Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on standard molecular diagnostic techniques (DNA extraction, PCR amplification, endpoint detection) and automated workflow. While it uses imaging software to interpret hybridization spots, there is no mention of AI/ML algorithms for this interpretation or for result determination. The result interpretation is described as automatic based on detected spots, not learned patterns.

Therapeutic

No
The device is an in vitro diagnostic (IVD) system intended for the qualitative detection of DNA from specific pathogens to aid in diagnosis, not for the direct treatment or therapy of a disease.

Diagnostic

Yes

The device is intended to "aid in the diagnosis of chlamydial urogenital disease, gonococcal urogenital disease and trichomoniasis in asymptomatic male individuals," which is a diagnostic purpose.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the system is comprised of an instrument with associated hardware and accessories, disposable cartridges, master mixes, and reagent components, in addition to the software. This indicates it is a hardware-based system with integrated software, not a software-only device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The document explicitly states in the "Intended Use / Indications for Use" section: "For In Vitro Diagnostic Use."

Furthermore, the description of the device and its intended use aligns perfectly with the definition of an IVD:

It is intended for the qualitative detection of DNA from specific pathogens (CT, NG, TV).
It uses biological specimens (male urine).
It is used to aid in the diagnosis of diseases (chlamydial urogenital disease, gonococcal urogenital disease, and trichomoniasis).
It is performed outside of the body ("In Vitro").

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Rheonix STI TriPlex™ Assay, as performed on the Rheonix Encompass MDx® Workstation, is an automated DNA extraction and multiplex PCR amplification test system intended for the direct, qualitative detection of DNA from Chlamydia trachomatis (CT), and/or Neisseria gonorrhoeae (NG), and/or Trichomonas vaginalis (TV) in male urine specimens collected with the Rheonix Urine Specimen Collection Kit. The test is indicated to aid in the diagnosis of chlamydial urogenital disease, gonococcal urogenital disease and trichomoniasis in asymptomatic or symptomatic male individuals.

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

QEP, OUY, MKZ, LSL, NSU

Device Description

The Rheonix Encompass MDx® Workstation and the Rheonix STT TriPlex™ Assay are comprised of an instrument with associated hardware and accessories, disposable microfluidic CARD cartridges, master mixes and reagent components used to extract, amplify, and detect DNA using end point PCR. In addition, all male urine specimens in this system must be collected using the Rheonix Urine Specimen Collection Kit. The process is fully automated with the user intervention required only for loading and unloading the samples and disposable assay components. The Rheonix Encompass MDx Workstation's software automatically interprets test results which may be called as POS (positive), NEG (negative), or IND (indeterminate) for each of the assay's three targets. In addition, if the instrument encounters an error during the performance of the assay, it will report an ERR code. If either an IND or ERR code results, the same specimen should be reanalyzed for the presence of the target for which the indeterminate or error code occurred. Each assay has a built-in process control that assures that the individual steps of the entire process occurred properly. The user may also include external positive and/or negative controls to monitor the assay performance.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Urogenital (male urine specimens)

Indicated Patient Age Range

14 years and older (male subjects)

Intended User / Care Setting

Not Found

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

Not Found

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

A multi-site and geographically diverse study was conducted to evaluate the Rheonix STT TriPlex Assay as performed on the Rheonix Encompass MDx Workstation using male urine specimens collected in the Rheonix Urine Specimen Collection Kit.
During the clinical study to evaluate the performance of the Rheonix STI TriPlex Assay, specimens from a total of 1627 male subjects (aged 14 years and older) were collected at 8 geographically distinct sites in US. The study enrolled both symptomatic and asymptomatic subjects. One first-catch urine specimen was collected from each subject. The collected specimen from each subject was subsequently aliquoted and transferred into a total of four different manufacturers' transport tubes (one from Rheonix Urine Specimen Collection kit and three others for reference methods). All processed urine specimens were shipped via overnight courier to a central laboratory for Patient Infection Status (PIS) testing and then distributed to one of three separate testing laboratories for test by Rheonix STI TriPlex assay.
Out of the total of 1627 subjects, 14 were excluded from test for reasons including incligibility issues (N = 1), Patient Withdrawal (N=3), sample mishandling/shipping issues (N=10). Of the remaining 1613 evaluable subjects, 1606 were evaluated for CT and NG (7 subjects were excluded from performance analysis due to testing noncompliance or unevaluable PIS testing results), while 1586 subjects were evaluated for TV (27 were excluded from performance analysis due to testing noncompliance or unevaluable PIS testing results). Of the 1586 subjects evaluated for TV, results from one subject were not included in the calculation of Sensitivity and Specificity for TV because of invalid test results in Rheonix STI TriPlex Assay. Therefore, results from a total of 1585 subjects were used to calculate the Sensitivity and Specificity of the Rheonix STI TriPlex Assay for the TV target while results from a total of 1606 subjects were used to calculate the Sensitivity and Specificity for the CT and NG targets.
The performance characteristics of the Rheonix STI TriPlex Assay was then compared to the PIS results determined from analysis of the comparator test results. For males the PIS testing for CT, NG and TV consisted of up to three different FDA cleared Nucleic Acid Amplification Tests (NAATs). When the first two NAATs yielded either two positive or two negative results, the PIS was defined as either PIS Positive or PIS Negatively. If the first two PIS tests were not concordant, then a third "tie-breaker" test was used to establish the PIS of the specimen whereby the PIS was considered either Positive or Negative based on the results of 2 out of the 3 results being either positive or negative, respectively.

