cobas CT/NG for use on cobas 6800/8800 systems

K173887 · Roche Molecular Systems, Inc. · LSL · Mar 21, 2018 · Microbiology

Device Facts

• Record ID: K173887
• Device Name: cobas CT/NG for use on cobas 6800/8800 systems
• Applicant: Roche Molecular Systems, Inc.
• Product Code: LSL · Microbiology
• Decision Date: Mar 21, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3390
• Device Class: Class 2
• Attributes: Real-World Evidence, Pediatric

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K173887 · Mar 21, 2018	cobas CT/NG for use on cobas 6800/8800 systems	Roche Molecular Systems, Inc.	Archived prospectively collected clinical specimens (urine, cervical, endocervical swabs) from a prior clinical study	Archived clinical specimens were used to supplement the prospective clinical study data to establish the Patient Infected Status (PIS) for Neisseria gonorrhoeae (NG) performance evaluation.	Archived clinical specimens; Patient Infected Status (PIS); Clinical performance evaluation

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Clinical Performance Evaluation (NG archived specimens); Retrospective analysis of archived prospectively collected clinical specimens	Female subjects from a prior clinical study (COB-CTNG-282); Sample Size: 371 archived specimens from 295 female subjects; Number of Sites: Not specified for archived portion	Not applicable for this study	Sensitivity, specificity, PPV, NPV for NG detection

Indications for Use

The cobas® CT/NG on the cobas® 6800/8800 system 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/or Neisseria gonorrhoeae (NG) DNA in male and female urine, clinician-instructed self-collected vaginal swab specimens (collected in a clinical setting), clinician-collected vaginal swab specimens, and endocervical swab specimens, all collected in cobas® PCR Media (Roche Molecular Systems, Inc.), and cervical specimens collected in PreservCyt® solution. This test is intended as an aid in the diagnosis of chlamydial and gonococcal disease in both symptomatic and asymptomatic individuals.

Device Story

Automated, qualitative in vitro nucleic acid diagnostic test; utilizes real-time PCR for direct detection of CT and NG DNA. Input: male/female urine, vaginal swabs, endocervical swabs, cervical specimens in PreservCyt. Process: fully automated nucleic acid extraction/purification; PCR amplification using target-specific primers/probes for CT cryptic plasmid/ompA gene and NG DR-9 region; DNA internal control monitors process. Output: qualitative results (positive/negative/invalid) displayed on system screen, exported, or printed. Used in clinical laboratory settings; operated by trained laboratory personnel. Healthcare providers use results to aid diagnosis of chlamydial and gonococcal disease; enables timely clinical decision-making and patient management.

Clinical Evidence

Multi-site, prospective clinical study (n=5,053 evaluable subjects). Performance compared to Patient Infected Status (PIS) based on combined results of FDA-cleared NAATs. Sensitivity for CT ranged from 92.5% to 100% across specimen types; specificity ranged from 98.8% to 99.8%. Sensitivity for NG ranged from 89.5% to 100%; specificity ranged from 99.7% to 100%. Reproducibility study confirmed high precision across sites, lots, and operators.

Technological Characteristics

Automated real-time PCR assay. Materials: magnetic glass particles for nucleic acid extraction. Energy: thermal cycling for PCR. Connectivity: integrated with cobas 6800/8800 Systems. Software: automated data management and result assignment. Sterilization: N/A (reagents). Detection: TaqMan-based fluorescence resonance energy transfer (FRET) using reporter/quencher probes.

Indications for Use

Indicated for the qualitative detection of Chlamydia trachomatis and/or Neisseria gonorrhoeae DNA in male and female urine, clinician-instructed self-collected vaginal swabs, clinician-collected vaginal swabs, endocervical swabs, and cervical specimens in PreservCyt solution. Intended for symptomatic and asymptomatic individuals as an aid in diagnosis.

Regulatory Classification

Identification

Neisseria spp. direct serological test reagents are devices that consist of antigens and antisera used in serological tests to identify Neisseria spp. from cultured isolates. Additionally, some of these reagents consist of Neisseria spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) which may be used to detect the presence of Neisseria spp. directly from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria belonging to the genus Neisseria, such as epidemic cerebrospinal meningitis, meningococcal disease, and gonorrhea, and also provides epidemiological information on diseases caused by these microorganisms. The device does not include products for the detection of gonorrhea in humans by indirect methods, such as detection of antibodies or of oxidase produced by gonococcal organisms.

Predicate Devices

• cobas® CT/NG v2.0 Test (K163184)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

A. 510(k) Number:

K173887

B. Purpose for Submission:

To determine substantial equivalence for the cobas CT/NG assay for use on the cobas 6800/8800 Systems for detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA from self-collected vaginal swabs, clinician-collected vaginal swabs, endocervical swabs, male and female urine, and cervical specimens collected in PreservCyt solution.

C. Measurand:

Chlamydia trachomatis and Neisseria gonorrhoeae DNA

D. Type of Test:

Nucleic acid extraction, purification and amplification assay (real-time polymerase chain reaction)

E. Applicant:

Roche Molecular Systems, Inc.

F. Proprietary and Established Names:

cobas CT/NG

cobas 6800/8800 Systems

G. Regulatory Information:

1. Regulation section:
21 CFR 866.3390 - Neisseria spp. direct serological test reagents
2. Classification:
Class II
3. Product code:
LSL - DNA-Reagents, Neisseria

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MKZ - DNA Probe, Nucleic Acid Amplification, Chlamydia
OOI - Real Time Nucleic Acid Amplification System

# 4. Panel:

Microbiology (83)

# H. Intended Use:

# 1. Intended use(s):

The cobas CT/NG on the cobas 6800/8800 system 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/or Neisseria gonorrhoeae (NG) DNA in male and female urine, clinician-instructed self-collected vaginal swab specimens (collected in a clinical setting), clinician-collected vaginal swab specimens, and endocervical swab specimens, all collected in cobas PCR Media (Roche Molecular Systems, Inc.), and cervical specimens collected in PreservCyt solution. This test is intended as an aid in the diagnosis of chlamydial and gonococcal disease in both symptomatic and asymptomatic individuals.

# Ancillary Collection Kits:

The cobas PCR Media Dual Swab Sample Kit is used to collect and transport endocervical and vaginal swab specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for gynecological specimens.

Note: This kit has been validated for use with the following tests:

- cobas CT/NG v2.0 Test
- cobas CT/NG for use on cobas 6800/8800 Systems

The cobas PCR Media Uni Swab Sample Kit is used to collect and transport human specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for human specimens.

Note: This kit has been validated for use with the following tests:

- cobas CT/NG v2.0 Test
- cobas CT/NG for use on cobas 6800/8800 Systems
- cobas Cdiff Test for use on the cobas 4800 System

The cobas PCR Urine Sample Kit is used to collect and transport urine specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for urine specimens. Use this collection kit only with either cobas CT/NG on cobas 6800/8800 Systems or the cobas CT/NG v2.0 Test.

# 2. Indication(s) for use:

Same as the Intended Use

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3. Special conditions for use statement(s):
For prescription use only

4. Special instrument requirements:
cobas 6800/8800 Systems

# I. Device Description:

The cobas CT/NG assay is a new fully automated, qualitative real-time PCR assay performed on the cobas 6800 System and cobas 8800 System. cobas CT/NG enables the detection of CT/NG DNA in endocervical and vaginal swabs, urine, and cervical specimens from infected female patients and urine specimens from infected male patients. Target-specific primers and two probes are used to detect but not discriminate between the CT cryptic plasmid and the ompA gene. Additionally, target-specific primers and two probes are used to detect but not discriminate between two conserved sequences in the NG DR-9 region. The DNA Internal Control, used to monitor the entire sample preparation and PCR amplification process, is introduced into each specimen during sample processing. In addition, the test utilizes a low titer positive and a negative control.

# J. Substantial Equivalence Information:

1. Predicate device name(s):
cobas CT/NG v2.0 Test

2. Predicate 510(k) number(s):
K163184

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Regulation | 866.3390 | 866.3390 |
| Intended Use | The cobas CT/NG on the cobas 6800/8800 system 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/or *Neisseria gonorrhoeae* (NG) DNA in male and female urine, clinician instructed self-collected vaginal swab specimens (collected in a clinical setting), clinician-collected vaginal swab | The cobas CT/NG v2.0 Test is an automated, in vitro nucleic acid amplification test for the qualitative detection of *Chlamydia trachomatis* (CT) and/or *Neisseria gonorrhoeae* (NG) DNA in urogenital specimens. The Test utilizes the Polymerase Chain Reaction (PCR) for the detection of *Chlamydia trachomatis* and *Neisseria gonorrhoeae* DNA in male and female urine, self-collected vaginal swab specimens (collected in a clinical setting), cliniciancollected vaginal swab |

