BD PROBETEC CHLAMYDIA TRACHOMATIS (CT) Q AMPLIFIED DNA ASSAY

K081824 · Becton, Dickinson & CO · MKZ · Dec 11, 2008 · Microbiology

Device Facts

• Record ID: K081824
• Device Name: BD PROBETEC CHLAMYDIA TRACHOMATIS (CT) Q AMPLIFIED DNA ASSAY
• Applicant: Becton, Dickinson & CO
• Product Code: MKZ · Microbiology
• Decision Date: Dec 11, 2008
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3120
• Device Class: Class 1

Indications for Use

The BD ProbeTecCT Q® Amplified DNA Assay, when tested with the BD Viper™ System in Extracted Mode, uses Strand Displacement Amplification technology for the direct, qualitative detection of Chlamydia trachomatis DNA in clinician-collected female endocervical and male urethral swab specimens, patient-collected vaginal swab specimens (in a clinical setting), and male and female urine specimens. The assay is indicated for use with asymptomatic and symptomatic individuals to aid in the diagnosis of chlamydial urogenital disease.

Device Story

The BD ProbeTec CT Q* Amplified DNA Assay is an in vitro diagnostic test for Chlamydia trachomatis DNA. It utilizes the BD Viper System for automated sample processing. Input specimens (swabs or urine) undergo chemical lysis and DNA binding to para-magnetic particles, followed by washing and elution. The system performs Strand Displacement Amplification (SDA) using amplification primers, a DNA polymerase, a restriction endonuclease, and a fluorescently-labeled detector probe. An Extraction Control (EC) is included to validate the extraction process. The system monitors fluorescence in real-time; an automated algorithm calculates peak fluorescence (MaxRFU) and compares it to a threshold to determine results (positive, negative, or EC failure). The device is used in clinical laboratory settings by trained personnel. Results assist clinicians in diagnosing chlamydial urogenital infections, enabling timely patient treatment and management.

Clinical Evidence

Clinical study evaluated 994 female and 472 male subjects across seven sites. Performance compared to a Patient Infected Status (PIS) algorithm using two reference NAATs. Total of 5388 results analyzed. Overall sensitivity 94.5% (95% CI: 92.6%-96.0%) and specificity 98.9% (95% CI: 98.6%-99.2%). Subgroup analysis provided for symptomatic/asymptomatic status and specimen types (endocervical, vaginal, urethral, urine). Reproducibility studies conducted at three sites using simulated specimens showed high concordance.

Technological Characteristics

Uses Strand Displacement Amplification (SDA) technology. Reagents provided in disposable microwells (priming and amplification). Detection via fluorescently-labeled oligonucleotide probes. Automated processing on the BD Viper System. Analyzes peak fluorescence (MaxRFU) against a threshold. Includes an internal Extraction Control (EC) for process validation.

Indications for Use

Indicated for symptomatic and asymptomatic male and female patients to aid in the diagnosis of chlamydial urogenital disease using endocervical, urethral, vaginal, or urine specimens.

Regulatory Classification

Identification

Chlamydia serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to chlamydia in serum. Additionally, some of these reagents consist of chlamydia antisera conjugated with a fluorescent dye used to identify chlamydia directly from clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria belonging to the genus Chlamydia and provides epidemiological information on these diseases. Chlamydia are the causative agents of psittacosis (a form of pneumonia), lymphogranuloma venereum (a venereal disease), and trachoma (a chronic disease of the eye and eyelid).

Predicate Devices

• BD ProbeTecET CT/GC Amplified DNA Assay (K984631)
• APTIMA Combo 2 Assay (K003395)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number: K081824

B. Purpose for Submission: To add the option for automated extraction of DNA from clinical specimens using the software accessory “BD ProbeTec™ Chlamydia trachomatis (CT) Q® Amplified DNA Assay” to direct the BD Viper™ System.

C. Measurand: Chlamydia trachomatis DNA

D. Type of Test: Strand displacement nucleic acid amplification assay

E. Applicant: BD Diagnostic System

F. Proprietary and Established Names: BD ProbeTec™ Chlamydia trachomatis (CT) Q® Amplified DNA Assay

G. Regulatory Information:

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| MKZ | Class I | 21CFR 866.3120 Chlamydia serological reagents | Microbiology (83) |

H. Intended Use:

1.) Intended use: The BD ProbeTecCT Q® Amplified DNA Assay, when tested with the BD Viper™ System in Extracted Mode, uses Strand Displacement Amplification technology for the direct, qualitative detection of Chlamydia trachomatis DNA in clinician-collected female endocervical and male urethral swab specimens, patient-collected vaginal swab specimens (in a clinical setting), and male and female urine specimens. The assay is indicated for use with asymptomatic and symptomatic individuals to aid in the diagnosis of chlamydial urogenital disease.

2.) Indications for use: Same as intended use

3) Special conditions for use statement(s): NA

4) Special instrument requirements: BD Viper™ System

I. Device Description: The BD ProbeTecCT Q® Amplified DNA Assay is based on the simultaneous amplification and detection of target DNA using amplification primers and a fluorescently-labeled detector probe (8, 9). The reagents for SDA are dried in two separate disposable microwells: the Priming Microwell contains the amplification primers, fluorescently-labeled detector probe, nucleotides and other reagents necessary

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for amplification, while the Amplification Microwell contains the two enzymes (a DNA polymerase and a restriction endonuclease) that are required for SDA. The BD Viper System pipettes a portion of the purified DNA solution from each Extraction Tube into a Priming Microwell to rehydrate the contents. After a brief incubation, the reaction mixture is transferred to a corresponding, pre-warmed Amplification Microwell which is sealed to prevent contamination and then incubated in one of the two thermally-controlled fluorescent readers. The presence or absence of C. trachomatis DNA is determined by calculating the peak fluorescence (Maximum Relative Fluorescent Units (MaxRFU)) over the course of the amplification process and by comparing this measurement to a predetermined threshold value.

In addition to the fluorescent probe used to detect amplified C. trachomatis target DNA, a second fluorescently-labeled oligonucleotide is incorporated in each reaction. The Extraction Control (EC) oligonucleotide is labeled with a different dye than that used for detection of the C. trachomatis-specific target and is used to confirm the validity of the extraction process. The EC is dried in the Extraction Tubes and is re-hydrated upon addition of the specimen and extraction reagents. At the end of the extraction process, the EC fluorescence is monitored by the BD Viper instrument and an automated algorithm is applied to both the EC and C. trachomatis-specific signals to report specimen results as positive, negative, or EC failure.