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

Study Type: Clinical Performance Study
Sample Size:

Male subjects: 1606 for CT and NG, 1585 for TV.
Key Results:
Clinical Performance for CT Detection (Male Urine):
Symptomatic (S): Sensitivity 95.4% (87.3% - 98.4%), Specificity 100.0% (98.9% - 100.0%)
Asymptomatic (A): Sensitivity 98.0% (92.9% - 99.4%), Specificity 100.0% (99.7% - 100.0%)
ALL: Sensitivity 96.9% (93.0% - 98.7%), Specificity 100.0% (99.7% - 100.0%)

Clinical Performance for NG Detection (Male Urine):

Symptomatic (S): Sensitivity 99% (94.6% - 99.8%), Specificity 100.0% (98.8% - 100.0%)
Asymptomatic (A): Sensitivity 100.0% (78.5% - 100.0%), Specificity 100.0% (99.7% - 100.0%)
ALL: Sensitivity 99.1% (95.2% - 99.8%), Specificity 100.0% (99.7% - 100.0%)

Clinical Performance for TV Detection (Male Urine):

Symptomatic (S): Sensitivity 100% (75.8% - 100.0%), Specificity 100% (99.0% - 100.0%)
Asymptomatic (A): Sensitivity 95.7% (79.0% - 99.2%), Specificity 99.9% (99.5% - 100.0%)
ALL: Sensitivity 97.1% (85.5% - 99.5%), Specificity 99.9% (99.6% - 100.0%)

Rate of non-reportable results (Initial/Unresolved):

Chlamydia trachomatis: Initial 1.3%, Unresolved 0.0%
Neisseria gonorrhoeae: Initial 1.0%, Unresolved 0.0%
Trichomonas vaginalis: Initial 1.5%, Unresolved 0.1%

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

CT (Chlamydia trachomatis):

Sensitivity: 96.9% (93.0% - 98.7%)
Specificity: 100.0% (99.7% - 100.0%)

NG (Neisseria gonorrhoeae):

Sensitivity: 99.1% (95.2% - 99.8%)
Specificity: 100.0% (99.7% - 100.0%)

TV (Trichomonas vaginalis):

Sensitivity: 97.1% (85.5% - 99.5%)
Specificity: 99.9% (99.6% - 100.0%)

Hypothetical Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for Male Urine at various prevalences:

Chlamydia trachomatis (96.9% Sen, 100% Spec):
- 1% prevalence: PPV 100%, NPV 100%
- 2% prevalence: PPV 100%, NPV 99.9%
- 5% prevalence: PPV 100%, NPV 99.8%
- 10% prevalence: PPV 100%, NPV 99.7%
- 15% prevalence: PPV 100%, NPV 99.5%
- 20% prevalence: PPV 100%, NPV 99.2%
- 25% prevalence: PPV 100%, NPV 99.0%
Neisseria gonorrhoeae (99.1% Sen, 100% Spec):
- 1% prevalence: PPV 100%, NPV 100%
- 2% prevalence: PPV 100%, NPV 100%
- 5% prevalence: PPV 100%, NPV 100%
- 10% prevalence: PPV 100%, NPV 99.9%
- 15% prevalence: PPV 100%, NPV 99.8%
- 20% prevalence: PPV 100%, NPV 99.8%
- 25% prevalence: PPV 100%, NPV 99.7%
Trichomonas vaginalis (97.1% Sen, 99.9% Spec):
- 1% prevalence: PPV 90.8%, NPV 100%
- 2% prevalence: PPV 95.2%, NPV 99.9%
- 5% prevalence: PPV 98.1%, NPV 99.9%
- 10% prevalence: PPV 99.1%, NPV 99.7%
- 15% prevalence: PPV 99.4%, NPV 99.5%
- 20% prevalence: PPV 99.6%, NPV 99.3%
- 25% prevalence: PPV 99.7%, NPV 99.0%

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

K182692

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

Not Found

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

Not Found

Regulation Number and Section

§ 866.3393 Device to detect nucleic acids from non-viral microorganism(s) causing sexually transmitted infections and associated resistance marker(s).

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

Summary

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Image /page/0/Picture/0 description: The image contains the logos of the U.S. Department of Health & Human Services and the U.S. Food & Drug Administration (FDA). The Department of Health & Human Services logo is on the left, featuring a stylized symbol. To the right is the FDA logo, with the acronym "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" and "ADMINISTRATION" in blue text.

Rheonix, Inc Richard Montagna Senior Vice President, Scientific & Clinical Affairs 2680 Grand Island Boulevard, Suite 1 Grand Island, New York 14072

Re: K193081

Trade/Device Name: Rheonix STI TriPlex Assay, Rheonix EncompassMDx Workstation Regulation Number: 21 CFR 866.3393 Regulation Name: Device To Detect Nucleic Acids From Non-Viral Microorganism(S) Causing Sexually Transmitted Infections And Associated Resistance Marker(S) Regulatory Class: Class II Product Code: QEP, NSU, LSL, OUY, MKZ Dated: March 31, 2020 Received: April 1, 2020

December 17, 2021

Dear Richard Montagna:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part

1

801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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

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

Sincerely,

Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K193081

Device Name Rheonix STI TriPlex Assay Rheonix Encompass MDx Workstation

Indications for Use (Describe)

For In Vitro Diagnostic Use.

The Rheonix STI TriPlex™ Assay, as performed on the Rheonix Encompass MDx® Workstation, is an automated DNA extraction and multiplex PCR amplification test system intended for the direct, qualitative detection of DNA from Chlamydia trachomatis (CT), and/or Neisseria gonorrhoeae (NG), and/or Trichomonas vaginalis (TV) in male urine specimens collected with the Rheonix Urine Specimen Collection Kit. The test is indicated to aid in the diagnosis of chlamydial urogenital disease, gonococcal urogenital disease and trichomoniasis in asymptomatic male individuals.