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| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | specimens, and endocervical swab specimens, all collected in cobas PCR Media (Roche Molecular Systems, Inc.), and cervical specimens collected in PreservCyt solution. This test is intended as an aid in the diagnosis of chlamydial and gonococcal disease in both symptomatic and asymptomatic individuals. Ancillary Collection Kits: The cobas PCR Media Dual Swab Sample Kit is used to collect and transport endocervical and vaginal swab specimens. The cobas® PCR Media serves as a nucleic acid stabilizing transport and storage medium for gynecological specimens. Note: This kit has been validated for use with the following tests: cobas CT/NG v2.0 Test cobas® CT/NG for use on cobas 6800/8800 Systems. The cobas PCR Media Uni Swab Sample Kit is used to collect and transport human specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for human specimens Note: This kit has been validated for use with the following tests: cobas CT/NG v2.0 Test cobas CT/NG for use on cobas 6800/8800 Systems cobas Cdiff Test for use on the cobas 4800 System The cobas PCR Urine Sample Kit is used to collect and transport urine specimens. The cobas PCR Media serves as a nucleic acid | specimens, and endocervical swab specimens, all collected in cobas PCR Media (Roche Molecular Systems, Inc.), and cervical specimens collected in PreservCyt solution. This test is intended as an aid in the diagnosis of chlamydial and gonococcal disease in both symptomatic and asymptomatic individuals. Ancillary Collection Kits: The cobas PCR Media Dual Swab Sample Kit is used to collect and transport endocervical and vaginal swab specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for gynecological specimens. Use this collection kit only with the cobas CT/NG v2.0 Test. The cobas PCR Media Uni Swab Sample Kit is used to collect and transport human specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for human specimens. Use this collection kit with cobas CT/NG v2.0 Test and cobas Cdiff Tests. The cobas PCR Urine Sample Kit is used to collect and transport urine specimens. The cobas PCR Media serves as a nucleic acid stabilizing transport and storage medium for urine specimens. Use this collection kit only with the cobas CT/NG v2.0 Test. |

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| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | stabilizing transport and storage medium for urine specimens Use this collection kit only with either cobas CT/NG on cobas 6800/8800 Systems or the cobas CT/NG v2.0 Test. | |
| Sample Types | Same | Male and female urine, Self-collected/clinician-collected vaginal swab specimens in cobas PCR Media, Endocervical swab specimens in cobas PCR Media, Cervical specimens in PreservCyt solution |
| Subject Status | Same | Asymptomatic and symptomatic |
| Sample Collection Devices | Same | cobas PCR Media Dual Swab Sample Kit cobas PCR Media Uni Swab Sample Kit cobas PCR Urine Sample Kit |
| CT Analyte targets | Same | CT cryptic plasmid DNA CT ompA gene |
| NG Analyte targets | Same | NG genomic DNA |
| Amplification Technology | Same | Real-time PCR |
| Detection Chemistry | Same | Paired reporter and quencher fluorescence labeled probes (TaqMan Technology) using fluorescence resonance energy transfer (FRET) |
| Result Analysis | Same | Based on PCR cycle threshold analysis |

| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Sample Preparation Procedure | Automated | Semi-automated |
| Analyzer | **cobas** 6800/8800 systems | **cobas** 4800 System |

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# K. Standard/Guidance Document Referenced (if applicable):

N/A

# L. Test Principle:

The cobas CT/NG assay is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas 6800/8800 software which assigns test results for all tests as positive, negative, or invalid. Results can be reviewed directly on the system screen, exported, or printed as a report.

Nucleic acid from patient samples and internal control DNA (DNA-IC), added into the specimens to monitor for inhibition of nucleic acid isolation and amplification, is simultaneously extracted. In summary, bacterial nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors are removed with subsequent wash steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature.

Selective amplification of target nucleic acid from the sample is achieved by the use of target-specific forward and reverse primers selected from highly conserved plasmid and genomic regions of CT and NG. A region on the CT cryptic plasmid and the ompA gene (dual target) and two conserved sequences of the NG DR-9 region are amplified. Selective amplification of DNA-IC is achieved by the use of sequence-specific forward and reverse primers which have no homology with either the CT or NG target regions. A thermostable DNA polymerase enzyme is used for PCR amplification. The target and DNA-IC sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP) instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicons from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master mix, during the first thermal cycling step. However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.

The cobas CT/NG master mix contains two detection probes specific for the CT target sequences, two detection probes specific for the NG target sequences and one for the DNA-IC. The probes are labeled with target specific fluorescent reporter dyes allowing simultaneous detection of CT targets, NG targets, and DNA-IC in three different channels. When not bound to the target sequence, the fluorescent signal of the intact probes is suppressed by a quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye increases concomitantly. Real-time detection and discrimination of PCR products is

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accomplished by measuring the fluorescence of the released reporter dyes for the CT and NG targets and DNA-IC, respectively.

# **M. Performance Characteristics (if/when applicable):**

1. Analytical performance:

a. Precision/Reproducibility:

Precision:

Precision of the cobas CT/NG assay was examined in-house using a panel composed of CT and NG cultures diluted into the following backgrounds: 1. negative endocervical swab specimen matrix collected in cobas PCR Media, 2. negative urine matrix mixed with cobas PCR Media, and 3. negative cervical specimen matrix collected in PreservCyt Solution. Endocervical swabs were intended to represent both swab specimen types (endocervical and vaginal) collected in cobas PCR Media. Four levels of each analyte were tested using CT serovar D and NG strain 2948 (ATCC 19424) as the target organisms.

The precision panel for each of the three matrices contained one negative panel member and panel members with high negative, low positive, and moderate positive concentrations of CT and NG, corresponding to ≤0.3X LoD, ~1X LoD, and ~3X LoD, for each panel matrix. Upon repeated measure, specimens with concentrations of 1X LoD are expected to yield positive results ≥95% of the time and specimens with concentrations of 3X LoD are expected to yield positive results ≥99% of the time. Testing was performed over 12 non-consecutive days with three lots of cobas CT/NG reagents and two runs per day using two instruments for a total of 24 runs. A total of 72 replicates were tested for each panel member.

Study results demonstrated the expected percent agreement for all panel members. All negative panel members tested negative throughout the study. Analysis of standard deviation and percent coefficient of variation (CV) of the Ct values from valid tests performed on positive panel members yielded overall CV (%) ranges from 1.59% to 4.05% for CT and from 1.17% to 3.55% for NG. Detailed results for this study are presented in the Tables 1-3 below.

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Table 1. Percent Agreement with Expected Results with 95% Confidence Intervals for CT and NG Positive Panel Members

| Panel | N Tested | Expected Hit Rate | CT Positive | CT Hit Rate | CT 95% CI | NG Positive | NG Hit Rate | NG 95% CI |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Endocervical Swab in cobas PCR Media | | | | | | | | |
| High Negative | 72 | 20-80% | 51 | 71% | 58.9%-81.0% | 32 | 44% | 32.7%-56.6% |
| Low Positive | 72 | ≥ 95% | 69 | 96% | 88.3%-99.1% | 68 | 94% | 86.4%-98.5% |
| Moderate Positive | 72 | ≥ 99% | 72 | 100% | 95.0%-100% | 72 | 100% | 95.0%-100% |
| Cervical Samples collected into PreservCyt Solution | | | | | | | | |
| High Negative | 72 | 20-80% | 38 | 53% | 40.7%-64.7% | 47 | 65% | 53.1%-76.1% |
| Low Positive | 72 | ≥ 95% | 72 | 100% | 95.0%-100% | 69 | 96% | 88.3%-99.1% |
| Moderate Positive | 72 | ≥ 99% | 72 | 100% | 95.0%-100% | 72 | 100% | 95.0%-100% |
| cobas PCR Media with Urine | | | | | | | | |
| High Negative | 72 | 20-80% | 56 | 78% | 66.4%-86.7% | 56 | 78% | 66.4%-86.7% |
| Low Positive | 72 | ≥ 95% | 71 | 99% | 92.5%-100% | 72 | 100% | 95.0%-100% |
| Moderate Positive | 72 | ≥ 99% | 72 | 100% | 95.0%-100% | 72 | 100% | 95.0%-100% |

Table 2. Overall Mean, Standard Deviations and Coefficients of Variation (%) for Cycle Threshold, CT Positive Panel Members

| Panel | Mean Ct | Between instrumen | | Between Lot | | Within Run | | Between Run | | Between Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV |
| Endocervical Swab in cobas PCR Media | | | | | | | | | | | | | |
| High Negative | 39.7 | 0.00 | 0.00 | 0.00 | 0.00 | 1.27 | 3.21 | 0.00 | 0.00 | 0.34 | 0.85 | 1.32 | 3.32 |
| Low Positive | 38.5 | 0.00 | 0.00 | 0.04 | 0.10 | 1.14 | 2.96 | 0.00 | 0.00 | 0.48 | 1.25 | 1.24 | 3.22 |
| Moderate Positive | 36.9 | 0.00 | 0.00 | 0.25 | 0.69 | 0.54 | 1.45 | 0.07 | 0.18 | 0.00 | 0.00 | 0.60 | 1.62 |
| Cervical Samples collected into PreservCyt Solution | | | | | | | | | | | | | |
| High Negative | 38.3 | 0.60 | 1.57 | 0.52 | 1.37 | 1.12 | 2.92 | 0.00 | 0.00 | 0.00 | 0.00 | 1.37 | 3.58 |
| Low Positive | 36.9 | 0.21 | 0.56 | 0.28 | 0.76 | 0.68 | 1.85 | 0.00 | 0.00 | 0.00 | 0.00 | 0.77 | 2.08 |
| Moderate Positive | 35.6 | 0.00 | 0.00 | 0.20 | 0.56 | 0.52 | 1.46 | 0.09 | 0.24 | 0.02 | 0.05 | 0.56 | 1.59 |
| cobas PCR Media with Urine | | | | | | | | | | | | | |
| High Negative | 38.9 | 0.00 | 0.00 | 0.12 | 0.30 | 1.25 | 3.22 | 0.39 | 1.01 | 0.00 | 0.00 | 1.32 | 3.39 |
| Low Positive | 38.3 | 0.11 | 0.28 | 0.00 | 0.00 | 1.52 | 3.97 | 0.00 | 0.00 | 0.29 | 0.77 | 1.55 | 4.05 |
| Moderate Positive | 37.1 | 0.00 | 0.00 | 0.00 | 0.00 | 1.05 | 2.84 | 0.00 | 0.00 | 0.28 | 0.77 | 1.09 | 2.94 |