## J. Substantial Equivalence Information:

a) Predicate device name (s):

BD ProbeTecET CT/GC Amplified DNA Assay

APTIMA Combo 2 Assay

b) Predicate Numbers (s): K984631, K003395

Comparison with predicate:

Comparison of Operating and Technological Characteristics: Assay

| BD ProbeTecCTQ Assay | BD ProbeTecCTQ Assay | BD ProbeTecET CT/GC Assay (K984361) |
| --- | --- | --- |
| Amplification Technology | Same as BD ProbeTecET CT/GC (K984631) | Strand Displacement Amplification |
| Priming Microwell | | |
| • Primers | Alternate region of cryptic CT plasmid | Region of cryptic CT plasmid |
| • Detector | Linear Detector
• Flourescein (fluorophore)
• Dabcyl (quencher) | Hairpin Detector
• Flourescein (fluorophore)
• Rhodamine (quencher) |

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| • Nucleotides | 4 of 4 nucleotides required for SDA | 1 of 4 nucleotides required for SDA |
| --- | --- | --- |
| • Non-specific reagents and cofactors | Same as BD ProbeTecET CT/GC (K984631) | Buffering components, magnesium ions, salt and stabilizing reagents |
| Amplification Microwell | | |
| • Restriction Enzyme | Same as BD ProbeTecET CT/GC (K984631) | BsoBI restriction enzyme |
| • Polymerase | Same as BD ProbeTecET CT/GC (K984631) | Bst DNA polymerase |
| • Nucleotides | 0 of 4 nucleotides required for SDA | 3 of 4 nucleotides required for SDA |
| • Non-specific reagents and cofactors | Same as BD ProbeTecET CT/GC (K984631) | Buffering components, magnesium ions, salt and stabilizing reagents |
| Assay Buffer | Bicine-potassium hydroxide-based | Potassium phosphate-based |

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

1. Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices, May 11, 2005. http://www.fda.gov/cdrh/ode/guidance/337.pdf
2. CLSI EP5-A2 “Evaluation of Precision Performance of Quantitative Measurement Methods”,
3. CLSI EP12-A “User Protocol for Evaluation of Qualitative Test Performance”.

L. Test Principle:

See Device Description

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

Reproducibility of the BD Viper System using the BD ProbeTecCT Q⁸ Assay was evaluated at three clinical sites on one BD Viper System per site. A panel of simulated specimens was tested that comprised CT and GC organisms seeded into swab diluent for the BD ProbeTecCT Q⁸ Assay. Simulated endocervical and urethral specimens contained a clean endocervical swab whereas the simulated urine and vaginal swab specimens did not. Un-inoculated swab diluent for the BD ProbeTecCT Q⁸ Assay was used for the CT negative samples. Nine replicates of each panel member were tested every day for five days on each BD Viper System. The data are summarized in the following table.

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Summary of Reproducibility Data on the BD Viper System for the CT Q⁺ Assay.

| | | | | | | Within Run | | Between Runs Within Site | | Between Site | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Specimen Type | CT EB's/mL | GC Cells/mL | % Correct | 95% CI | MaxRF UMean | SD | %CV | SD | %CV | SD | %CV |
| Endocervical / Urethral | 0 | 0 | 98.5% (133/135) | (94.8-99.8%) | 29.9 | 233.0 | 778.5 | 0.0 | 0.0 | 33.9 | 113.4 |
| | 30 | 0 | 100.0% (135/135) | (97.3-100.0%) | 2011.2 | 114.1 | 5.7 | 0.0 | 0.0 | 14.8 | 0.7 |
| | 0 | 100 | 100.0% (135/135) | (97.3-100.0%) | 1.4 | 6.0 | 442.7 | 1.0 | 76.9 | 0.0 | 0.0 |
| | 30 | 250 | 100.0% (135/135) | (97.3-100.0%) | 1991.9 | 118.0 | 5.9 | 17.6 | 0.9 | 10.4 | 0.5 |
| | 75 | 100 | 100.0% (135/135) | (97.3-100.0%) | 1954.8 | 169.4 | 8.7 | 0.0 | 0.0 | 0.0 | 0.0 |
| Urine/Vaginal | 0 | 0 | 100.0% (135/135) | (97.3-100.0%) | 0.9 | 5.0 | 542.4 | 0.0 | 0.0 | 0.0 | 0.0 |
| | 30 | 0 | 100.0% (135/135) | (97.3-100.0%) | 1999.8 | 131.8 | 6.6 | 34.2 | 1.7 | 0.0 | 0.0 |
| | 0 | 100 | 100.0% (135/135) | (97.3-100.0%) | 0.8 | 3.4 | 442.4 | 0.0 | 0.0 | 0.0 | 0.0 |
| | 30 | 250 | 100.0% (135/135) | (97.3-100.0%) | 1995.2 | 125.8 | 6.3 | 33.1 | 1.7 | 52.9 | 2.7 |
| | 75 | 100 | 100.0% (135/135) | (97.3-100.0%) | 2014.4 | 109.5 | 5.4 | 0.0 | 0.0 | 0.0 | 0.0 |

A second study was conducted internally to characterize the reproducibility of test results (i.e., proportion positive or negative) at target levels below the analytical Limit of Detection (LOD) of the BD ProbeTecCT Q⁺ Assay. A panel of simulated specimens was tested that comprised CT and GC organisms seeded into Q⁺ swab diluent at two different levels each of which was below the respective analytical LOD for the organisms (1:10, 1:100). These levels were selected to fall within the dynamic range of the analytical LOD curve of the assay. Fifteen replicates of each panel member were tested every day for five days across three BD Viper Systems. The data are summarized in the following table.

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Characterization of System Reproducibility at Target Levels below the Analytical Limit of Detection for the CT Q⁺ Assay.

| Specimen Type | Dilution of Analytical LOD | % Positive | 95% CI (Positive) | Max RFU Mean (Positive) | % Negative | 95% CI (Negative) | Max RFU Mean (Negative) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Endocervical/ Urethral | 1:10 | 70.2 (158/225) | (63.8, 76.1) | 1794.2 | 29.8 (67/225) | (23.9, 36.2) | 2.6 |
| Endocervical/ Urethral | 1:100 | 10.2 (23/225) | (6.6,14.9) | 1643.8 | 89.8 (202/225) | (85.1, 93.4) | 1.6 |
| Urine/Vaginal | 1:10 | 64.4 (145/225) | (57.8, 70.7) | 1733.9 | 35.6 (80/225) | (29.3, 42.2) | 4.6 |
| Urine/Vaginal | 1:100 | 10.7 (24/225) | (7.0, 15.5) | 1666.6 | 89.3 (201/225) | (84.5, 93.0) | 2.4 |

b. Linearity/assay reportable range: NA

c. Traceability, Stability, Expected values (controls, calibrators, or methods): The recommended Positive, Negative and Internal Control material were tested a sufficient number of times with acceptable results on all testing days.

d. Detection limit: The Limits of Detection (LODs) for the CT Q⁺ Assay with C. trachomatis serovar H in urine and swab specimens when extracted on the BD Viper System were determined to be < 15 CT elementary bodies (EB) per mL for neat and Q⁺ UPT urine and < 30 CT EB per mL for expressed vaginal and endocervical swab specimens. A correlation of EB to Inclusion-forming units (IFU) suggests that the CT Q⁺ assay LODs with serovar H in urine and swab specimens correspond to < 1 IFU per mL (15). The CT Q⁺ Assay on the BD Viper System in extracted mode was able to detect 16 isolates representing 15 CT serovars (A, B, Ba, C, D, E (2)*, F, G, H, I, J, K, LGV1, LGV2, and LGV3) with ≥ 95% proportion positive at a concentration of 15 EB per mL in CT/GC Q⁺ Swab Diluent.

* Testing with CT serovar E included the nvCT strain, a new variant with a deletion in the cryptic plasmid. (21)

e. Analytical specificity:

1. Cross Reactivity: DNA from 141 organisms listed in the following table was extracted on the BD Viper System and tested with the BD ProbeTec CT Q⁺ Amplified DNA Assay. All potential cross-reactive species were tested at >1×10⁸ cells/mL except where noted. The CT Q⁺ Assay did not cross-react with any of the organisms tested.