Type of Use (Select one or both, as applicable)
-------------------------------------------------	--

X Prescription Use (Part 21 CFR 801 Subpart D)

| Over-The-Counter Use (21 CFR 801 Subpart C)

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3

Image /page/3/Picture/1 description: The image shows the logo for Rheonix. The logo consists of the word "Rheonix" in a bold, sans-serif font, with a horizontal line above the "e". Below the word is a graphic of a molecule or network of connected circles. The circles are green and connected by gray lines.

CORPORATE HEADQUARTERS 10 Brown Road Ithaca, NY 14850 Phone: 607-257-1242 Fax: 607-257-0979

WESTERN NY OFFICES (SENDER'S ADDRESS) 2680 Grand Island Blvd, Ste 1 Grand Island, NY 14072 Phone: 716-773-4232 Fax: 607-257-0979 Email: Rmontagna@rheonix.com

510(k) Summary Date of Summary: December 02, 2021

A. APPLICANT

Rheonix, Inc. 10 Brown Road Ithaca, NY 14805 Phone: 607-257-1242 Fax: 607-257-0979

B. CONTACT PERSON

Richard A. Montagna, PhD, FACB Senior Vice President, Scientific & Clinical Affairs Direct line: 716-773-4232, Ext 11. Email: Rmontagna@rheonix.com

C. MEASURAND

Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) DNA.

D. TYPE OF TEST

Nucleic acid amplification assay (end point polymerase chain reaction)

4

E. TRADE NAME OF DEVICE(S)

Rheonix STI TriPlex™ Assay on Rheonix Encompass MDx® Workstation

F. IDENTIFICATION OF LEGALLY MARKETED PREDICATE DEVICE

BD MAX CTGCTV2 (assay) and BD MAX™ System (instrument) 510(k) Number: K182692

G. REGULATORY INFORMATION

1. REGULATION SECTION:

21 CFR 866.3393 - Nucleic acid detection system for non-viral microorganism(s) causing sexually transmitted infections

2. CLASSIFICATION:

Class II

3. PRODUCT CODE:

QEP: Nucleic acid detection system for non-viral microorganism(s) causing sexually transmitted infections OUY: Trichomonas vaginalis Nucleic Acid Amplification Test System MKZ: DNA Probe, Nucleic Acid Amplification, Chlamydia LSL: DNA-Reagents, Neisseria

NSU: Instrumentation for Clinical Multiplex Test Systems

1. PANEL:
  Microbiology (83)

H. INTENDED USE

1. INTENDED USE(S):

The Rheonix STI TriPlex™ Assay, as performed on the Rheonix Encompass MDx® Workstation, is an automated DNA extraction and multiplex PCR amplification test system intended for the direct, qualitative detection of DNA from Chlamydia trachomatis (CT), and/or Neisseria gonorrhoeae (NG), and/or Trichomonas vaginalis (TV) in male urine specimens collected with the Rheonix Urine Specimen Collection Kit. The test is indicated to aid in the diagnosis of chlamydial urogenital disease, gonococcal urogenital disease and trichomoniasis in asymptomatic or symptomatic male individuals.

5

INDICATION(S) FOR USE: 2.

Same as Intended Use

3. SPECIAL CONDITIONS FOR USE STATEMENT(S):

For prescription use only.

4. SPECIAL INSTRUMENT REQUIREMENTS:

The Rheonix STI TriPlex Assay must be performed on the Rheonix Encompass MDx® Workstation

5. SPECIAL SPECIMEN COLLECTION REQUIREMENTS:

Specimens analyzed using the Rheonix STI TriPlex Assay as performed using the Rheonix Encompass MDx Workstation must be collected in the Rheonix Urine Specimen Collection Kit.

I. DEVICE DESCRIPTION

The Rheonix Encompass MDx® Workstation and the Rheonix STT TriPlex™ Assay are comprised of an instrument with associated hardware and accessories, disposable microfluidic CARD cartridges, master mixes and reagent components used to extract, amplify, and detect DNA using end point PCR. In addition, all male urine specimens in this system must be collected using the Rheonix Urine Specimen Collection Kit. The process is fully automated with the user intervention required only for loading and unloading the samples and disposable assay components. The Rheonix Encompass MDx Workstation's software automatically interprets test results which may be called as POS (positive), NEG (negative), or IND (indeterminate) for each of the assay's three targets. In addition, if the instrument encounters an error during the performance of the assay, it will report an ERR code. If either an IND or ERR code results, the same specimen should be reanalyzed for the presence of the target for which the indeterminate or error code occurred. Each assay has a built-in process control that assures that the individual steps of the entire process occurred properly. The user may also include external positive and/or negative controls to monitor the assay performance.