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**Table 3. Overall Mean, Standard Deviations and Coefficients of Variation (%) for Cycle Threshold, NG Positive Panel Members**

| Panel | Mean Ct | Between instrumen | | Between Lot | | Within Run | | Between Run | | Between Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV |
| **Endocervical Swab in cobas PCR Media** | | | | | | | | | | | | | |
| High Negative | 39.1 | 0.00 | 0.00 | 0.31 | 0.79 | 0.84 | 2.14 | 0.72 | 1.85 | 0.57 | 1.46 | 1.28 | 3.28 |
| Low Positive | 38.1 | 0.00 | 0.00 | 0.00 | 0.00 | 1.27 | 3.34 | 0.00 | 0.00 | 0.00 | 0.00 | 1.27 | 3.34 |
| Moderate Positive | 36.5 | 0.00 | 0.00 | 0.24 | 0.67 | 0.69 | 1.89 | 0.00 | 0.00 | 0.15 | 0.40 | 0.74 | 2.04 |
| **Cervical Samples collected into PreservCyt Solution** | | | | | | | | | | | | | |
| High Negative | 39.0 | 0.34 | 0.87 | 0.00 | 0.00 | 1.11 | 2.85 | 0.08 | 0.20 | 0.45 | 1.16 | 1.25 | 3.21 |
| Low Positive | 38.0 | 0.00 | 0.00 | 0.00 | 0.00 | 1.25 | 3.28 | 0.00 | 0.00 | 0.00 | 0.00 | 1.25 | 3.28 |
| Moderate Positive | 35.8 | 0.00 | 0.00 | 0.28 | 0.78 | 0.76 | 2.13 | 0.00 | 0.00 | 0.00 | 0.00 | 0.81 | 2.27 |
| **cobas PCR Media with Urine** | | | | | | | | | | | | | |
| High Negative | 39.1 | 0.00 | 0.00 | 0.26 | 0.66 | 1.35 | 3.46 | 0.00 | 0.00 | 0.18 | 0.45 | 1.39 | 3.55 |
| Low Positive | 36.7 | 0.14 | 0.38 | 0.16 | 0.42 | 0.71 | 1.92 | 0.00 | 0.00 | 0.00 | 0.00 | 0.74 | 2.00 |
| Moderate Positive | 34.9 | 0.00 | 0.00 | 0.16 | 0.47 | 0.37 | 1.06 | 0.06 | 0.18 | 0.00 | 0.00 | 0.41 | 1.17 |

# Reproducibility:

A Reproducibility Study was performed across different sites, lots, operators/batches, and days, for cobas CT/NG using three panels of pooled negative or contrived specimens prepared from swabs and urine in cobas PCR Media and cervical specimens in PreservCyt Solution. Testing was performed at one in-house and two external sites. One panel consisted of the three sample matrices, with six concentrations per matrix, and three replicates per concentration for a total of 54-samples in one panel. Concentrations utilized were as follows, with positive panel members prepared by spiking CT serovar D and/or NG 19424 into the CT/NG-negative background:

| Panel Member | CT Level | NG Level |
| --- | --- | --- |
| 1 | Negative | Negative |
| 2 | ~ 0.3 × LoD (High negative) | ~ 0.3 × LoD (High negative) |
| 3 | ~ 1.0 × LoD | Negative |
| 4 | Negative | ~ 1.0 × LoD |
| 5 | ~ 3.0 × LoD | ~ 1.0 × LoD |
| 6 | ~ 1.0 × LoD | ~ 3.0 × LoD |

CT = *Chlamydia trachomatis*; LoD = limit of detection; NG = *Neisseria gonorrhoeae*

A batch was comprised of one 54-sample panel and two controls (one positive control and one negative control). Two operators at each site tested one batch each per day with each lot. Two valid batches had to be completed within a 24-hour period. Each site received two of the three reagent lots and performed 6 days of testing per reagent lot for a total of

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12 days of testing. For each combination of concentrations noted, a total of 216 replicates were tested in each specimen type (urine, swab, and PreservCyt) for a total of 3,888 tests performed, with only two failed tests each from PreservCyt.

No false positive results for either CT or NG were observed in any of the three matrices; thus the negative percent agreement was 100% for both analytes. For panel members with concentrations at or near the limit of detection (e.g., 1x LoD) of the test, the lower limit of the 2-sided 95% CI of the percentage of correct test results was at least 97.4% for CT and 95.3% for NG.

For panel members with concentrations 3-times above the limit of detection (e.g., 3x LoD) of the test, the lower limit of the 2-sided 95% CI of the percentage of correct test results was 98.3% for both CT and NG.

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**Table 4. Percent Agreement for Panel Members With Concentration at or Near the LoD (1x LoD) or 3x LoD**

| Media Type | Panel Member | CT | | NG | |
| --- | --- | --- | --- | --- | --- |
| | | Percent Agreement | Percent Agreement 95% CI | Percent Agreement | Percent Agreement 95% CI |
| PCR Media/Urine | 1.0x LoD CT, Negative NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | Negative CT, 1.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | 3.0x LoD CT, 1.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | 1.0x LoD CT, 3.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| PCR Media/Swab | 1.0x LoD CT, Negative NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | Negative CT, 1.0x LoD NG | 100 (216/216) | (98.3, 100) | 98.1 (212/216) | (95.3, 99.5) |
| | 3.0x LoD CT, 1.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | 1.0x LoD CT, 3.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| PreservCyt/Cervical | 1.0x LoD CT, Negative NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | Negative CT, 1.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | 3.0x LoD CT, 1.0x LoD NG | 100 (216/216) | (98.3, 100) | 100 (216/216) | (98.3, 100) |
| | 1.0x LoD CT, 3.0x LoD NG | 99.5 (214/215*) | (97.4, 99.9) | 100 (215/215) | (98.3, 100) |

Notes: LoD= Limit of Detection; CT= *Chlamydia trachomatis*; NG = *Neisseria gonorrhoeae*.

*assessed based on valid test results

For each positive panel member, precision was evaluated by sample type in terms of lot, site, day, operator/batch within site, lot and day, and within-batch components on the corresponding analyte cycle threshold (Ct) values of cobas CT/NG.

# *Chlamydia trachomatis* results:

The range of the total coefficient of variation, among positive panel members, was from 0.9% to 3.2%. The maximum total coefficient of variation was observed in the high negative panel members (0.3x LoD CT, 0.3x LoD NG) and most of that variability (98.6% for urine, 100% for swab and 81.7% for cervical) was explained by random error.

Detailed results from testing are presented in the Tables 5-8 as follows:

11

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Table 5. CT: Overall Mean, Attributable Percentage of Total Variance, Total Precision Standard Deviation, and CV(%) of Cobas CT/NG Cycle Threshold (Ct) Values by CT Positive Panel Member for Each Media Type

| Panel Member | | Mean CT Value | | Percentage of Total Variance (CV[%]) | | | | | Total Precision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Media Type | Concentration | \(N^a\) | Mean Estimateb | Site | Lot | Day | Operator /Batch | Within-Batch | SDc | CV(%)d |
| PCR Media/ Urine | 0.3xLoD CT, 0.3xLoD NG | 154 | 39.2 | 1.4% (0.4) | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 98.6% (3.0) | 1.20 | 3.1 |
| | 1xLoD CT, Negative NG | 216 | 36.8 | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 100.0% (1.5) | 0.54 | 1.5 |
| | 3xLoD CT, 1xLoD NG | 216 | 35.4 | 2.4% (0.1) | 0.0% (0.0) | 21.1% (0.4) | 0.0% (0.0) | 76.5% (0.8) | 0.33 | 0.9 |
| | 1xLoD CT, 3xLoD NG | 216 | 36.9 | 0.0% (0.0) | 0.0% (0.0) | 10.3% (0.5) | 4.4% (0.3) | 85.3% (1.5) | 0.59 | 1.6 |
| PCR Media/ Swab | 0.3xLoD CT, 0.3xLoD NG | 128 | 39.5 | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 100.0% (3.2) | 1.26 | 3.2 |
| | 1xLoD CT, Negative NG | 216 | 37.2 | 0.0% (0.0) | 1.6% (0.2) | 6.6% (0.5) | 0.0% (0.0) | 91.8% (1.7) | 0.66 | 1.8 |
| | 3xLoD CT, 1xLoD NG | 216 | 35.5 | 4.7% (0.2) | 0.0% (0.0) | 9.0% (0.3) | 4.8% (0.2) | 81.6% (0.9) | 0.37 | 1.0 |
| | 1xLoD CT, 3xLoD NG | 216 | 37.2 | 0.0% (0.0) | 0.0% (0.0) | 3.6% (0.4) | 0.0% (0.0) | 96.4% (2.3) | 0.87 | 2.3 |
| PreservCyt/ Cervical | 0.3xLoD CT, 0.3xLoD NG | 92 | 39.9 | 0.0% (0.0) | 0.0% (0.0) | 18.3% (1.4) | 0.0% (0.0) | 81.7% (2.9) | 1.29 | 3.2 |
| | 1xLoD CT, Negative NG | 216 | 37.0 | 12.0% (0.6) | 1.9% (0.2) | 0.0% (0.0) | 0.0% (0.0) | 86.2% (1.5) | 0.60 | 1.6 |
| | 3xLoD CT, 1xLoD NG | 216 | 35.6 | 0.6% (0.1) | 3.7% (0.2) | 0.0% (0.0) | 6.3% (0.3) | 89.3% (0.9) | 0.36 | 1.0 |
| | 1xLoD CT, 3xLoD NG | 214 | 36.8 | 13.1% (0.6) | 3.7% (0.3) | 5.3% (0.4) | 2.3% (0.3) | 75.6% (1.5) | 0.63 | 1.7 |

Note: The table only includes results with detectable analyte. SD = standard deviation. CV(%) = percent Coefficient of variation.