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Potential Cross-reacting Microorganisms

| Acinetobacter calcoaceticus | Epstein Barr Virus *** | Peptostreptococcus productus | Neisseria elongata subsp. nitroreduscens (2) |
| --- | --- | --- | --- |
| Acinetobacter lwoffii | Escherichia coli | Plesiomonas shigelloides | Neisseria elongata |
| Actinomyces israelii | Flavobacterium meningosepticum | Propionibacterium acnes | Neisseria flava (4) |
| Adenovirus*** | Gardnerella vaginalis | Providencia stuartii | Neisseria flavescens (4) |
| Aeromonas hydrophilia | Gemella haemolysans | Pseudomonas aeruginosa | Neisseria gonorrhoeae |
| Alcaligenes faecalis* | Haemophilus influenzae | Salmonella minnesota | Neisseria lactamica (7) |
| Bacillus subtilis* | Herpes Simplex Virus ** | Salmonella typhimurium | Neisseria meningitidis (12) |
| Bacteroides fragilis | Human papillomavirus (16 and 18)*** | Staphylococcus aureus | Neisseria mucosa (5) |
| Candida albicans* | Kingella kingae | Staphylococcus epidermidis | Neisseria perflava (8) |
| Candida glabrata* | Klebsiella pneumoniae | Streptococcus agalactiae | Neisseria polysaccharea (2) |
| Candida tropicalis* | Lactobacillus acidophilus* | Streptococcus mitis | Neisseria sicca (5) |
| Chlamydia pneumoniae*** | Lactobacillus brevis | Streptococcus mutans | Neisseria subflava (15) |
| Chlamydia psittaci* | Lactobacillus jensenii* | Streptococcus pneumoniae* | Neisseria weaverii (3) |
| Citrobacter freundii | Listeria monocytogenes | Streptococcus pyogenes | |
| Clostridium perfringens | Mobiluncus mulieris | Streptomyces griseus** | |
| Corynebacterium renale | Moraxella lacunata* | Trichomonas vaginalis** | |
| Cryptococcus neoformans* | Moraxella osloensis | Veillonella parvula | |
| | | | |

Cytomegalovirus** Morganella morganii Vibrio

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| | | parahaemolyticus |
| --- | --- | --- |
| Edwardsiella tarda | Mycobacterium gordonae | Yersinia enterocolitica |
| Enterobacter cloacae | Mycobacterium smegmatis | Branhamella catarrhalis (5) |
| Enterococcus faecalis | Peptostreptococcus anaerobius | Neisseria cinerea (2) |
| Enterococcus faecium | Peptostreptococcus asaccharolyticus | Neisseria elongata subsp. glycolytica |

(n) number of strains tested in the BD ProbeTecCT Qx Assay

* Tested at > 1×10⁷ cells or EB per mL; **Tested at > 1×10⁶ cells or viral particles per mL; ***Tested at ≥ 1×10⁶ genomic equivalents per mL; *** tested at ≥ 1×10⁵ TCID₅₀/mL

2. Interference: The performance of the BD ProbeTecCT Qx Assay on the BD Viper System in extracted mode was evaluated in the presence of potential interfering substances which may be encountered in swab and/or urine specimens. Potential interfering substances were spiked into Qx UPT urine and vaginal swab specimen matrices in both the presence and the absence of CT elementary bodies (30 CT EB/mL in urine matrix and 90 CT EB/mL in swab matrix). Results are summarized in the following table.

GC Qx Interfering Substances

| Interpretation | Swab | Urine |
| --- | --- | --- |
| No Interference Observed | Blood (≤60%)
Seminal Fluid
Mucus
Over The Counter vaginal products and contraceptives
Hemorrhoidal cream
Prescription vaginal treatments
Leukocytes (1×10⁶ cells/mL)
1×10⁶ EB/mL C. trachomatis | Blood (≤1%)
Seminal fluid
Mucus
Antibiotics
Analgesics
Over The Counter deodorant sprays and powders
Hormones
Leukocytes
Albumin <1 mg/mL
Glucose
Acidic urine (pH 4.0)
Alkaline urine (pH 9.0)
Bilirubin
1×10⁶ EB/mL C. trachomatis
Organisms associated with Urinary Tract Infections |
| May cause extraction control (EC) failures | Blood (>60%) | Not applicable |

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f. Assay cut-off: NA

g. Cross Contamination and Carryover

An internal study was conducted to evaluate the risk of producing a false positive result in either the same run on the BD Viper System in extracted mode (within run cross-contamination) or in a subsequent run (between run carryover). Testing was conducted using negative and positive samples on three BD Viper Systems. Negative samples consisted of CT/GC Q⁴ Swab Diluent. Positive samples consisted of a representative analyte (at 10⁵ CT EB/mL) spiked into CT/GC Q⁴ Swab Diluent. The overall rate of cross-contamination (i.e., with alternating columns of positive and negative samples and a prevalence of 50%) was 0.41% (9/2208). The overall rate of carryover contamination (i.e., carryover between successive runs when the prevalence was 50% in the previous run) was 0.36% (8/2208). Cross-contamination and carryover rates across the three BD Viper Systems are summarized in table.

Cross Contamination and Carryover Contamination

| Assay Dispense Mode Selected | BD Viper System | Cross-Contamination | | | Carryover Contamination | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | n | Positive Results | Percent Positive | n | Positive Results | Percent Positive |
| Dual Assay | 1 | 736 | 5 | 0.68 | 736 | 1 | 0.14 |
| | 2 | 736 | 0 | 0.00 | 736 | 3 | 0.41 |
| | 3 | 736 | 4 | 0.54 | 736 | 4 | 0.54 |
| | Overall | 2208 | 9 | 0.41 | 2208 | 8 | 0.36 |
| Single Assay | 1 | 190 | 0 | 0.00 | 186 | 0 | 0.00 |
| | 2 | 188 | 1 | 0.53 | 186 | 1 | 0.54 |
| | 3 | 188 | 0 | 0.00 | 186 | 0 | 0.00 |
| | Overall | 566 | 1 | 0.18 | 568 | 1 | 0.18 |

2. Comparison studies:

a. Method comparison with predicate method: See below under Performance Characteristics
b. Matrix comparison: NA

3. Clinical studies:

a. Clinical Sensitivity: NA
b. Clinical specificity: NA

Performance Characteristics

Clinician-collected endocervical and male urethral swab specimens, patient-collected vaginal swab specimens (in a clinical setting), and male and female Q⁴ UPT and neat

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urine specimens were collected from 1059 female subjects and 479 male subjects attending OB/GYN, sexually transmitted disease (STD) and family planning clinics at seven geographically diverse clinical sites in North America. Subjects were classified as symptomatic if they reported symptoms such as dysuria, urethral discharge, coital pain/difficulty/bleeding, testicular or scrotum pain/swelling, abnormal vaginal discharge, or pelvic/uterine/adnexal pain. Subjects were classified as asymptomatic if they did not report symptoms. Sixty five female subjects and 7 male subjects were excluded from the data analysis due to age requirement violations, antibiotic treatment in the last 21 days, opting to withdraw from the study after initially consenting, failure to obtain paired swab and urine specimens, urine quantity less than 20 mL, or transport and storage errors related to specimen collection. Therefore, the final data analysis included 994 compliant female subjects and 472 compliant male subjects.

