total number of valid tests for the panel member.

#### CLINICAL PERFORMANCE EVALUATION 6.

#### 6.1. Clinical study

The clinical utility and performance of cobas® liat CT/NG was established in a multi-site, prospective study by comparing the results to a Patient Infected Status (PIS) or a Composite Comparator Algorithm (CCA) derived from a combination of FDA-cleared NAATs for the 2 analytes. A result for PIS (for male urine) or a CCA (for vaginal swabs) was generated for CT or NG. Male urine, and vaginal swabs were collected and tested at 13 geographically diverse intended use clinical sites across the US. There were 48 operators that took part in cobas® liat CT/NG testing, of which, 43 represented CLIA-waived operators. Five of the 48 operators represented experienced laboratorians in a moderate complexity laboratory.

A total of 4852 subjects (2512 females and 2340 males) were enrolled in the study and provided specimens for collection. Note, two subjects, declared male at birth, provided vaginal swab specimens. Of these subjects, 72 were non-evaluable due to protocol deviations and incidents

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

(18), invalid cobas and/or final comparator result (45), or sample collection incidents (9). Of the evaluable subjects, 2304 male subjects provided 2302 male urine specimens (2 subjects provided vaginal swab specimens) and 2476 females provided 1240 clinician-collected vaginal swabs and 1236 self-collected vaginal swabs for evaluation in the clinical study.

Prospectively enrolled female subjects provided 4 vaginal swab specimens, three for comparator tests and one for the cobas® liat CT/NG/MG nucleic acid test. Vaginal swab specimen for the cobas® liat CT/NG/MG nucleic acid test was either collected by clinician or self-collected.

Prospectively enrolled male subjects provided a urine specimen that was aliquoted into the respective manufacturers' collection devices and cobas® PCR Media.

Specimens were tested for CT and NG with the investigational and the reference comparator NAATs. All tests were run according to the respective IFU.

The clinical performance of cobas® liat CT/NG was evaluated by comparing the results from collected specimen types to a pre-specified PIS algorithm. The PIS/CCA result for each analyte was derived from a combination of 3 reference NAATs (NAAT1, NAAT2, and NAAT3). If NAAT1 and NAAT2 are concordant, then the final PIS/CCA result for the respective analyte is the concordant result obtained from NAAT1 and NAAT2. If NAAT1 and NAAT2 are discordant, then NAAT3 is performed to be the tiebreaker between the first 2 discordant results. Table 15 below shows the PIS and CCA algorithm for each analyte.

| NAAT 1 | NAAT 2 | NAAT 3 (if needed) | Patient Infected<br>Statusª | Composite<br>Comparator<br>Algorithm |
|---------|---------|--------------------|-----------------------------|--------------------------------------|
| + | + | N/A | Infected | Positive |
| + | - | + | Infected | Positive |
| - | + | + | Infected | Positive |
| - | - | N/A | Not Infected | Negative |
| + | - | - | Not Infected | Negative |
| - | + | - | Not Infected | Negative |
| - | Invalid | + | Indeterminate | Indeterminate |
| - | Invalid | - | Not Infected | Negative |
| Invalid | - | + | Indeterminate | Indeterminate |
| Invalid | - | - | Not Infected | Negative |
| + | Invalid | - | Indeterminate | Indeterminate |

Table 15: Determination of the PIS/CCA result for CT and NG, respectively

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

| NAAT 1 | NAAT 2 | NAAT 3 (if needed) | Patient Infected<br>Statusa | Composite<br>Comparator<br>Algorithm |
|---------|---------|--------------------|-----------------------------|--------------------------------------|
| Invalid | + | - | Indeterminate | Indeterminate |
| + | Invalid | + | Infected | Positive |
| Invalid | + | + | Infected | Positive |
| Invalid | Invalid | N/A | Indeterminate | Indeterminate |

N/A: not applicable; NAAT: nucleic acid amplification test.

a The results from NAAT1 and NAAT2 determined if NAAT3 needed to be performed. The "Infected" or "Not Infected" patient infected status was derived from the total combination of results obtained from the reference NAATs.

The sample types of male urine and vaginal swab were used to create the PIS and CCA results, respectively, for men and women. The cobas® liat CT/NG results of…