J. SUBSTANTIAL EQUIVALENCE INFORMATION

1. PREDICATE DEVICE NAME(S):

BD MAX CTGCTV2 (assay) and BD MAX™ System (instrument)

2. PREDICATE 510(K) NUMBER: K182692

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3. COMPARISON WITH PREDICATE

| Item | Device under investigation | Predicate Device
(K182692) |
|--------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | The Rheonix STI
TriPlex™ Assay, as
performed on the
Rheonix Encompass
MDx® Workstation, is an
automated DNA
extraction and multiplex
PCR amplification test
system intended for the
direct, qualitative
detection of DNA from
Chlamydia trachomatis (CT),
and/or Neisseria gonorrhoeae
(NG), and/or Trichomonas
vaginalis (TV) in male urine
specimens collected with
the Rheonix Urine
Specimen Collection Kit.
The test is indicated to aid
in the diagnosis of
chlamydial urogenital
disease, gonococcal
urogenital disease and
trichomoniasis in
asymptomatic or
symptomatic male
individuals. | The BD MAX CTGCTV2
assay, as performed using
the BD MAX System
incorporates automated
DNA extraction and real-
time polymerase chain
reaction (PCR) for the
direct, qualitative detection
of DNA from Chlamydia
trachomatis (CT), Neisseria
gonorrhoeae (NG) and/or
Trichomonas vaginalis (TV).
The assay may be used for
detection of CT and/or
NG DNA in male urine
specimens, and the
detection of CT, NG,
and/or TV DNA in female
urine specimens, clinician-
collected female
endocervical swab
specimens and patient-
collected vaginal swab
specimens (in a clinical
setting). The assay is
indicated for use to aid in
the diagnosis of chlamydial
urogenital disease,
gonococcal urogenital
disease and/or
trichomoniasis in
asymptomatic and
symptomatic individuals. |
| Assay Results | Qualitative | Qualitative |
| Organisms Detected | CT/NG/TV | CT/NG/TV |
| Instrument | Rheonix Encompass
MDx Workstation | BD MAX™ System |
| Item | Device under investigation | Predicate Device (K182692) |
| Technology | NAAT using end point PCR with detection via colorimetric detection | NAAT using Real-time multiplex PCR with detection via fluorescence. |
| Specimen Types | Male urine | Male and female urine, Endocervical swab, and patient-collected vaginal swabs, Liquid-Based Cytology (LBC) specimens |

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K. STANDARD/GUIDANCE DOCUMENT REFERENCED (IF APPLICABLE)

Establishing the Performance Characteristics of In Vitro Diagnostic Devices for Chlamydia trachomatis and/or Neisseria gonorrhea: Screening and Diagnostic Testing - Draft Guidance for Industry and FDA Staff, May 11, 2011.

Class II Special Controls Guideline: Nucleic Acid Amplification Assays for the detection of Trichomonas vaginalis - Guideline for Industry and FDA Staff, August 4, 2015.

EP7-A2, 2005 - Interference Testing in Clinical Chemistry, CLSI Approved Guideline.

EP12-A2, 2008 – User Protocol for Evaluation of Qualitative Test Performance; CLSI Approved Guideline.

EP17-A2, 2012 - Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline-Second Edition

L. TEST PRINCIPLE

The Rheonix STI TriPlex Assay, as performed on the Rheonix Encompass MDx Workstation consists of automated DNA extraction and end-point PCR for the qualitative detection of CT/NG/TV DNA from urogenital specimens. The specimens collected using the Rheonix Urine Specimen Collection Kit are loaded directly onto the Rheonix Encompass MDx Workstation to which the various disposable consumables contained within the Rheonix STI TriPlex Assay have also been loaded by the user. The user interface of the Rheonix Encompass MDx Workstation guides users regarding the proper placement of the various disposable assay consumables and patient specimens. The workstation automates sample preparation, including target organism lysis, DNA extraction and concentration, reagent rehydration and amplification of target DNA using

8

polymerase chain reaction (PCR). Detection of the various targets and controls is achieved by end point hybridization of the resulting amplicons with DNA probes located on microarray filters contained within the disposable microfluidic CARD cartridges. The Rheonix Encompass MDx Workstation performs results interpretation automatically.

All assay steps are automatically performed within the microfluidic CARD cartridges and include cell lysis, extraction and purification of DNA, multiplex PCR and finally hybridization of the resulting target and control amplicons with their corresponding probes located on the integrated DNA array. The control and target amplicons are each generated using biotinylated primer sets and the hybridization spots are detected by the sequential addition of streptavidinylated horseradish peroxidase and its substrate, 3, 3, 5, 5' tetramethylbenzidine (TMB). The resulting blue colored hybridization spots are detected by the Workstation's CMOS camera and the instrument's imaging software interprets the location and intensity of the various hybridization spots. The results are reported as POS (positive), NEG (negative) or IND (indeterminate), based on how the intensity of the hybridization spots corresponds to the threshold intensities established to differentiate a POS from a NEG result. Specimens that yield signal intensities between the POS and NEG thresholds are reported as IND and should be repeated using the same specimen.

M. PERFORMANCE CHARACTERISTICS (IF/WHEN APPLICABLE)

1. ANALYTICAL PERFORMANCE:

A. PRECISION

Within-laboratory precision was evaluated for the Rheonix STI TriPlex assay at one site. Testing was performed by three operators performing 2 runs/day over 3 nonconsecutive days over a 12day period with each sample tested in duplicate. The blinded Precision Test Panel (PTP) contained negative male urine specimen matrix individually spiked with CT, NG or TV at the concentrations specified below.:

True Negative (TN): no targets
High Negative (HN): 0.25x LoD
Low Positive (LP): 2x LoD
Moderate Positive (MP): 5x LoD

Precision study results for the Rheonix STI TriPlex Assay are described in Table 1.

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| Panel

Member	Percent (%) Observed versus Expected
	C. trachomatis
Urine	N. gonorrhoeae
Urine	T. vaginalis
Urine
aTN	97.2%
(35/36)
85.8% - 99.5%	100%
(36/36)
90.4% - 100%	100%
(36/36)
90.4% - 100%
bHN	33.3%
(12/36)
20.2% - 49.7%	85.3%
(29/34)
69.9% - 93.6%	72.2%
(26/36)
56.0% - 84.2%
LP	91.7%
(33/36)
78.2% - 97.1%	100%
(36/36)
90.4% - 100%	100%
(36/36)
90.4% - 100%
MP	97.2%
(35/36)
85.8% - 99.5%	97.2%
(35/36)
85.8% - 99.5%	100%
(36/36)
90.4% - 100%

Table 1: Overall Precision Study Results Using One Lot of the Rheonix STI TriPlex Assay Kit

a For the True Negative (TN) category, the reported agreement of negative results. b For the High Negative (HN) category, the reported agreement indicates the percent of positive results.