Five specimens were collected from each of the 994 eligible female subjects. A urine specimen was collected and split into Q⁴ UPT, neat urine and the two reference urine specimen collection devices followed by a vaginal swab specimen and three randomized endocervical swab specimens. Up to four specimens were collected from each of the 472 eligible male subjects. Up to three randomized urethral swab specimens were collected followed by a urine specimen that was split into Q⁴ UPT, neat urine and the two reference urine specimen collection devices. BD ProbeTec CT Q⁸ assay results were generated from the Q⁴ UPT and neat urine specimens, the vaginal swab specimen, one endocervical swab specimen and one male urethral swab specimen. The remaining two endocervical swab specimens, up to two male urethral swab specimens, and the two reference urine specimens for each male and female subject were tested using two reference methods: the BD ProbeTec ET CT/AC assay and another commercially available NAAT (Nucleic Acid Amplification Test). Specimen testing was conducted either at the site of specimen collection or at a designated BD Viper testing site.

All performance calculations were based on the total number of BD ProbeTec CT Q⁸ assays results for endocervical, vaginal and male urethral swab specimens, and male and female Q⁴ UPT and neat urine specimens compared to a patient infected status (PIS) algorithm for each gender. In the algorithm, the designation of a subject as being infected with CT or not was based on endocervical swab and urine specimen results from the commercially available BD ProbeTec ET CT/AC assay and another commercially available NAAT. Subjects were considered infected with CT if two of the four endocervical swab and urine specimens (or two of the three or four urethral swab and urine specimens) tested positive in the BD ProbeTec ET CT/AC assay and the other reference NAAT (one specimen testing positive in each NAAT). Subjects were considered non-infected if less than two reference NAAT results were positive. A total of 5388 BD ProbeTec CT Q⁸ assay results was used to calculate sensitivity and specificity. Sensitivity and specificity by specimen type and symptomatic status are presented in the table below.

Performance of the assay with endocervical swabs, patient collected vaginal swab specimens (in a clinical setting), female UPT and neat urine was assessed in the clinical study. Separate performance was calculated for specimens collected from pregnant females. Sensitivity compared to patient infected status for FS was 62.5% (5/8): the test and reference NAAT swab specimens were negative; the test and reference NAAT urine

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specimens were positive yielding a PIS positive result. FV sensitivity was 75% (6/8): the test and reference NAAT swab specimens were negative; the test and reference NAAT urine specimens were positive yielding a PIS positive result. FNU and FUPT sensitivity were 100% (8/8). Specificity was 94.7% (18/19) for FS, FV, FNU, and FUPT separately.

Tables below summarize the results from symptomatic and asymptomatic subjects designated as infected or non-infected with CT according to the PIS algorithm.

CT Q+ Assay Performance Compared to Patient Infected Status (by specimen type and symptomatic status)

| Specimen Type | Symptomatic | N | Sensitivity | 95% C.I. | Specificity | 95% C.I. | PPV% | NPV% | Error Initial/Final |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| FS | N | 450 | 93.0% (53/57) | (83.0% - 98.1%) | 98.0% (385/393) | (96.0% - 99.1%) | 86.9 | 99.0 | 2/0 |
| | Y | 543 | 89.7% (52/58) | (78.8% - 96.1%) | 98.6% (478/485) | (97.0% - 99.4%) | 88.1 | 98.8 | 1/1 |
| | Total | 993 | 91.3% (105/115) | (84.6% - 95.8%) | 98.3% (863/878) | (97.2% - 99.0%) | 87.5 | 98.9 | 3/1 |
| FV | N | 449 | 98.2% (56/57) | (90.6% - 100.0%) | 99.5% (390/392) | (98.2% - 99.9%) | 96.6 | 99.7 | 0/0 |
| | Y | 544 | 94.8% (55/58) | (85.6% - 98.9%) | 99.0% (481/486) | (97.6% - 99.7%) | 91.7 | 99.4 | 0/0 |
| | Total | 993 | 96.5% (111/115) | (91.3% - 99.0%) | 99.2% (871/878) | (98.4% - 99.7%) | 94.1 | 99.5 | 0/0 |
| FN | N | 450 | 93.0% (53/57) | (83.0% - 98.1%) | 100.0% (393/393) | (99.1% - 100.0%) | 100.0 | 99.0 | 0/0 |
| | Y | 543 | 93.1% (54/58) | (83.3% - 98.1%) | 99.0% (480/485) | (97.6% - 99.7%) | 91.5 | 99.2 | 0/0 |
| | Total | 993 | 93.0% (107/115) | (86.8% - 96.9%) | 99.4% (873/878) | (98.7% - 99.8%) | 95.5 | 99.1 | 0/0 |
| FUPT | N | 450 | 94.7% (54/57) | (85.4% - 98.9%) | 99.5% (391/393) | (98.2% - 99.9%) | 96.4 | 99.2 | 0/0 |
| | Y | 543 | 91.4% (53/58) | (81.0% - 97.1%) | 99.0% (480/485) | (97.6% - 99.7%) | 91.4 | 99.0 | 0/0 |

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| Specimen Type | Symptomatic | N | Sensitivity | 95% C.I. | Specificity | 95% C.I. | PPV% | NPV% | Error Initial/Final |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Total | 993 | 93.0% (107/115) | (86.8% - 96.9%) | 99.2% (871/878) | (98.4% - 99.7%) | 93.9 | 99.1 | 0/0 |
| MS | N | 215 | 88.6% (31/35) | (73.3% - 96.8%) | 98.9% (178/180) | (96.0% - 99.9%) | 93.9 | 97.8 | 1/0 |
| | Y | 257 | 93.9% (62/66) | (85.2% - 98.3%) | 97.9% (187/191) | (94.7% - 99.4%) | 93.9 | 97.9 | 1/0 |
| | Total | 472 | 92.1% (93/101) | (85.0% - 96.5%) | 98.4% (365/371) | (96.5% - 99.4%) | 93.9 | 97.9 | 2/0 |
| MN | N | 215 | 100.0% (35/35) | (90.0% - 100.0%) | 98.9% (178/180) | (96.0% - 99.9%) | 94.6 | 100.0 | 0/0 |
| | Y | 257 | 97.0% (64/66) | (89.5% - 99.6%) | 99.5% (190/191) | (97.1% - 100.0%) | 98.5 | 99.0 | 0/0 |
| | Total | 472 | 98.0% (99/101) | (93.0% - 99.8%) | 99.2% (368/371) | (97.7% - 99.8%) | 97.1 | 99.5 | 0/0 |
| MUPT | N | 215 | 100.0% (35/35) | (90.0% - 100.0%) | 98.9% (178/180) | (96.0% - 99.9%) | 94.6 | 100.0 | 0/0 |
| | Y | 257 | 97.0% (64/66) | (89.5% - 99.6%) | 97.4% (186/191) | (94.0% - 99.1%) | 92.8 | 98.9 | 0/0 |
| | Total | 472 | 98.0% (99/101) | (93.0% - 99.8%) | 98.1% (364/371) | (96.2% - 99.2%) | 93.4 | 99.5 | 0/0 |
| Total | | 5388 | 94.5% (721/763) | (92.6% - 96.0%) | 98.9% (4575/4625) | (98.6% - 99.2%) | 93.5 | 99.1 | 5/1 |

Note: For female subjects, infections localized to the endocervix or urethra have been reported in the literature (16-20). Analyses were performed on the endocervical swab specimens and female UPT and neat urine specimens to further characterize the ten negative female endocervical swabs (105/115) and the eight negative female UPT and neat urine specimens (107/115).