\( ^{a} \) Number of valid tests with detectable analyte.

\( ^{b} \) Calculated using SAS MIXED procedure.

\( ^{c} \) Calculated using the total variability from the SAS MIXED procedure.

\( ^{d} \) CV(%) = (SD/Mean) * 100.

LoD= Limit of Detection; CT= Chlamydia trachomatis ; NG = Neisseria gonorrhoeae.

Table 6. CT: Percent Agreement by Panel Member for Lot, Site and Day - cobas PCR Media/Urine

| | | | CT Percent Agreementa | | |
| --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | Siteb | Day |

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| | | | CT Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site^{b} | | | Day | | |
| Negative CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 0.3x LoD CT, 0.3x LoD NG | 1.20 | 3.1 | 1 | 76.4 | 55/72 | 1 | 68.1 | 49/72 | 1 | 80.6 | 29/36 |
| | | | 2 | 70.8 | 51/72 | 2 | 73.6 | 53/72 | 2 | 77.8 | 28/36 |
| | | | 3 | 66.7 | 48/72 | 3 | 72.2 | 52/72 | 3 | 66.7 | 24/36 |
| | | | | | | | | | 4 | 77.8 | 28/36 |
| | | | | | | | | | 5 | 69.4 | 25/36 |
| | | | | | | | | | 6 | 55.6 | 20/36 |
| | | | | | | | | | | | |
| 1x LoD CT, Negative NG | 0.54 | 1.5 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| Negative CT, 1x LoD NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |

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| | | | CT Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site^{b} | | | Day | | |
| 3x LoD CT, 1x LoD NG | 0.33 | 0.9 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 1x LoD CT, 3x LoD NG | 0.59 | 1.6 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

$^{a}$ For CT Negative samples, Percent Agreement = (number of CT negative results/total valid results) x 100.

For CT Positive samples, Percent Agreement = (number of CT positive results/total valid results) x 100.

$^{b}$ Site 1, Site 2, and Site 3, respectively.

Ct=Cycle threshold; SD=Standard Deviation; CV=Coefficient of Variation; LoD= Limit of Detection.

CT= Chlamydia trachomatis; NG = Neisseria gonorrhoeae; n/a= not applicable.

Table 7. CT: Percent Agreement by Panel Member for Lot, Site and Day - cobas PCR Media/Swab

| | | | CT Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site^{b} | | | Day | | |
| Negative CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 0.3x LoD CT, 0.3x LoD NG | 1.26 | 3.2 | 1 | 61.1 | 44/72 | 1 | 56.9 | 41/72 | 1 | 50.0 | 18/36 |
| | | | 2 | 59.7 | 43/72 | 2 | 61.1 | 44/72 | 2 | 63.9 | 23/36 |
| | | | 3 | 56.9 | 41/72 | 3 | 59.7 | 43/72 | 3 | 55.6 | 20/36 |
| | | | | | | | | | 4 | 61.1 | 22/36 |

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| | | | CT Percent Agreement\( ^a \) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site\( ^b \) | | | Day | | |
| | | | | | | | | | 5 | 66.7 | 24/36 |
| | | | | | | | | | 6 | 58.3 | 21/36 |
| | | | | | | | | | | | |
| 1x LoD CT, Negative NG | 0.66 | 1.8 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| Negative CT, 1x LoD NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 3x LoD CT, 1x LoD NG | 0.37 | 1.0 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 1x LoD CT, 3x LoD NG | 0.87 | 2.3 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

\( ^{a} \) For CT Negative samples, Percent Agreement = (number of CT negative results/total valid results) x 100.

For CT Positive samples, Percent Agreement = (number of CT positive results/total valid results) x 100.

\( ^{b} \) Site 1, Site 2, and Site 3, respectively.

Ct=Cycle threshold; SD=Standard Deviation; CV=Coefficient of Variation; LoD= Limit of Detection.

CT= Chlamydia trachomatis; NG = Neisseria gonorrhoeae; n/a= not applicable.

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Table 8. CT: Percent Agreement by Panel Member for Lot, Site and Day - Preservcyt/Cervical

| | | | CT Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site^{b} | | | Day | | |
| Negative CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 0.3x LoD CT, 0.3x LoD NG | 1.29 | 3.2 | 1 | 38.9 | 28/72 | 1 | 34.7 | 25/72 | 1 | 40.0 | 14/35 |
| | | | 2 | 47.9 | 34/71 | 2 | 48.6 | 35/72 | 2 | 52.8 | 19/36 |
| | | | 3 | 41.7 | 30/72 | 3 | 45.1 | 32/71 | 3 | 38.9 | 14/36 |
| | | | | | | | | | 4 | 47.2 | 17/36 |
| | | | | | | | | | 5 | 41.7 | 15/36 |
| | | | | | | | | | 6 | 36.1 | 13/36 |
| | | | | | | | | | | | |
| 1x LoD CT, Negative NG | 0.60 | 1.6 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| Negative CT, 1x LoD NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |
| 3x LoD CT, 1x LoD NG | 0.36 | 1.0 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| | | | | | | | | | | | |

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| | | | CT Percent Agreement\( ^{a} \) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site\( ^{b} \) | | | Day | | |
| 1x LoD CT, 3x LoD NG | 0.63 | 1.7 | 1 | 98.6 | 71/72 | 1 | 98.6 | 71/72 | 1 | 97.2 | 35/36 |
| | | | 2 | 100.0 | 71/71 | 2 | 100.0 | 71/71 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 35/35 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

\( ^{a} \) For CT Negative samples, Percent Agreement = (number of CT negative results/total valid results) x 100.

For CT Positive samples, Percent Agreement = (number of CT positive results/total valid results) x 100.

\( ^{b} \) Site 1, Site 2, and Site 3, respectively.

Ct=Cycle threshold; SD=Standard Deviation; CV=Coefficient of Variation; LoD= Limit of Detection.

CT= Chlamydia trachomatis; NG = Neisseria gonorrhoeae; n/a= not applicable.

### Neisseria gonorrhoeae results:

The range of the total coefficient of variation, among positive panel members, was from 1.0% to 3.1%. The maximum total coefficient of variation was observed in the lowest concentration of positive panel members (0.3x LoD CT, 0.3x LoD NG) and most of that variability (98.7% for urine, 98.1% for swab and 85.3% for cervical) was explained by random error (within-batch).

Detailed results from testing are presented in Tables 9-12.

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Table 9. NG: Overall Mean, Attributable Percentage of Total Variance, Total Precision Standard Deviation, and CV(%) of cobas CTNG Cycle Threshold (Ct) Values by NG Positive Panel Member for Each Media Type