B. REPRODUCIBILITY

Site-to-Site reproducibility of the Rheonix STI TriPlex Assay was evaluated at three sites (two external and one internal). Each site was provided with the same panels as described for the Precision study above. The testing was performed by two operators per site who performed two runs per day (each specimen analyzed in duplicate) over 5 non-consecutive days. The results for Site-to-Site Reproducibility study are shown in Tables 2-5 as below.

Table 2: Rheonix STI TriPlex Assay Site-to-Site Reproducibility Study Results

Panel Member	Percent (%) Observed versus Expected
	C. trachomatis	N. gonorrhoeae	T. vaginalis
	Urine	Urine	Urine
aTN	100%	100%	100%
	(120/120)	(120/120)	(120/120)
	96.9% - 100%	96.9% - 100%	96.9% - 100%
bHN	44.1%	79.2%	78.8%
	(52/118)	(95/120)	(93/118)
	35.4% - 53.1%	71.1% - 85.5%	70.6% - 85.2%
LP	100%	100%	100%
	(120/120)	(120/120)	(120/120)
	96.9% - 100%	96.9% - 100%	96.9% - 100%
MP	100%	100%	100%
	(120/120)	(120/120)	(120/120)
	96.9% - 100%	96.9% - 100%	96.9% - 100%

a For the True Negative (TN) category, the reported agreement indicates the pervent of negative results. bFor the High Negative (HN) category, the reported agreement indicates the percent of positive results.

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The qualitative reproducibility across sites and by target is presented below in Tables 3 – 5.

Table 3 Percent Agreement with Expected Result by Site for Qualitative CT Results in
Urine

| Sample ID | Site | Sample
Size | Number
Positive | Percent
Positive Tests | 95% CI |
|-----------|------|----------------|--------------------|---------------------------|-----------------|
| TN | 1 | 40 | 0 | 0.0% | (0.0%, 8.8%) |
| | 2 | 40 | 0 | 0.0% | (0.0%, 8.8%) |
| | 3 | 40 | 0 | 0.0% | (0.0%, 8.8%) |
| HN | 1 | 40 | 18 | 45.0% | (30.7%, 60.17%) |
| | 2 | 40 | 20 | 50.0% | (35.2%, 64.8%) |
| | 3 | 38 | 14 | 36.8% | (23.4%, 52.7%) |
| LP | 1 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 2 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 3 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| MP | 1 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 2 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 3 | 40 | 40 | 100.0% | (91.2%, 100.0%) |

Table 4 Percent Agreement with Expected Result by Site for Qualitative NG Results in Urine

| Sample ID | Site | Sample
Size | Number
Positive | Percent
Positive Tests | 95% CI |
|-----------|------|----------------|--------------------|---------------------------|-----------------|
| TN | 1 | 40 | 0 | 0.0% | (0.0%, 8.8%) |
| | 2 | 40 | 0 | 0.0% | (0.0%, 8.8%) |
| | 3 | 40 | 0 | 0.0% | (0.0%, 9.0%) |
| HN | 1 | 40 | 34 | 85.0% | (70.9%, 92.9%) |
| | 2 | 40 | 31 | 77.5% | (62.5%, 87.7%) |
| | 3 | 40 | 30 | 75.0% | (59.8%, 85.8%) |
| LP | 1 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 2 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 3 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| MP | 1 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 2 | 40 | 40 | 100.0% | (91.2%, 100.0%) |
| | 3 | 40 | 40 | 100.0% | (91.2%, 100.0%) |

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Sample ID	Site	Sample Size	Number Positive	Percent Positive Tests	95% CI
TN	1	40	0	0.0%	(0.0%, 8.8%)
	2	40	0	0.0%	(0.0%, 8.8%)
	3	40	0	0.0%	(0.0%, 9.0%)
HN	1	40	32	80.0%	(65.2%, 89.5%)
	2	40	34	85.0%	(70.9%, 92.9%)
	3	40	27	67.5%	(52.0%, 79.9%)
LP	1	40	40	100.0%	(91.2%, 100.0%)
	2	40	40	100.0%	(91.2%, 100.0%)
	3	40	40	100.0%	(91.2%, 100.0%)

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Table 5 Percent Agreement with Expected Result by Site for Qualitative TV Results in Urine

For the Lot-to-Lot reproducibility, a single operator completed two separate runs per day on a single instrument for each of three separate lots of Rheonix STI TriPlex Assay kits for 3 non-consecutive days over a 12 day period. The test panel used was the same as described for the Precision study. The results for Lot-to-Lot reproducibility are shown below.