- Of the 115 female subjects defined as positive by the PIS algorithm, ten had infections localized to the urethra as indicated by the urine reference result (i.e., BD ProbeTec ET CT/AC assay and other NAAT endocervical swab specimens were negative, BD ProbeTec ET CT/AC assay and other NAAT urine specimens were positive). The BD ProbeTec CT Q⁵ assay was negative for nine of the ten endocervical swab specimens from these subjects.

- Of the 115 female subjects defined as positive by the PIS algorithm, three had infections localized to the endocervix as indicated by the endocervical reference result (i.e., BD ProbeTec ET CT/AC assay and other NAAT urine specimens were negative, BD ProbeTec ET CT/AC assay and other NAAT endocervical swab

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specimens were positive.) The BD ProbeTec CT Q' assay was negative for UPT and neat urine for these three subjects.

CT Q* Assay Performance Compared to Patient Infected Status (by clinical site).

| Specimen Type | Clinical Site | Prevalence | n | Sensitivity | 95% C.I. | Specificity | 95% C.I. | # CT (+) and GC (+) | PPV% | NPV% |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| FS1 | 1 | 16.1% | 15 5 | 96.0% (24/25) | (79.6% - 99.9%) | 96.2% (125/130) | (91.3% - 98.7%) | 5 | 82.8% | 99.2% |
| | 2 | 11.0% | 15 5 | 88.2% (15/17) | (63.6% - 98.5%) | 98.6% (136/138) | (94.9% - 99.8%) | 6 | 88.2% | 98.6% |
| | 3 | 12.3% | 73 | 88.9% (8/9) | (51.8% - 99.7%) | 100.0% (64/64) | (94.4% - 100.0%) | 2 | 100.0 % | 98.5% |
| | 4 | 18.1% | 10 5 | 89.5% (17/19) | (66.9% - 98.7%) | 100.0% (86/86) | (95.8% - 100.0%) | 6 | 100.0 % | 97.7% |
| | 5 | 10.0% | 70 | 100.0% (7/7) | (59.0% - 100.0%) | 96.8% (61/63) | (89.0% - 99.6%) | 0 | 77.8% | 100.0 % |
| | 6 | 8.5% | 36 5 | 90.3% (28/31) | (74.2% - 98.0%) | 98.5% (329/334) | (96.5% - 99.5%) | 3 | 84.8% | 99.1% |
| | 7 | 10.0% | 70 | 85.7% (6/7) | (42.1% - 99.6%) | 98.4% (62/63) | (91.5% - 100.0%) | 0 | 85.7% | 98.4% |
| FV2 | 1 | 16.1% | 15 5 | 100.0% (25/25) | (86.3% - 100.0%) | 97.7% (127/130) | (93.4% - 99.5%) | 5 | 89.3% | 100.0 % |
| | 2 | 11.0% | 15 5 | 100.0% (17/17) | (80.5% - 100.0%) | 99.3% (137/138) | (96.0% - 100.0%) | 6 | 94.4% | 100.0 % |
| | 3 | 12.3% | 73 | 77.8% (7/9) | (40.0% - 97.2%) | 100.0% (64/64) | (94.4% - 100.0%) | 2 | 100.0 % | 97.0% |
| | 4 | 18.1% | 10 5 | 94.7% (18/19) | (74.0% - 99.9%) | 100.0% (86/86) | (95.8% - 100.0%) | 6 | 100.0 % | 98.9% |
| | 5 | 10.0% | 70 | 100.0% (7/7) | (59.0% - 100.0%) | 96.8% (61/63) | (89.0% - 99.6%) | 0 | 77.8% | 100.0 % |
| | 6 | 8.5% | 36 5 | 96.8% (30/31) | (83.3% - 99.9%) | 100.0% (334/334) | (98.9% - 100.0%) | 3 | 100.0 % | 99.7% |
| | 7 | 10.0% | 70 | 100.0% (7/7) | (59.0% - 100.0%) | 98.4% (62/63) | (91.5% - 100.0%) | 0 | 87.5% | 100.0 % |
| FNU3 | 1 | 16.1% | 15 5 | 92.0% (23/25) | (74.0% - 99.0%) | 97.7% (127/130) | (93.4% - 99.5%) | 5 | 88.5% | 98.4% |

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| Specimen Type | Clinical Site | Prevalence | n | Sensitivity | 95% C.I. | Specificity | 95% C.I. | # CT (+) and GC (+) | PPV% | NPV% |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 2 | 11.0% | 15
5 | 82.4%
(14/17) | (56.6% - 96.2%) | 100.0%
(138/138) | (97.4% - 100.0%) | 6 | 100.0 % | 97.9% |
| | 3 | 12.3% | 73 | 88.9% (8/9) | (51.8% - 99.7%) | 100.0%
(64/64) | (94.4% - 100.0%) | 2 | 100.0 % | 98.5% |
| | 4 | 18.3% | 10
4 | 100.0%
(19/19) | (82.4% - 100.0%) | 97.6%
(83/85) | (91.8% - 99.7%) | 6 | 90.5% | 100.0 % |
| | 5 | 10.0% | 70 | 100.0%
(7/7) | (59.0% - 100.0%) | 100.0%
(63/63) | (94.3% - 100.0%) | 0 | 100.0 % | 100.0 % |
| | 6 | 8.5% | 36
6 | 93.5%
(29/31) | (78.6% - 99.2%) | 100.0%
(335/335) | (98.9% - 100.0%) | 3 | 100.0 % | 99.4% |
| | 7 | 10.0% | 70 | 100.0%
(7/7) | (59.0% - 100.0%) | 100.0%
(63/63) | (94.3% - 100.0%) | 0 | 100.0 % | 100.0 % |
| FUPT^{4} | 1 | 16.1% | 15
5 | 96.0%
(24/25) | (79.6% - 99.9%) | 97.7%
(127/130) | (93.4% - 99.5%) | 5 | 88.9% | 99.2% |
| | 2 | 11.0% | 15
5 | 82.4%
(14/17) | (56.6% - 96.2%) | 99.3%
(137/138) | (96.0% - 100.0%) | 6 | 93.3% | 97.9% |
| | 3 | 12.3% | 73 | 88.9% (8/9) | (51.8% - 99.7%) | 100.0%
(64/64) | (94.4% - 100.0%) | 2 | 100.0 % | 98.5% |
| | 4 | 18.3% | 10
4 | 100.0%
(19/19) | (82.4% - 100.0%) | 100.0%
(85/85) | (95.8% - 100.0%) | 6 | 100.0 % | 100.0 % |
| | 5 | 10.0% | 70 | 100.0%
(7/7) | (59.0% - 100.0%) | 100.0%
(63/63) | (94.3% - 100.0%) | 0 | 100.0 % | 100.0 % |
| | 6 | 8.5% | 36
6 | 93.5%
(29/31) | (78.6% - 99.2%) | 99.4%
(333/335) | (97.9% - 99.9%) | 3 | 93.5% | 99.4% |
| | 7 | 10.0% | 70 | 85.7% (6/7) | (42.1% - 99.6%) | 98.4%
(62/63) | (91.5% - 100.0%) | 0 | 85.7% | 98.4% |
| MS^{5} | 1 | 24.1% | 20
3 | 87.8%
(43/49) | (75.2% - 95.4%) | 98.1%
(151/154) | (94.4% - 99.6%) | 9 | 93.5% | 96.2% |
| | 2 | 22.4% | 76 | 100.0%
(17/17) | (80.5% - 100.0%) | 98.3%
(58/59) | (90.9% - 100.0%) | 10 | 94.4% | 100.0 % |
| | 4 | 23.8% | 10
1 | 95.8%
(23/24) | (78.9% - 99.9%) | 100.0%
(77/77) | (95.3% - 100.0%) | 11 | 100.0 % | 98.7% |
| | 5 | 15.5% | 71 | 90.9%
(10/11) | (58.7% - 99.8%) | 96.7%
(58/60) | (88.5% - 99.6%) | 3 | 83.3% | 98.3% |

22 of the 115 FUPT positive subjects were co-infected with GC.
33 of the 101 MS positive subjects were co-infected with GC.