| Panel Member | | Mean CT value | | Percentage of Total Variance (CV[%]) | | | | | Total Precision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Media Type | Concentration | \(N^a\) | Mean Estimateb | Site | Lot | Day | Operator /Batch | Within-Batch | SDc | CV(%)d |
| PCR Media/ Urine | 0.3xLoD CT, 0.3xLoD NG | 159 | 39.3 | 0.7% (0.3) | 0.0% (0.0) | 0.6% (0.2) | 0.0% (0.0) | 98.7% (3.0) | 1.20 | 3.0 |
| | Negative CT, 1xLoD NG | 216 | 36.7 | 0.0% (0.0) | 0.5% (0.1) | 6.9% (0.5) | 0.0% (0.0) | 92.6% (1.7) | 0.63 | 1.7 |
| | 3xLoD CT, 1xLoD NG | 216 | 36.6 | 0.0% (0.0) | 2.5% (0.3) | 8.3% (0.5) | 0.0% (0.0) | 89.2% (1.6) | 0.61 | 1.7 |
| | 1xLoD CT, 3xLoD NG | 216 | 35.1 | 0.0% (0.0) | 0.0% (0.0) | 14.0% (0.4) | 0.0% (0.0) | 86.0% (1.0) | 0.37 | 1.0 |
| PCR Media/ Swab | 0.3xLoD CT, 0.3xLoD NG | 113 | 39.8 | 0.0% (0.0) | 0.0% (0.0) | 1.9% (0.4) | 0.0% (0.0) | 98.1% (3.1) | 1.25 | 3.1 |
| | Negative CT, 1xLoD NG | 212 | 38.2 | 0.0% (0.0) | 0.1% (0.1) | 1.8% (0.4) | 6.5% (0.7) | 91.6% (2.6) | 1.04 | 2.7 |
| | 3xLoD CT, 1xLoD NG | 216 | 36.9 | 0.0% (0.0) | 0.0% (0.0) | 6.3% (0.6) | 0.0% (0.0) | 93.7% (2.1) | 0.82 | 2.2 |
| | 1xLoD CT, 3xLoD NG | 216 | 35.7 | 0.0% (0.0) | 3.8% (0.3) | 14.4% (0.5) | 0.0% (0.0) | 81.8% (1.3) | 0.50 | 1.4 |
| PreservCyt/ Cervical | 0.3xLoD CT, 0.3xLoD NG | 112 | 39.5 | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 14.7% (1.0) | 85.3% (2.4) | 1.04 | 2.6 |
| | Negative CT, 1xLoD NG | 216 | 35.7 | 7.2% (0.4) | 4.9% (0.3) | 0.0% (0.0) | 0.0% (0.0) | 87.9% (1.3) | 0.49 | 1.4 |
| | 3xLoD CT, 1xLoD NG | 216 | 36.3 | 0.0% (0.0) | 0.0% (0.0) | 0.0% (0.0) | 9.6% (0.5) | 90.4% (1.6) | 0.61 | 1.7 |
| | 1xLoD CT, 3xLoD NG | 215 | 34.6 | 2.3% (0.2) | 0.0% (0.0) | 5.8% (0.2) | 12.0% (0.3) | 79.8% (0.9) | 0.34 | 1.0 |

Note: The table only includes results with detectable analyte. SD = standard deviation. CV(%) = percent coefficient of variation.

\( ^{a} \) Number of valid tests with detectable analyte.

\( ^{b} \) Calculated using SAS MIXED procedure.

\( ^{c} \) Calculated using the total variability from the SAS MIXED procedure.

\( ^{d} \) CV(%) = (SD/Mean) * 100.

LoD= Limit of Detection; CT = Chlamydia trachomatis ; NG = Neisseria gonorrhoeae.

18

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**Table 10. NG: Percent Agreement by Panel Member for Lot, Site and Day - cobas PCR Media/Urine**

| Panel Member | Ct SD | Ct CV % | NG Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Lot | | | Site^{b} | | | Day | | |
| Negative CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| 0.3x LoD CT, 0.3x LoD NG | 1.20 | 3.0 | 1 | 79.2 | 57/72 | 1 | 70.8 | 51/72 | 1 | 77.8 | 28/36 |
| | | | 2 | 73.6 | 53/72 | 2 | 76.4 | 55/72 | 2 | 75.0 | 27/36 |
| | | | 3 | 68.1 | 49/72 | 3 | 73.6 | 53/72 | 3 | 72.2 | 26/36 |
| | | | | | | | | | 4 | 80.6 | 29/36 |
| | | | | | | | | | 5 | 61.1 | 22/36 |
| | | | | | | | | | 6 | 75.0 | 27/36 |
| 1x LoD CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| Negative CT, 1x LoD NG | 0.63 | 1.7 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| 3x LoD CT, 1x LoD NG | 0.61 | 1.7 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

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| | | | NG Percent Agreement\( ^{a} \) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site\( ^{b} \) | | | Day | | |
| | | | | | | | | | | | |
| 1x LoD CT, 3x LoD NG | 0.37 | 1.0 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

\( ^{a} \) For NG Negative samples, Percent Agreement = (number of NG negative results/total valid results) x 100.

For NG Positive samples, Percent Agreement = (number of NG positive results/total valid results) x 100.

\( ^{b} \) Site 1, Site 2, and Site 3, respectively.

Ct=Cycle threshold; SD=Standard Deviation; CV=Coefficient of Variation; LoD=Limit of Detection.

CT= Chlamydia trachomatis; NG = Neisseria gonorrhoeae; n/a= not applicable.

Table 11. NG: Percent Agreement by Panel Member for Lot, Site and Day - cobas PCR Media/Swab

| | | | NG Percent Agreement\( ^{a} \) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site\( ^{b} \) | | | Day | | |
| Negative CT,Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| 0.3x LoD CT,0.3x LoD NG | 1.25 | 3.1 | 1 | 50.0 | 36/72 | 1 | 50.0 | 36/72 | 1 | 52.8 | 19/36 |
| | | | 2 | 51.4 | 37/72 | 2 | 52.8 | 38/72 | 2 | 55.6 | 20/36 |
| | | | 3 | 55.6 | 40/72 | 3 | 54.2 | 39/72 | 3 | 44.4 | 16/36 |
| | | | | | | | | | 4 | 55.6 | 20/36 |
| | | | | | | | | | 5 | 52.8 | 19/36 |
| | | | | | | | | | 6 | 52.8 | 19/36 |

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| | | | NG Percent Agreement\( ^{a} \) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site\( ^{b} \) | | | Day | | |
| 1x LoD CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| Negative CT, 1x LoD NG | 1.04 | 2.7 | 1 | 100.0 | 72/72 | 1 | 97.2 | 70/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 94.4 | 68/72 | 3 | 97.2 | 70/72 | 3 | 97.2 | 35/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 97.2 | 35/36 |
| | | | | | | | | | 6 | 94.4 | 34/36 |
| 3x LoD CT, 1x LoD NG | 0.82 | 2.2 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| 1x LoD CT, 3x LoD NG | 0.50 | 1.4 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

\( ^{a} \) For NG Negative samples, Percent Agreement = (number of NG negative results/total valid results) x 100.

For NG Positive samples, Percent Agreement = (number of NG positive results/total valid results) x 100.

\( ^{b} \) Site 1, Site 2, and Site 3, respectively.

Ct=Cycle threshold; SD=Standard Deviation; CV=Coefficient of Variation; LoD=Limit of Detection.

CT= Chlamydia trachomatis; NG = Neisseria gonorrhoeae; n/a= not applicable.

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Table 12. NG: Percent Agreement by Panel Member for Lot, Site and Day - Preservcyt/Cervical

| | | | NG Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site^{b} | | | Day | | |
| Negative CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| 0.3x LoD CT, 0.3x LoD NG | 1.04 | 2.6 | 1 | 63.9 | 46/72 | 1 | 59.7 | 43/72 | 1 | 54.3 | 19/35 |
| | | | 2 | 47.9 | 34/71 | 2 | 52.8 | 38/72 | 2 | 55.6 | 20/36 |
| | | | 3 | 44.4 | 32/72 | 3 | 43.7 | 31/71 | 3 | 47.2 | 17/36 |
| | | | | | | | | | 4 | 55.6 | 20/36 |
| | | | | | | | | | 5 | 52.8 | 19/36 |
| | | | | | | | | | 6 | 47.2 | 17/36 |
| 1x LoD CT, Negative NG | n/a | n/a | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| Negative CT, 1x LoD NG | 0.49 | 1.4 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |
| 3x LoD CT, 1x LoD NG | 0.61 | 1.7 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 72/72 | 2 | 100.0 | 72/72 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 36/36 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

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| | | | NG Percent Agreement^{a} | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member | Ct SD | Ct CV % | Lot | | | Site^{b} | | | Day | | |
| 1x LoD CT, 3x LoD NG | 0.34 | 1.0 | 1 | 100.0 | 72/72 | 1 | 100.0 | 72/72 | 1 | 100.0 | 36/36 |
| | | | 2 | 100.0 | 71/71 | 2 | 100.0 | 71/71 | 2 | 100.0 | 36/36 |
| | | | 3 | 100.0 | 72/72 | 3 | 100.0 | 72/72 | 3 | 100.0 | 36/36 |
| | | | | | | | | | 4 | 100.0 | 35/35 |
| | | | | | | | | | 5 | 100.0 | 36/36 |
| | | | | | | | | | 6 | 100.0 | 36/36 |

$^{a}$ For NG Negative samples, Percent Agreement = (number of NG negative results/total valid results) x 100.

For NG Positive samples, Percent Agreement = (number of NG positive results/total valid results) x 100.

$^{b}$ Site 1, Site 2, and Site 3, respectively.

Ct=Cycle threshold; SD=Standard Deviation; CV=Coefficient of Variation; LoD= Limit of Detection.

CT= Chlamydia trachomatis; NG = Neisseria gonorrhoeae; n/a= not applicable.

b. Linearity/assay reportable range:

Not applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

# Controls:

External controls are provided in the cobas CT/NG Positive Control Kit and the cobas Buffer Negative Control Kit, and are required to be included in each run. Validation of results is performed automatically by the cobas 6800/8800 software based on negative and positive control performance. Invalid batches require repeat testing of the entire batch. The CT/NG Positive Control contains non-infectious DNA plasmids of both CT and NG sequences and is used as a run control to monitor target amplification and detection. The cobas Buffer Negative Control Kit contains buffer with no nucleic acid.

In addition, a DNA Internal Control is introduced into each specimen during sample processing and monitors specimens for substances that may interfere with nucleic acid isolation and PCR amplification. The DNA Internal Control contains a non-CT/NG related DNA construct containing primer and probe specific sequence regions.