100.0%

(91.2%, 100.0%)

| Target | Sample
Type | Panel
Member
ID | Correct | Total | % Correct | 95% CI |
|--------|----------------|-----------------------|---------|-------|-----------|---------------|
| CT | | TN* | 36 | 36 | 100 | 90.4% - 100% |
| | | HN** | 14 | 36 | 38.9 | 24.8% - 55.1% |
| | | LP | 36 | 36 | 100 | 90.4% - 100% |
| | | MP | 35 | 36 | 97.2 | 85.8% - 99.5% |
| NG | Urine | TN* | 36 | 36 | 100 | 90.4% - 100% |
| | | HN** | 7 | 35 | 20.0 | 10.0% - 35.9% |
| | | LP | 36 | 36 | 100 | 90.4% - 100% |
| | | MP | 36 | 36 | 100 | 90.4% - 100% |
| TV | | TN* | 36 | 36 | 100 | 90.4% - 100% |
| | | HN** | 10 | 36 | 27.8 | 15.9% - 44.0% |
| | | LP | 36 | 36 | 100 | 90.4% - 100% |
| | | MP | 36 | 36 | 100 | 90.4% - 100% |

Table 3: Lot-to-Lot Reproducibility

1

2

3

MP

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*TN samples do not contain any target analytes. Therefore "% Correct" refers to the pervent of negative test results. **HN samples, the "% Correct" refers to the percent of positive results.

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C. CARRY OVER/CROSS-CONTAMINATION STUDIES

A study was conducted to investigate within-run carryover and between-run carryover while processing samples with high microbial loads of Chlamydia trachomatis, Neiseria gonorrhoeae, and Trichomonas vaginalis in the Rheonix STI TriPlex Assay. Two different composite samples were tested. One panel member consisted of all three targets at high concentration (Chlanydia trachomats, serovar D at 10° IFU/ml, Neisseria gonorrhoeae, ATCC strain 49226, at 10° CFU/ml and Trichomonas vaginalis, ATCC strain 30236 at 10° trophozoites/mL) and a second panel member consisted of only the matrix without any spiked targets. The high positive samples were run in a CARD cartridge lane immediately adjacent to lane that contained the negative sample. A total of 144 alternating samples were run across nine days and six runs on a single Rheonix Encompass MDx Workstation. In all cases, the high positive sample vielded positive results for all targets in each run while the negative sample yielded negative results for all targets in each run. Therefore, no carry over or cross contamination was observed.

D. LINEARITY/ASSAY REPORTABLE RANGE

Not applicable.

E. TRACEABILITY, STABILITY, EXPECTED VALUES (CONTROLS, CALIBRATORS, OR METHODS)

Controls:

External controls are not provided by the manufacturer, but commercially available positive and negative controls were used throughout the clinical studies. Contrived positive controls can also be used by spiking Chlamydia trachomatis (Serovar H, ATCC VR-879 or Serovar D, ZeptoMetrix Z054), Neisseria gonorrhoeae (ATCC 19424 or ATCC 49226) and Trichomonas vaginalis (ATCC 30236 or ATCC 50143) into negative matrix. Use of a previously characterized clinical sample known to be positive may also be used. Similarly, a previously characterized clinical sample known to be negative may also be used as a negative control.

An internal process control (a chimeric plasmid containing sequences recognized by each of the three sets of PCR primer pairs) is present in the buffer system used in the Rheonix STI TriPlex assay and therefore can confirm if steps from extraction to signal detection of the assay perform properly.

Specimen Stability Studies:

Male urine specimens must be transferred from the collection cup into the transport buffer immediately after collection and are stable for 120 days when stored at 2-8°C or 75 days when stored at 30°C. (Table 7).

	Transport and/or Storage Temperature
Specimen Type	2-8 °C	30 °C
Male urine	120 days	75 days

Table 4: Sample Stability

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Device Stability Studies

1. Rheonix STI TriPlex Assay

When properly stored under recommended conditions at room temperature (i.e., 15 °C -30 °C), the Rheonix STI TriPlex Assay kit is stable for 24 months.

2. Rheonix Urine Specimen Collection Kit

When properly stored under recommended conditions at room temperature (i.e., 15 °C -30 °C), the Rheonix Urine Specimen Collection Kit is stable for 6 months.

F. DETECTION LIMITS

The Limit of Detection (LoD) for the Rheonix STI TriPlex Assay in male urine was determined by testing two representative strains of each target organism detected by the assay. Each target organism was prepared and quantified prior to testing and inoculated into pooled male unine matrix at multiple concentrations. The samples were then loaded into the Rheonix Encompass MDx Workstation in a manner identical to that used for authentic clinical specimens. Each matrix suspension was tested using at least 20 replicates per LoD concentration. Once the preliminary LoD was determined, the LoD was confirmed by evaluating 44 replicates at the presumed LoD concentration. The LoD was defined as the lowest concentration at which at least 95% of all replicates gave a positive test result. The final LoDs for urine matrix are presented in Table 8.

| Organism | Strain | Specimen | LoD Concentration
(units/ml)* |
|------------------------------|---------------|------------|----------------------------------|
| Chlamydia trachomatis | Serovar D | | 19 IFU/ml |
| Chlamydia trachomatis | Serovar H | | 26 IFU/ml |
| Neisseria gonorrhoeae | ATCC 49226 | Male Urine | 180 CFU/ml |
| Neisseria gonorrhoeae | ATCC 19424 | | 110 CFU/ml |
| Trichomonas vaginalis | ATCC30236** | | 4 Trophozoites/ml |
| Trichomonas vaginalis | ATCC 50143*** | | 5 Trophozoites/ml |

Table 5: Limit of Detection (LoD) for the Rheonix STI TriPlex Assay

IFU refers to Inclusion Forming Units and CFU refers to colony forming units. ** Metronidazole (MTZ) sensitive

***MTZ resistant

Inclusivity

Once the LoD was established, an additional 13 serovars of CT, 30 strains of NG and 6 strains of TV were tested in 20 replicates at the most challenging LoDs noted above for each target in pooled urine matrix. If the initial concentration tested yielded lower than 95% positive results for a particular strain, then additional replicates were tested at higher target concentration.