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Analysis of CT Positive/Negative Specimens from Female Subjects Based on Patient Infected Status

| | NAAT 1 | | NAAT 2 | | BD ProbeTec CT Q® Amplified DNA Assay | | | | Symptomatic Status | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PIS CT | Endocervical Swab | Urine | Endocervical Swab | Urine | Q® Endocervical Swab | Q® Vaginal Swab | Neat Urine | Q® UPT Urine | A | S | Total |
| + | - | + | - | + | - | - | + | + | 0 | 2 | 2 |
| | - | + | - | + | - | + | - | - | 1 | 0 | 1 |
| | - | + | - | + | - | + | + | + | 2 | 4 | 6 |
| | - | + | - | + | + | - | + | + | 1 | 0 | 1 |
| | - | + | + | + | - | + | + | + | 1 | 0 | 1 |
| | - | + | + | + | + | + | + | + | 1 | 3 | 4 |
| | + | NA | + | + | + | + | + | + | 0 | 1 | 1 |
| | + | - | + | - | + | + | - | - | 1 | 2 | 3 |
| | + | - | + | + | + | - | - | - | 0 | 1 | 1 |
| | + | - | + | + | + | + | - | - | 0 | 1 | 1 |
| | + | - | + | + | + | + | - | + | 1 | 0 | 1 |
| | + | - | + | + | + | + | + | - | 1 | 0 | 1 |
| | + | - | + | + | + | + | + | + | 0 | 2 | 2 |
| | + | + | - | + | + | + | + | + | 1 | 0 | 1 |
| | + | + | + | + | + | + | - | + | 1 | 0 | 1 |
| + | + | + | + | + | + | + | - | 0 | 1 | 1 | |
| + | + | + | + | + | + | + | + | 46 | 41 | 87 | |
| Total PIS Positive | | | | | | | | | 57 | 58 | 115 |
| - | NA | - | - | - | - | - | - | - | 11 | 2 | 13 |
| | NA | - | - | + | - | - | - | - | 1 | 0 | 1 |
| | - | NA | - | - | - | - | NA | NA | 0 | 1 | 1 |
| | - | NA | - | - | - | - | - | - | 1 | 0 | 1 |

33 of the 101 MNU positive subjects were co-infected with GC.

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15

| | NAAT 1 | | NAAT 2 | | BD ProbeTec CT Q² Amplified DNA Assay | | | | Symptomatic Status | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PIS CT | Endocervical Swab | Urine | Endocervical Swab | Urine | Q² Endocervical Swab | Q² Vaginal Swab | Neat Urine | Q² UPT Urine | A | S | Total |
| | - | I | - | - | - | - | - | - | 5 | 1 | 6 |
| | - | - | NA | - | - | - | - | - | 1 | 2 | 3 |
| | - | - | - | - | LE | - | - | - | 0 | 1 | 1 |
| | - | - | - | - | - | NA | - | - | 1 | 0 | 1 |
| | - | - | - | - | - | - | - | - | 362 | 456 | 818 |
| | - | - | - | - | - | - | - | + | 1 | 2 | 3 |
| | - | - | - | - | - | - | + | - | 0 | 2 | 2 |
| | - | - | - | - | - | + | - | - | 0 | 1 | 1 |
| | - | - | - | - | + | - | - | - | 6 | 2 | 8 |
| | - | - | - | + | - | - | - | - | 0 | 4 | 4 |
| | - | - | - | + | + | + | - | - | 0 | 1 | 1 |
| | - | - | + | - | - | - | - | - | 1 | 3 | 4 |
| | - | - | + | - | - | + | - | + | 1 | 0 | 1 |
| | - | - | + | - | + | - | - | - | 1 | 1 | 2 |
| | - | - | + | - | + | + | - | - | 1 | 0 | 1 |
| | - | - | + | + | - | - | + | - | 0 | 1 | 1 |
| | - | - | + | + | + | + | - | + | 0 | 1 | 1 |
| | - | - | + | + | + | + | + | + | 0 | 1 | 1 |
| | - | + | - | - | - | - | - | - | 0 | 1 | 1 |
| | + | - | - | - | - | - | - | - | 0 | 2 | 2 |
| + | + | - | - | + | + | + | + | 0 | 1 | 1 | |
| Total PIS Negative | | | | | | | | | 393 | 486 | 879 |

I Indeterminate
LE Liquid Level Error

Analysis of CT Positive/Negative Specimens from Male Subjects Based on Patient Infected Status

| | NAAT 1 | | NAAT 2 | | BD ProbeTec CT Q² Amplified DNA Assay | | | Symptomatic Status | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PIS CT | Urethral Swab | Urine | Urethral Swab | Urine | Q² Urethral Swab | Neat Urine | Q² UPT Urine | A | S | Total |
| + | NA | + | + | + | + | + | + | 0 | 6 | 6 |
| | - | + | - | + | - | + | + | 3 | 2 | 5 |
| | - | + | + | - | - | - | - | 0 | 1 | 1 |
| | - | + | + | + | + | + | + | 1 | 4 | 5 |
| | + | - | + | + | - | + | + | 0 | 1 | 1 |
| | + | - | + | + | + | - | - | 0 | 1 | 1 |
| | + | + | NA | + | + | + | + | 2 | 5 | 7 |
| | + | + | - | + | + | + | + | 0 | 1 | 1 |
| | + | + | + | - | + | + | + | 0 | 1 | 1 |
| | + | + | + | + | - | + | + | 1 | 0 | 1 |

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| | + | + | + | + | + | + | + | 28 | 44 | 72 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Total PIS Positive | | | | | | | | 35 | 66 | 101 |
| - | NA | - | - | - | - | - | - | 4 | 12 | 16 |
| | NA | - | - | - | - | - | + | 0 | 1 | 1 |
| | - | I | NA | - | - | - | - | 1 | 0 | 1 |
| | - | I | - | - | - | - | - | 4 | 1 | 5 |
| | - | - | NA | - | - | - | - | 10 | 20 | 30 |
| | - | - | - | - | - | - | - | 157 | 146 | 303 |
| | - | - | - | - | - | - | + | 0 | 2 | 2 |
| | - | - | - | - | + | - | - | 0 | 2 | 2 |
| | - | - | - | - | + | - | + | 0 | 1 | 1 |
| | - | - | - | + | - | - | - | 0 | 3 | 3 |
| | - | - | + | - | - | - | - | 1 | 1 | 2 |
| | - | - | + | - | + | - | - | 0 | 1 | 1 |
| | - | - | + | - | + | + | + | 1 | 0 | 1 |
| | - | - | + | + | - | + | + | 0 | 1 | 1 |
| | + | - | - | - | - | - | - | 1 | 0 | 1 |
| | + | + | NA | - | + | + | + | 1 | 0 | 1 |
| Total PIS Negative | | | | | | | | 180 | 191 | 371 |

## Neat and Qᵉ UPT Urine Stability

Pools of CT negative male and female urine specimens were used in analytical experiments to support the urine storage and transport stability claims.