# Specimen Stability:

Specimen stability during storage was evaluated for the following four specimen types:

- Endocervical swabs collected in cobas PCR media
- Vaginal swabs collected in cobas PCR media
- Urine stabilized by cobas PCR media
- Cervical specimens collected in PreservCyt Solution (primary and secondary containers)

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Samples were prepared for each specimen matrix by combining prescreened negative individual specimens to generate ten unique pools. Five pools of each specimen type were used as CT/NG negative samples and five pools were used to prepare CT/NG positive samples. Positive samples were prepared by spiking individual CT and NG positive specimens into the negative samples to approximately 3 X LoD. Testing was performed on the day of sample preparation and at several time points during the storage time period. Study results demonstrated that positive samples remained positive for a minimum of 12 months when stored at both 2-8°C and 32°C. Matrix-only samples yielded the expected negative results for the same storage conditions. PreservCyt samples transferred to secondary containers demonstrated stability for up to 31 days when stored at both 2-8°C and 32°C.

# d. Detection limit:

Analytical sensitivity (Limit of Detection or LoD) of the cobas CT/NG Test for use on the cobas 6800/8800 Systems was determined by analyzing a dilution series of quantified cultures of Chlamydia trachomatis (serovars D and I) and Neisseria gonorrhoeae isolates 2948 (ATCC 19424) and 891. CT and NG cultures were diluted into a matrix of pooled negative specimens for each specimen type:

- Endocervical swabs collected in cobas PCR media
- Vaginal swabs collected in cobas PCR media
- Urine stabilized by cobas PCR media
- Cervical specimens collected in PreservCyt Solution

CT serovars and NG strains were tested at five concentration levels across three reagent lots, with 21-30 replicates for each positive level per reagent lot. The claimed LoD as established by the 95% Hit Rate Analysis represents the lowest concentration level which was detected in ≥ 95% of tested replicates and for which all higher concentration levels also had ≥95% detection. The LoD for CT serovars D and I and NG strains 2948 and 891 for each matrix are as follows:

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Table 13. Analytical Sensitivity (Limit of Detection)

| Specimen Types | C. trachomatis | | | | N. gonorrhoeae | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Serovar D | | Serovar I | | Strain 2948 | | Strain 891 | |
| | LoD (IFU/mL) | Mean Ct Value | LoD (IFU/mL) | Mean Ct Value | LoD (CFU/mL) | Mean Ct Value | LoD (CFU/mL) | Mean Ct Value |
| Endocervical Swab in cobas PCR Media | 0.3 | 36.6 | 1.4 | 37.1 | 0.4 | 36.3 | 0.08 | 37.5 |
| Vaginal Swab in cobas PCR | 0.3 | 37.3 | 1.4 | 37.0 | 0.4 | 36.3 | 0.08 | 37.0 |
| cobas PCR Media with Urine | 0.2 | 37.8 | 1.3 | 37.1 | 0.2 | 36.3 | 0.04 | 38.3 |
| Cervical Samples collected into PreservCyt Solution | 0.6 | 37.4 | 2.9 | 37.4 | 0.2 | 36.7 | 0.08 | 37.5 |
| IFU = Inclusion Forming Unit; quantification of the same C. trachomatis culture using DFA method equates 1 IFU to 6.6 signal generating units (SGU) for Serovar D, and 13.9 SGU for serovar I, where SGU includes Elementary Bodies as well as Reticulate Bodies of C. trachomatisCFU = Colony Forming Units | | | | | | | | |

#### Inclusivity:

Inclusivity and verification of the LoD were performed for 13 additional CT serovars, the Swedish new variant strain (nvCT) and an additional 43 independently isolated strains of NG using one lot of reagents. Testing was performed using CT and NG cultures diluted into negative background prepared for each specimen type by pooling pre-screened specimens negative for both CT and NG. Specimen types evaluated were: 1. vaginal swab specimens collected in cobas PCR media (intended to represent both endocervical and vaginal swab specimens), 2. urine mixed with cobas PCR media, and 3. cervical specimens collected in PreservCyt Solution. CT serovars and NG strains were spiked to concentrations that were near the highest LoD levels determined in the Limit of Detection study, and were tested concurrently when feasible. Twenty replicates were tested for each strain in each specimen type. Additional dilution levels and replicates were tested for those strains which did not demonstrate a \(\geq 95\%\) hit rate for the previously established LoDs. Results are shown in the Tables 14 and 15 below. For NG, all strains with the same LoD and hit rate for a given specimen type are presented as a group, shown in Table 15 in the columns labeled "Numbers of NG Strains."

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Table 14. Inclusivity Testing for CT Serovars

| Serovar Type or Variant | Swab* Specimens | | Urine Specimens | | PreservCyt Specimens | |
| --- | --- | --- | --- | --- | --- | --- |
| | IFU/mL | % Pos | IFU/mL | % Pos | IFU/mL | % Pos |
| A | 1.4 | 100 | 0.7 | 100 | 1.4 | 100 |
| B | 5.9 | 100 | 2.9 | 100 | 5.9 | 100 |
| Ba | 18.3 | 100 | 9.1 | 100 | 18.3 | 100 |
| C | 0.6 | 100 | 0.3 | 100 | 0.6 | 100 |
| E | 6.4 | 100 | 3.2 | 100 | 6.4 | 100 |
| F | 3.2 | 100 | 1.6 | 100 | 3.2 | 100 |
| G | 2.9 | 100 | 1.5 | 100 | 2.9 | 100 |
| H | 9.7 | 100 | 4.8 | 100 | 9.7 | 100 |
| J | 1.4 | 100 | 0.7 | 100 | 1.4 | 100 |
| K | 2.0 | 100 | 1.0 | 100 | 2.0 | 100 |
| LGV Type 1 | 5.9 | 100 | 3.0 | 100 | 5.9 | 100 |
| LGV Type 2 | 12.8 | 100 | 6.4 | 100 | 12.8 | 100 |
| LGV Type 3 | 0.7 | 100 | 0.4 | 100 | 0.7 | 100 |
| nvCT | 0.7 | 100 | 0.3 | 100 | 0.7 | 100 |

* Vaginal swab samples were used as a representative swab sample type for vaginal and endocervical swab specimens.

IFU = Inclusion Forming Unit

Table 15. Inclusivity Testing for NG Strains

| Numbers of NG Strains | Swab* Specimens | |
| --- | --- | --- |
| | CFU/mL | % Pos |
| 39 | 0.4 | ≥95 |
| 4 | 1.0 | ≥95 |
| Total = 43 | | |
| Numbers of NG Strains | Urine Specimens | |
| | CFU/mL | % Pos |
| 41 | 0.2 | ≥95 |
| 2 | 0.5 | 100 |
| Total = 43 | | |
| Numbers of NG Strains | PreservCyt Specimens | |
| | CFU/mL | % Pos |
| 42 | 0.4 | ≥95 |
| 1 | 1.0 | 100 |
| Total = 43 | | |

* Vaginal swab samples were used as a representative swab sample type for vaginal and endocervical swab specimens.

CFU = Colony Forming Units

In summary, based upon study results, the cobas CT/NG assay for use on the cobas 6800/8800 Systems detects the known serovars of CT and 43 strains of NG.

e. Analytical specificity and microbial interference:

A panel of 149 bacteria, fungi, and viruses, including those commonly found in the male and female urogenital tract, 20 representatives of non-gonorrhoeae Neisseria strains and

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other phylogenetically unrelated organisms, were tested with the cobas CT/NG assay to assess analytical specificity/cross-reactivity (see Table 17). Samples were spiked at concentrations of approximately \( 1 \times 10^{6} \) units/mL for bacteria and approximately \( 1 \times 10^{5} \) units/mL \( ^{1} \) for viruses into negative background prepared for each specimen type by pooling pre-screened specimens negative for both CT and NG. Specimen types evaluated were: 1. vaginal swab specimens collected in cobas PCR media (intended to represent both endocervical and vaginal swab specimens), 2. urine mixed with cobas PCR media, and 3. cervical specimens collected in PreservCyt Solution. Testing was performed with each potential interfering organism alone as well as with each organism mixed with CT and NG cultures at approximately 3x LoD. Results indicated that none of these organisms interfered with the detection of CT and NG or produced false positive results in the CT/NG negative matrices.