For CT: of all 13 additional strains tested, 11 were detected at 3xLoD or lower target concentrations, and strains L1 and Ba were detected at higher concentrations with ≥95% posstivity, as shown in the table below.

For NG: all 30 strains of NG were detected at 95% positive rate or higher at 110 CFU/mL in urine matrix.

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For TV: all six strains of TV were detected at 95% positive rate or higher at 4 trophozoites/mL in urine matrix.

The results of the inclusivity study are shown below.

CT Serovar	IFU/mL	x LoD	%Pos
A	11	0.6	100
B	11	0.6	100
C	33	1.7	100
E, nvCT	22	1.2	100
I	22	1.2	100
J	55	2.9	100
K	33	1.7	95
G	33	1.7	100
L1	154	8.1	100
L2	11	0.6	100
L3	55	2.9	100
Ba	110	5.8	95

Table 9. Results for Additional Strains of CT

Table 10. Results for Additional Strains of NG

| NG
Strain | CFU/ml | x LoD | %Pos | NG
Strain | CFU/ml | x LoD | %Pos |
|--------------|--------|-------|------|--------------|--------|-------|------|
| Z423 | 110 | 1 | 100 | Z438 | 110 | 1 | 100 |
| Z424 | 110 | 1 | 100 | Z439 | 110 | 1 | 100 |
| Z425 | 110 | 1 | 100 | Z440 | 110 | 1 | 100 |
| Z426 | 110 | 1 | 100 | Z441 | 110 | 1 | 100 |
| Z427 | 110 | 1 | 100 | Z442 | 110 | 1 | 100 |
| Z428 | 110 | 1 | 95 | Z443 | 110 | 1 | 100 |
| Z429 | 110 | 1 | 100 | Z444 | 110 | 1 | 100 |
| Z430 | 110 | 1 | 100 | Z445 | 110 | 1 | 100 |
| Z431 | 110 | 1 | 100 | Z446 | 110 | 1 | 100 |
| Z432 | 110 | 1 | 100 | Z448 | 110 | 1 | 100 |
| Z433 | 110 | 1 | 100 | Z449 | 110 | 1 | 100 |
| Z434 | 110 | 1 | 100 | Z450 | 110 | 1 | 100 |
| Z435 | 110 | 1 | 100 | Z451 | 110 | 1 | 100 |
| Z436 | 110 | 1 | 100 | Z452 | 110 | 1 | 100 |
| Z437 | 110 | 1 | 100 | Z466 | 110 | 1 | 100 |

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TV Strain	Trophozoites/ml	x LoD	%Pos
Z070	4	1	100
CDC252*	4	1	100
Z158	4	1	100
Z159	4	1	100
ATCC 30238	4	1	100
ATCC 30001	4	1	100

Table 11. Results for Additional Strains of TV

*MTZ resistant

G. ANALYTICAL SPECIFICITY

The Rheonix STI TriPlex Assay was performed on samples containing phylogenetically related species and other microorganisms that potentially could be found in urogenital specimens to determine if the other microorganisms might cross-react with any of the three targets of the assay. The bacteria, yeasts, and parasites were tested in the urine matrix at 1 x 106 cells/ml and viruses were tested in the same matrix at 1 x 105 viral particles/ml. Each individual microorganism was tested in triplicate and considered potentially cross-reactive if one or more test replicates yielded a positive result. Of the 156 non-target organisms tested (Table 12), 154 gave negative results for all three replicates. Herpes Simplex Virus, Type I gave one positive result out of three replicates for TV while Neisseria meningitides serogroup D gave one positive result out of three replicates for NG. Upon retest in triplicate, however, all replicates for both microorganisms gave negative results.