For neat urine, pools were co-spiked with CT serovar H and GC strain ATCC 19424 at 45 EB per mL and 150 cells per mL, respectively. Neat urine specimens were stored at either 2-8°C for 1, 3 or 7 days; or at 30°C for 8, 24 or 30 h; or at -20°C for 60 days. At each time point, samples were removed from storage and tested with the BD ProbeTecCT Qᵉ Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Qᵉ assay under all conditions tested.

For Qᵉ UPT urine, pooled specimens were co-spiked with CT serovar H and GC strain ATCC 19424 at 45 EB per mL and 150 cells per mL, respectively. The spiked urine specimen pools were then stored at either 2-8°C for 24 h or 30°C for 8 h prior to transfer into Qᵉ UPT tubes. The Qᵉ UPT specimen pools were then stored either at 2-8°C for 14, 21 or 30 days; or at 30°C for 14, 21 or 30 days; or at -20°C for 60 days. At each time point Qᵉ UPT specimens were removed from storage and tested with the BD ProbeTecCT Qᵉ Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Qᵉ assay under all conditions tested.

## Vaginal Dry and Expressed Swab Stability

Pools of CT negative vaginal swab matrix were used in analytical experiments to support the storage and transport stability claims for dry vaginal swab specimens. Pools were co-spiked with CT serovar H and GC strain ATCC 19424 to achieve 90 EB per mL and 300 cells per mL, respectively, when seeded onto swabs and expressed in CT/GC Qᵉ Swab

16

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Diluent. Seeded dry swabs were stored at 2-8°C for 3, 7, or 14 days; or at 30°C for 3, 7 or 14 days; or at -20°C for 30 or 60 days. At each time point, dry swabs were removed from storage and expressed into 2 mL of CT/GC Qx Swab Diluent and evaluated with the BD ProbeTec CT Qx Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Qx assay under all conditions tested.

Pools of CT negative vaginal swab matrix were used in analytical experiments to support the storage and transport stability claims for expressed vaginal swab specimens. Pools were spiked with CT serovar H and GC strain ATCC 19424 to achieve 90 EB per mL and 300 cells per mL, respectively. The spiked swab matrix was stored at 2-8°C for 7, 14 or 30 days; or at 30°C for 7, 14 or 30 days; or at -20°C for 30 or 60 days. At each time point, samples were removed from storage and tested with the BD ProbeTec CT Qx Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Qx assay under all conditions tested.

## Endocervical and Urethral Swab Specimen Stability

Pools of CT negative endocervical swab matrix were used in analytical experiments to support the storage and transport stability claims for endocervical and urethral swab specimens. Pools of swab matrix were spiked with CT serovar H and GC strain ATCC 19424 at 90 EB per mL and 300 cells per mL, respectively. The pools were dispensed in 2 mL volumes into BD sample tubes to simulate "wet" endocervical specimens and stored at either 2-8°C for 7, 14 or 30 days; or at 30°C for 7, 14 or 30 days; or at -20°C for 30 or 60 days. At each time point, samples were removed from storage and tested with the BD ProbeTec CT Qx Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Qx assay under all conditions tested.

## Post Pre-warm Specimen Stability

Pools of male and female CT negative neat urine were used in analytical experiments to support the storage stability claims for pre-warmed neat and Qx UPT urine specimens. Pooled specimens were spiked with CT serovar H and GC strain ATCC 19424 at 45 EB per mL and 150 cells per mL, respectively and either added to Qx UPT tubes or left untreated as neat urine. Both specimen types were pre-warmed at 114°C for 15 min, and cooled for 15 min. After the pre-warm process, specimen tubes were stored at either 2-8°C for 1, 3 or 7 days; or at 30°C for 1, 3 or 7 days; or at -20°C for 30 days. At each time point samples were removed from storage and tested with the BD ProbeTecCT Qx Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Qx assay under all conditions tested.

Pools of CT negative vaginal and endocervical swab specimen matrices in CT/GC Qx Swab Diluent were used in analytical experiments to support the storage stability claims for pre-warmed expressed vaginal, endocervical, and male urethral swab specimens. For both types of matrix, pooled specimens were spiked with CT serovar H and GC strain ATCC 19424 at 90 EB per mL and 300 cells per mL, respectively and aliquotted into 2 mL volumes in BD specimen tubes. The tubes were pre-warmed at 114°C for 15 min and

17

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cooled for $15\mathrm{min}$ . After the pre-warm process, the specimen tubes were stored either at $2 - 8^{\circ}\mathrm{C}$ for 3 or 7 days; or at $30^{\circ}\mathrm{C}$ for 3 or 7 days; or at $-20^{\circ}\mathrm{C}$ for 30 days. At each time point, samples were removed from storage and tested with the BD ProbeTecCT Q $^{\mathrm{x}}$ Assay on the BD Viper System in extracted mode. Thirty-two assay replicates were generated for each condition (sample type/temperature/duration). The expected results were obtained with the CT Q $^{\mathrm{x}}$ assay under all conditions tested.

# 4. MaxRFU Frequency Distribution:

A total of 5388 CT Q $^x$ Assay results was evaluated at seven geographically diverse clinical sites. A frequency distribution of the initial MaxRFU values for the CT Q $^x$ assay is shown in Figure A. The distribution of MaxRFU values from CT Q $^x$ true positive, true negative, false positive and false negative specimens (i.e. from those specimens that yielded results which were discordant with the patient infected status (PIS)) is shown in the table below.

Figure A: Frequency Distribution of MaxRFU for the CT Q $^x$ Assay.

CT Q $^x$ MaxRFU Ranges for False Negative, False Positive, True Negative and True Positive Results

| | MaxRFU range | 0-49 | 50-99 | 100-124 | 125-149 | 150-199 | 200-249 | 250-349 | 350-499 | 500-799 | ≥800 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| n | | 4590 | 26 | 1 | 1 | 3 | 3 | 0 | 5 | 4 | 755 |
| FN | FNU | 8 | 0 | 0 | | | | | | | |
| | FS | 10 | 0 | 0 | | | | | | | |
| | FUPT | 8 | 0 | 0 | | | | | | | |
| | FV | 4 | 0 | 0 | | | | | | | |
| | MNU | 2 | 0 | 0 | | | | | | | |