Table 17. Microorganisms tested for analytical specificity/cross-reactivity

| Achromobacter xerosis | Gemella haemolysans | Neisseria subflava |
| --- | --- | --- |
| Acinetobacter calcoaceticus | Haemophilus ducreyi | Neisseria weaverii |
| Acinetobacter lwoffii | Haemophilus influenzae | Pantoea agglomerans |
| Actinomyces israelii | Helicobacter pylori | Paracoccus denitrificans |
| Aerococcus viridans | Herpes simplex virus I | Peptostreptococcus anaerobius |
| Aeromonas hydrophila | Herpes simplex virus II** | Peptostreptococcus asaccharolyticus |
| Alcaligenes faecalis | HPV16* | Peptostreptococcus magnus |
| Atopobium vaginae | Kingella denitrificans | Plesiomonas shigelloides |
| Bacillus subtilis | Kingella kingae | Propionibacterium acnes |
| Bacteriodes fragilis | Klebsiella oxytoca | Proteus mirabilis |
| Bacteroides caccae | Klebsiella pneumoniae | Proteus penneri |
| Bacteroides ureolyticus | Lactobacillus acidophilus | Proteus vulgaris |
| Bergeriella denitrificans | Lactobacillus brevis | Providencia rettgeri |
| Bifidobacterium adolescentis | Lactobacillus crispatus | Providencia stuartii |
| Bifidobacterium breve | Lactobacillus jensenii | Pseudomonas aeruginosa |
| Bifidobacterium longum | Lactobacillus lactis | Pseudomonas fluorescens |
| Blautia product | Lactobacillus leichmannii | Pseudomonas putida |
| Branhamella catarrhalis | Lactobacillus oris | Rahnella aquatilis |
| Brevibacterium linens | Lactobacillus parabuchnerri | Rhizobium radiobacter |
| Campylobacter coli | Lactobacillus reuteri | Rhodospirillum rubrum |
| Campylobacter jejuni | Lactobacillus vaginalis | Saccharomyces cerevisiae |
| Candida albicans | Lactococcus lactis cremoris | Salmonella choleraesuis |

\( ^{1} \) All bacteria were quantified as Colony Forming Units (CFU) except Chlamydophila pneumoniae and Chlamydophila psittaci quantified as Elementary Bodies (EB). All viruses were quantified as units/mL as determined by TCID \( _{50} \) Endpoint Dilution Assay. Trichomonas vaginalis and HPV16 were quantified as cells/mL

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| Candida glabrata | Legionella pneumophila | Salmonella minnesota |
| --- | --- | --- |
| Candida parapsilosis | Leuconostoc paramesenteroides | Salmonella typhimurium |
| Candida tropicalis | Listeria monocytogenes | Serratia denitrificans |
| Chlamydophila pneumoniae | Micrococcus luteus | Serratia marcescens |
| Chlamydophila psittaci | Moraxella lacunata | Shigella dysenteriae |
| Chromobacter violaceum | Moraxella osloensis | Staphylococcus aureus |
| Citrobacter freundii | Morganella morganii | Staphylococcus epidermidis |
| Clostridium difficile | Mycobacterium smegmatis | Staphylococcus saprophyticus |
| Clostridium perfringens | Mycoplasma genitalium*** | Streptococcus agalactiae |
| Corynebacterium genitalium | Mycoplasma hominis | Streptococcus anginosus |
| Corynebacterium xerosis | Neisseria cinerea | Streptococcus bovis |
| Cryptococcus neoformans | Neisseria elongata subsp. elongata | Streptococcus dysgalactiae |
| Cytomegalovirus** | Neisseria elongata subsp. nitroreducens | Streptococcus equinis |
| Deinococcus radiodurans | Neisseria flava | Streptococcus mitis |
| Derxia gummosa | Neisseria flavescens | Streptococcus mutans |
| Eikenella corrodens | Neisseria kochi | Streptococcus pneumoniae |
| Enterobacter aerogenes | Neisseria lactamica | Streptococcus pyogenes |
| Enterobacter cloacae | Neisseria macacae | Streptococcus salivarius |
| Enterococcus avium | Neisseria meningitidis Serogroup A | Streptococcus sanguis |
| Enterococcus casseliflavus | Neisseria meningitidis Serogroup B | Streptomyces griseinus |
| Enterococcus faecalis | Neisseria meningitidis Serogroup C | Trichomonas vaginalis |
| Enterococcus faecium | Neisseria meningitidis Serogroup D | Trueperella pyogenes |
| Erysipelothrix rhusiopathiae | Neisseria meningitidis Serogroup W135 | Ureaplasma urealyticum |
| Escherichia coli | Neisseria meningitidis Serogroup Y | Veillonela parvula |
| Escherichia fergusonii | Neisseria mucosa | Vibrio cholerae |
| Flavobacterium meningosepticum | Neisseria perflava | Vibrio parahaemolyticus |
| Fusobacterium nucleatum | Neisseria polysaccharea | Yersinia enterocolitica |
| Gardnerella vaginalis | Neisseria sicca | - |

* HPV16 was tested as CaSki cells.

** Organism was tested at a concentration of 1 x \(10^{4}\) Units/mL.

***Organism was tested at a concentration of 1 x \(10^{5}\) CFU/mL.

#### f. Assay cut-off:

An assay cut-off of Ct value = 50, where 50 is the last PCR profile cycle, was established for the cobas CT/NG for use on the cobas 6800/8800 Systems. Negative results occur when amplification does not occur or fails to result in adequate signal, and no Ct value is reported. Acceptability of the cut-off was verified through analysis of CT and NG cycle threshold (Ct) value distribution for all specimens from the US clinical study and selected

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analytical studies, as evidenced by a clear distinction between positive results and negative results/the end of the PCR profile. Analytical studies were included in this assessment to enrich the dataset with results from samples with a low target concentration. Stability and interference studies data were not included in this analysis, since those studies are designed to stress the system thereby potentially producing invalid results.

Assessed for these studies as described, the difference between the latest Ct value in positive specimens and assay cut-off was 5.5 cycles for CT and 3.5 cycles for NG. For specimens from the clinical study only, broken out to assess performance in a typical patient population, the difference between the latest Ct value in positive specimens and assay cut-off was 7.8 cycles for CT and 3.5 cycles for NG (shown below). The following graphs illustrate the distribution of Ct values from the clinical study.

Ct Distributions - CT positive results from US Clinical Trial


Ct Distributions - NG positive results from US Clinical Trial


### g. Contamination:

Sample-to-sample and run-to-run cross-contamination studies were performed to evaluate potential cross contamination on the cobas 6800/8800 Systems using the cobas CT/NG assay. Contrived sample backgrounds consisting of clean media with HCT-15 cells were used to represent swab samples collected in cobas PCR media (endocervical and vaginal swabs) and cervical specimens collected in PreservCyt Solution. Pooled CT/NG negative urine specimens were included in the study. Positive samples were prepared by co-spiking a portion of the negative samples with CT and NG cultures to a concentration that generated a Ct value ~20 cycles representing the Ct value lower than that observed in 95% of positive results in the intended use population. Three runs consisting of a checkerboard pattern of positive CT and NG samples alternating with negative samples were performed, followed by a full run of CT/NG negative samples to assess run-to-run contamination. Two CT/NG negative urine samples out of 144 total negative samples tested positive for both CT and NG within one of the checkerboard runs (2/144, 1.4%). The total sample-to-sample cross-contamination rate of cobas CT/NG for all sample types for the three checkerboard runs was determined to be 0.5% (2/432). Run-to-run cross-contamination was not observed (0/282).

### h. Competitive Inhibition Studies:

To assess competitive inhibition between CT and NG, contrived specimens were tested with low and moderate concentrations of one target mixed with very high concentrations

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of the opposite target. Low and moderate concentrations were defined as ~1x LoD and ~3x LoD, respectively, and high concentrations were defined as generating a Ct value that was lower than the value observed in 95% of CT and NG positive specimens. Positive test samples were prepared by spiking CT serovar D and NG strain 2948 cultures into negative background matrix prepared for each specimen type by pooling pre-screened specimens negative for both CT and NG. Specimen types evaluated were: 1. vaginal swab collected in cobas PCR media (intended to represent both endocervical and vaginal swab samples), 2. urine mixed with cobas PCR media, and 3. cervical specimens collected in PreservCyt Solution. Five replicates of each panel member were tested. Additional samples were prepared for the vaginal swab matrix containing CT at a high concentration and NG at 0.4 CFU/ml, 0.65 CFU/ml, and 1.95 CFU/ml. These concentrations of NG represent LoD as determined by the observed 95% hit rate, the upper limit for the Probit estimate of the LoD, and 3X of the upper limit for the Probit estimate of the LoD, respectively.

Results indicate that CT was detected at or above the LoD in all specimen types even when NG was present at a high concentration. When CT was present at high concentrations, NG was detected in 100% of specimens (all types) at moderate levels (~3X LoD), and for cervical and urine specimens NG was also detected in 100% of specimens at low levels (~1X LoD). For vaginal swab specimens, when CT was present at high concentrations, NG was detected in 35% (7 of 20) of replicates at 0.4 CFU/ml, 60% (12 of 20) of replicates at 0.65 CFU/ml, and 100% (20 of 20) of replicates at 1.95 CFU/ml.

A limitation is included in the package insert describing the identified competitive inhibition.

# i. Interferences:

# Testing of Exogenous Substances:

Performance of the cobas CT/NG assay for use on the cobas 6800/8800 Systems was evaluated in the presence of potentially interfering exogenous substances including over-the counter products and prescription drugs that may be present in urogenital specimens. Specimen types evaluated were: 1. vaginal swabs collected in cobas PCR media (intended to represent both endocervical and vaginal swab samples), 2. urine mixed with cobas PCR media, and 3. cervical specimens collected in PreservCyt Solution. Testing was performed using negative background matrix prepared for each specimen type by pooling pre-screened specimens negative for both CT and NG. The negative matrices were spiked with potential interferents at levels expected from normal patient usage. Interferents were tested in CT/NG negative specimen pools as well as in specimen pools co-spiked with serovars D or I of Chlamydia trachomatis and strains 2948 or 891 of Neisseria gonorrhoeae at ~3x LoD.