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19

| | MaxRFU range | 0-49 | 50-99 | 100-124 | 125-149 | 150-199 | 200-249 | 250-349 | 350-499 | 500-799 | ≥800 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| n | | 4590 | 26 | 1 | 1 | 3 | 3 | 0 | 5 | 4 | 755 |
| | MS | 8 | 0 | 0 | | | | | | | |
| | MUPT | 2 | 0 | 0 | | | | | | | |
| | Total | 42 | 0 | 0 | | | | | | | |
| FP | FNU | | | | 0 | 1 | 0 | 0 | 0 | 0 | 4 |
| | FS | | | | 0 | 0 | 0 | 0 | 2 | 2 | 11 |
| | FUPT | | | | 0 | 0 | 2 | 0 | 0 | 0 | 5 |
| | FV | | | | 0 | 1 | 0 | 0 | 0 | 0 | 6 |
| | MNU | | | | 0 | 0 | 0 | 0 | 0 | 1 | 2 |
| | MS | | | | 0 | 1 | 0 | 0 | 0 | 0 | 5 |
| | MUPT | | | | 1 | 0 | 1 | 0 | 0 | 0 | 5 |
| | Total | | | | 1 | 3 | 3 | 0 | 2 | 3 | 38 |
| TN | FNU | 868 | 5 | 0 | | | | | | | |
| | FS | 857 | 6 | 0 | | | | | | | |
| | FUPT | 866 | 5 | 0 | | | | | | | |
| | FV | 866 | 4 | 1 | | | | | | | |
| | MNU | 368 | 0 | 0 | | | | | | | |
| | MS | 364 | 1 | 0 | | | | | | | |
| | MUPT | 359 | 5 | 0 | | | | | | | |
| | Total | 4548 | 26 | 1 | | | | | | | |
| TP | FNU | | | | 0 | 0 | 0 | 0 | 2 | 1 | 104 |
| | FS | | | | 0 | 0 | 0 | 0 | 1 | 0 | 104 |
| | FUPT | | | | 0 | 0 | 0 | 0 | 0 | 0 | 107 |
| | FV | | | | 0 | 0 | 0 | 0 | 0 | 0 | 111 |
| | MNU | | | | 0 | 0 | 0 | 0 | 0 | 0 | 99 |
| | MS | | | | 0 | 0 | 0 | 0 | 0 | 0 | 93 |
| | MUPT | | | | 0 | 0 | 0 | 0 | 0 | 0 | 99 |
| | Total | | | | 0 | 0 | 0 | 0 | 3 | 1 | 717 |

Controls: During the clinical evaluation, there were no CT Q⁴ positive control failures from 253 CT Q⁴ plate runs. For the CT Q⁴ negative control, a failure was observed in 1 of 253 CT Q⁴ plate runs. The CT/GC Q⁴ positive and negative control MaxRFU values observed in the clinical trials are shown in the table below.

Distribution of MaxRFU Results for the CT Q⁴ Assay Positive and Negative Controls

| | Max RFU | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Control | n | Range | 5th Percentile | Mean | Median | 95th Percentile |
| CT Q⁴ Negative Control | 252 | 0 - 41 | 0.0 | 0.7 | 0.0 | 4.3 |
| CT Q⁴ Positive Control | 253 | 629 - 2378 | 1597.7 | 1939.4 | 1968.8 | 2184.0 |

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# 5. Expected values/Reference range:

A. Prevalence: The prevalence of positive C. trachomatis specimens in patient populations depends upon: clinic type, age, risk factors, gender, and test method. The prevalence observed with the CT Q $^4$ Assay during a multi-center clinical trial ranged from $8.5\%$ to $18.3\%$ for female specimens and $0\%$ to $24.1\%$ for male specimens.

B. Positive and Negative Predictive Value: Hypothetical positive and negative predictive values (PPV &amp; NPV) for the CT Q $^4$ Assay are shown in Table 5. These calculations are based on hypothetical prevalence and overall sensitivity and specificity (compared to the patient infected status) of $94.5\%$ and $98.9\%$ , respectively. In addition, PPV and NPV based on actual prevalence, sensitivity and specificity are shown in Tables 8 and 9. PPV was calculated using: (Sensitivity * Prevalence) / (Sensitivity*Prevalence + (1 - Specificity) * (1 - Prevalence)). NPV was calculated using: (Specificity * (1 - Prevalence) / (1-Sensitivity) * Prevalence + Specificity * (1-Prevalence)).

CT Hypothetical Positive and Negative Predictive Values Compared to Patient Infected Status.

| Prevalence (%) | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) |
| --- | --- | --- | --- | --- |
| 2 | 94.5 | 98.9 | 64.1 | 99.9 |
| 5 | 94.5 | 98.9 | 82.1 | 99.7 |
| 10 | 94.5 | 98.9 | 90.7 | 99.4 |
| 20 | 94.5 | 98.9 | 95.6 | 98.6 |
| 30 | 94.5 | 98.9 | 97.4 | 97.7 |
| 40 | 94.5 | 98.9 | 98.3 | 96.4 |
| 50 | 94.5 | 98.9 | 98.9 | 94.7 |

# N. Instrument:

BD Viper™ System in extracted mode with the addition of the CTQ and GCQ Assays: The BD Viper System with the capability of automated nucleic acid extraction is the third generation of the BD Viper robotic platform for amplified DNA analysis. The system builds upon its predecessors, the BD Viper Instrument (K023955) and the BD Viper System (K052481).

Comparison of Operating and Technological Characteristics

| | BD Viper System: Extracted mode of operation | BD Viper System Non-extracted mode of operation (K052481) |
| --- | --- | --- |
| Thermal Module: • Priming Temperature • Priming to Amplification Transfer Temperature • Amplification | • Same as BD Viper System K052481 • 70°C • Same as BD Viper System K052481 | • 70°C • 70°C to 52.5°C • 52.5°C |

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| Temperature | | |
| --- | --- | --- |
| Amplification Plate Sealing | • Same as BD Viper System K052481 | • Automated sealing of amplification plate |
| Optical Module: Fluorescent readers | • Same as BD Viper System K052481 • Same as BD Viper System K052481 • Extraction control read on ROX | • Automatic amplification plate transfer into resident readers (2 readers) • Target read on FAM channel • ROX channel not used |
| Software Module | | |
| Sample log-in | • Same as BD Viper System K052481 • Patient Sample Location Identification (optional) | • Bar-code or key entered accession numbers |
| Algorithm | • MaxRFU – Maximum (normalized) Relative Fluorescence Units | • MOTA – Method Other Than Acceleration (area under the curve) |
| Menu | • Same as BD Viper System K052481 • Added: CTQ, GCQ and CTQ/GCQ | • CT or CT/GC |
| Controls and System Checks | | |
| Assay Controls | • Positive and negative run controls • Automated control rehydration • Extraction control • Specimen processing control procedure | • Positive and negative run controls • Manual control rehydration • Specimen processing control procedure |
| System Checks | • Same as BD Viper System K052481 • Extraction reagent checks • Liquid Waste reservoir check | • Consumables, Pipettor, Thermal, Optical, Fluid Volumes |
| Waste and Waste Disposal | | |
| • Waste Composition • Liquid Waste Disposal Container • Liquid Waste Neutralizer | • Same as BD Viper System K052481 and liquid waste (extraction reagents) • Modified waste disposal bottle • Neutralization pouch for liquid waste | • Solid waste (pipette tips) • Waste disposal bottle resident, not used except in aborted run condition • NA |

# O. System Descriptions:

Viewing the BD Viper System from the perspective of assay workflow, the level of automation added to enable automated nucleic acid extraction on the existing BD Viper System includes the following:

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(1) Chemical lysis of organisms in clinical specimens,
(2) Chemical extraction and purification of DNA using paramagnetic particles facilitated by employment of an extractor block containing a movable magnet assembly;
(3) Elution of extracted DNA into SDA-compatible buffer; and
(4) Transfer of the eluate from the extraction tube to the assay priming microwells.

Beyond these additions to the existing BD Viper System’s workflow, the following processing functions are common to both systems (extracted and non-extracted):

(5) Priming microwell heat spike;
(6) Transfer of sample from priming microwells to prewarmed amplification microwells located directly on the reader stage/heater;
(7) Amplification microwell plate sealing and movement of the sealed amplification microwells into the fluorescent reader;
(8) Amplification temperature control and fluorescent photodetection; and
(9) Calculation and result interpretation.

Software: The FDA has reviewed applicant’s Hazard Analysis and Software development processes for the device

P. Other Supportive Device and Instrument Information: NA

Q. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

R. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.