Five replicates of each sample for each specimen type were tested with the potential interferents, except for RepHresh Odor Eliminating Vaginal Gel And RepHresh Clean Balance Gel for which two replicates each were tested to verify interference observed with Replens, a product with similar formulation. In addition, one sample for each specimen type was tested without the addition of potentially interfering products.

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Results of the study demonstrate that 14 of the 18 exogenous substances tested did not interfere with the performance of the assay for detection of CT and NG. Metronidazole Vaginal Gel, RepHresh Odor Eliminating Vaginal Gel, RepHresh Clean Balance Gel and Replens Vaginal Moisturizer showed interference leading to false negative and invalid results in urogenital specimens at levels that may be present in patient specimens. Limitations are included in the package insert describing assay interference.

Table 18. Substance Concentrations That Do Not Interfere with Test Performance in Urogenital Specimens

| Product Name | Vaginal Swabs | Urine | PreservCyt Solution |
| --- | --- | --- | --- |
| | mg/mL | mg/mL | mg/mL |
| Clindamycin Phosphate Vaginal Cream | 7.1 | 3.4 | 1.6 |
| Equate tioconazole 1 | 3.7 | 1.7 | 0.8 |
| Equate Vagicaine Anti-Itch Cream | 4.1 | 2.0 | 0.9 |
| Estrace | 3.8 | 2.0 | 1.0 |
| K-Y Ultra Gel | 5.7 | 2.7 | 1.2 |
| Metronidazole Vaginal Gel | 0.1* | 0.1* | 0.2* |
| Monistat 3 Vaginal Antifungal Combination Pack | 3.7 | 1.7 | 0.7 |
| Monistat Complete Care Itch Relief Cream | 3.7 | 1.8 | 0.9 |
| 7 Day Vaginal Cream | 3.9 | 1.8 | 0.8 |
| Norforms Suppositories | 3.4 | 1.7 | 0.7 |
| Premarin | 6.1 | 3.1 | 1.4 |
| Replens Long-Lasting Vaginal Moisturizer | 0.05* | 0.05* | 0.2* |
| Summer's Eve Feminine Deodorant Spray | 6.4 | 3.1 | 2.0 |
| VCF - Vaginal Contraceptive Foam | 2.1 | 1.0 | 0.4 |
| Yeast Gard Advanced | 3.7 | 1.7 | 1.0 |
| Azo Standard (urine only) | N/A | 0.1 | N/A |
| RepHresh Odor Eliminating Vaginal Gel | ** | ** | ** |
| RepHresh Clean Balance Gel | ** | ** | ** |

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

** Concentrations of product that did not interfere with test performance could not be determined.

### Testing of Endogenous Substances:

Performance of cobas CT/NG was evaluated in the presence of potentially-interfering endogenous substances including whole blood, peripheral blood mononuclear cells (PBMC), and mucus. Sample types included in the study were: 1. endocervical swabs collected in cobas PR media (intended to represent both endocervical and vaginal swab samples), 2. urine mixed with cobas PCR media, and 3. cervical specimens collected in PreservCyt Solution. Interferents were tested in CT/NG negative specimen pools as well as in specimen pools spiked with serovars D or I of Chlamydia trachomatis and strains 2948 or 891 of Neisseria gonorrhoeae at ~ 3x LoD. The endogenous substances were introduced to the negative and positive samples at levels that may be present in typical clinical specimens. Five replicates of each sample for each specimen type were tested with each potentially interfering substance, and one sample for each specimen type was tested without the addition of the potentially interfering substance.

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Mucus at a concentration of approximately 1% (w/v) caused false negative results for NG in endocervical samples. Whole blood caused interference at 10% (v/v) in CT/NG negative and positive urine samples and in CT/NG positive PreservCyt samples.

Limitations are included in the package insert describing endogenous substances that demonstrated interference with cobas CT/NG. A summary of endogenous substances concentrations that do not interfere with cobas CT/NG assay performance is shown in Table 19.

Table 19. Endogenous Substances Concentrations That Do Not Interfere with cobas CT/NG for Use on the 6800/8800 Systems

| Interferent | Endocervical Swab | PreservCyt | Urine |
| --- | --- | --- | --- |
| Albumin (% w/v) | NT | NT | 5% |
| Bilirubin (% w/v) | NT | NT | 0.5% |
| Mucus (% w/v) | 0.5% | 1.0% | 0.5% |
| Glucose (% w/v) | NT | NT | 1.0% |
| Peripheral Blood Mononuclear Cells (PBMCs as | 1.0E+06 | 1.0E+06 | 1.0E+06 |
| pH (acidic and alkaline) | NT | NT | pH 4 and pH 9 |
| Semen (% w/v) | 1.5% | 1.5% | NA |
| Whole Blood (% v/v) | 10% | 5% | 5% |

NT = Not Tested

# 2. Comparison studies:

a. Method comparison with predicate device:

Not applicable

b. Matrix comparison:

Not applicable

# 3. Clinical studies:

The clinical performance of cobas CT/NG was established in a multi-site, prospective collection study by comparing the results to a Patient Infected Status (PIS) that used a combination of FDA-cleared NAATs for urogenital specimens in each patient. Female and male urogenital specimens were collected at nine geographically diverse sites in the US, which included family planning and obstetrics/gynecology (OB/GYN) clinics and sexually transmitted disease clinics. The testing was performed at four laboratory testing sites (three external and one internal).

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Prospectively enrolled female subjects provided the following urogenital specimens: first-void urine, three vaginal swabs, one endocervical swab in cobas PCR Media, and one cervical sample in PreservCyt Solution. If the female was in the clinician-collected vaginal swab arm of the study, two of the vaginal swabs were placed in the respective manufacturers' collection device and one in cobas PCR Media. If the female subject was in the self-collected vaginal swab arm of the study, then one vaginal swab was self-collected first and placed into cobas PCR Media and then followed by the two clinician-collected vaginal swabs placed in the two respective manufacturers' collection devices.

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

Subjects were classified as symptomatic if they self-reported symptoms indicative of a CT or NG infection, as listed below. Subjects were classified as asymptomatic if they did not report any of these symptoms.

- Dysuria (pain during urination)
Coital pain, difficulty or bleeding
Pelvic pain
Abnormal vaginal discharge
Pelvic, uterine or ovarian pain
- Urethral discharge
Testicular pain
Scrotal pain or swelling

Specimens were tested for CT and NG using cobas CT/NG and FDA-cleared commercially available NAATs. All tests were run according to the respective manufacturers' Instructions For Use.

The clinical performance of cobas CT/NG was evaluated by comparing the results from collected specimen types to a pre-specified PIS (Patient Infected Status) algorithm as determined by the combined results from two FDA-cleared NAATs for females and three FDA-cleared NAATs for males. The PIS algorithms for Female and Male subjects are shown in the Tables 20 and 21.

Archived prospectively collected female urine, cervical specimens in PreservCyt, and endocervical swabs were obtained for the clinical study for cobas CT/NG v2 test on the cobas 4800 System. The PIS of these specimens were already determined during the clinical study for cobas CT/NG v2 test on the cobas 4800 System. NG PIS infected female subjects who had specimens with adequate volume for NG testing with cobas CT/NG assay were included in the current study. In addition, a random subset of specimens from NG PIS non-infected females was also included. Stability of archived specimens was evaluated. Some archived specimens were not tested due to volume limitation, therefore, in addition, Ct value distributions from the original study for archived tested specimens and specimens not tested due to volume limitations were reviewed. This analysis supported that the use of the archived specimens will provide an unbiased estimation of

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the test performance

**Table 20. Determination of Female Patient Infected Status (PIS) for Urogenital Specimens$^{a}$**

| NAAT1 Urine/Vaginal | NAAT2 Urine/Vaginal | Patient Infected Status (PIS)^{b} |
| --- | --- | --- |
| +/+ | +/+ | Infected |
| +/+ | +/- or -/+ | Infected |
| +/- or -/+ | +/+ | Infected |
| +/- | -/+ | Infected |
| -/+ | +/- or -/+ | Infected |
| +/- | +/- | Infected (Urine) Non-Infected (Vaginal) |
| +/- or -/+ | -/- | Not Infected |
| +/+ | -/- | Not Infected |
| -/- | +/+ | Not Infected |
| -/- | +/- or -/+ | Not Infected |
| -/- | -/- | Not Infected |

$^{a}$ One or more positives in each NAAT (NAAT1 and NAAT2) designates the PIS as Infected. Any other combination of results defines the PIS as Not Infected.

$^{b}$ In the scenario where one or more of the sample types are invalid, the remaining sample types with valid results from NAAT1 and NAAT2 must have concordant positive or concordant negative results to determine the PIS as Infected or Not Infected, respectively. For all other cases where one or more of the sample types are invalid, the PIS is indeterminate.

**Table 21. Determination of Male Patient Infected Status (PIS) for Urine Specimens**

| NAAT1 Urine | NAAT2 Urine/ | NAAT3 Urine | Patient Infected Status (PIS)^{a} |
| --- | --- | --- | --- |
| + | + | + | Infected |
| + | + | - | Infected |
| + | - | + | Infected |
| - | + | + | Infected |
| - | - | + | Not Infected |
| - | + | - | Not Infected |
| + | - | - | Not Infected |
| - | - | - | Not Infected |

$^{a}$ If at least 2 out of the 3 test results are concordant positive or negative then the PIS can be considered as Infected or Not Infected, respectively. If one test result is invalid/missing and t…