Microorganisms Tested
Achromobacter xerosis	Chlamydophila (Chlamydia) psittaci
Acinetobacter calcoaceticus	Chlamydophila (Chlamydia) psittaci
Acinetobacter lwoffii	Chromobacterium violaceum
Actinomyces israelii	Citrobacter freundii
Actinomyces pyogenes (Trueperella pyogenes)	Clostridium difficile
Aerococcus viridans	Clostridium perfringens
Aeromonas hydrophila	Corynebacterium genitalium
Alcaligenes faecalis	Corynebacterium xerosis
Atopobium vaginae	Cryptococcus neoformans
Bacillus subtilis	Cytomegalovirus (CMV)
Bacteroides fragilis	Deinococcus radiodurans
Bergeriella (Neisseria) denitrificans	Derxia gummosa
Bifidobacterium adolescentis	Eikenella corrodens
Bifidobacterium breve	Elizabethkingia meningoseptica (Flavobacterium melingosepticum)
Blautia producta (Peptostreptococcus productus)	Enterobacter aerogenes
Brevibacterium linens	Enterobacter cloacae
Campylobacter jejuni	Enterococcus avium
Campylobacter ureolyticus (Bacteroides ureolyticus)	Enterococcus faecalis
Candida albicans	Enterococcus faecium
Candida glabrata	Erysipelothrix rhusiopathiae
Candida parapsilosis	Escherichia coli
Candida tropicalis	Fusobacterium nucleatum
Chlamydophila pneumoniae	Gardnerella vaginalis
Microorganisms Tested
Gemella haemolysans	Neisseria cinerea
Haemophilus ducreyi	Neisseria cinerea
Haemophilus influenzae	Neisseria elongata
Herpes Simplex Virus , type I (HSV1)*	Neisseria elongata
Herpes Simplex Virus , type II (HSV2)	Neisseria elongata
HIV type 1	Neisseria flavescens
HPV 16	Neisseria flavescens
Weissella paramesenteroides (Leuconostoc paramensenteroides)	Neisseria lactamica
Human Papilloma Virus 6	Neisseria lactamica
Kingella denitrificans	Neisseria lactamica
Kingella kingae	Neisseria lactamica
Klebsielia oxytoca	Neisseria lactamica
Klebsiella pneumoniae	Neisseria lactamica
Lactobacillus acidophilus	Neisseria lactamica
Lactobacillus brevis	Neisseria lactamica
Lactobacillus crispatus	Neisseria lactamica
Lactobacillus jensenii	Neisseria meningitidis serogroup A
Lactobacillus debrueckii (lactis)	Neisseria meningitidis serogroup B
Lactobacillus vaginalis	Neisseria meningitidis serogroup C
Legionella pneumophila	Neisseria meningitidis serogroup C
Legionella pneumophila	Neisseria meningitidis serogroup C
Listeria monocytogenes	Neisseria meningitidis serogroup C
Micrococcus luteus	Neisseria meningitidis serogroup D*
Mobiluncus curtisii	Rhodospirillum rubrum
Moraxella (Branhamella) catarrhalis	Saccharomyces cerevisiae
Moraxella lacunata	Yersinia enterocolitica
Moraxella osloensis	Neisseria meningitidis serogroup W135
Morganella morganii	Neisseria meningitidis serogroup Y
Mycobacterium smegmatis	Neisseria mucosa
Mycoplasma genitalium	Neisseria mucosa
Mycoplasma hominis	Neisseria mucosa
Neisseria cinerea	Neisseria polysaccharea
Neisseria cinerea	Neisseria sicca
Neisseria sicca	Pseudomonas aeruginosa
Neisseria sicca	Pseudomonas fluorescens
Neisseria subflava biovar flava	Pseudomonas putida
Neisseria subflava biovar flava	Rahnella aquatilis
Neisseria subflava biovar perflava	Rhizobium (Agrobacterium) radiobacter
Neisseria subflava biovar perflava	Salmonella enterica minnesota
Neisseria subflava biovar perflava	Salmonella typhimurium
Neisseria subflava biovar perflava	Serratia marcescens
Neisseria subflava biovar perflava	Staphylococcus aureus
Neisseria subflava biovar perflava	Staphylococcus epidermidis
Neisseria subflava biovar subflava	Staphylococcus saprophyticus
Pantoea agglomerans (Erwinia herbicola)	Streptococcus agalactiae
Paracoccus denitrificans	Streptococcus bovis
Pentatrichomonas hominis	Streptococcus mitis
Peptostreptococcus anaerobius	Streptococcus mutans
Peptostreptococcus magnus (Finegoldia magna)	Streptococcus pneumoniae
Plesiomonas shigelloides	Streptococcus pyogenes
Prevotella bivia	Streptococcus salivarius
Propionibacterium acnes	Streptococcus sanguinis
Proteus mirabilis	Trichomonas tenax
Proteus vulgaris	Ureaplasma urealyticum
Providencia stuartii	Vibrio parahaemolyticus

Table 6: Rheonix STI TriPlex Assay Specificity Results (Bacteria, Yeasts, and Viruses)

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17

INTERFERING SUBSTANCES

A total of 26 potentially interfering substances that could be present in urogenital tract specimens were evaluated at concentrations selected to be medically relevant. The testing was performed by analyzing pooled male urine that contained a mixture of the three targets spiked at 1.5 x their respective LoDs. Since a small percentage of samples analyzed at 1.5 x LoD would be expected to return negative results, even in the absence of any interfering substances, if potential interference was observed at 1.5 x LoD, the potential interfering substance was retested using matrix spiked with 3 x their respective LoDs. None of the substances tested yielded interference at the concentrations noted in the following tables.

| Table 13. Results for Interfering Substances Studies

Substances Tested in Urine Matrix
Interfering substance	Concentration
Whole Blood	2% (v/v)
Semen	5% (v/v)
Hormones	0.48 ng/mL 17-α-Ethinylestradiol
AntiProtozoal (Metronidazole)	48 µg/mL
Glucose	0.48 mg/mL
Acetylsalicylic Acid	260.8 µg/mL
Azithromycin	4.8 µg/mL
Phenazopyridine Hydrochloride	80 µg/mL
Norithindrone	8 ng/mL
4-Acetaminophenol	80 µg/mL
Naproxen	200 µg/mL
Ibuprofen	200 µg/mL
Amoxicillin Trihydrate	30.08 µg/mL
Tetracycline Hydrochloride	6 µg/mL
Ceftriaxone	324.4 µg/mL
Sulfamethoxazole	160 µg/mL
Trimethoprim	16 µg/mL L
Erythromycin	24 µg/mL
Human Serum Albumin	0.4 mg/mL
Leukocytes	106 cells/mL
Feminine Deodorant Spray	0.68% v/v
Talcum Powdera	0.6% w/v
Bilirubin	0.08 mg/mL
Biotin	3500 ng/ml
Urine (high pH)	pH 9
Urine (low pH 4)	pH 4

Table 13. Results for Interfering Substances Studies

"May interfere with the Rheonix STI TriPlex assay when at concentrations higher than shown.

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MIXED INFECTION/COMPETITIVE INTERFERENCE

The mixed infection/competitive interference study was designed to evaluate the ability of the Rheonix STI TriPlex Assay to detect low positive results in the presence of the other targets at high concentrations in urine matrix. Two (2) organisms (Neisseria gonorrhoeae and Trichomonas vaginalis) were individually prepared at 1.5X their respective LoD and Chlamatis was prepared at 1.7X its respective LoD to serve as a low target in the Rheonix STI Collection Buffer. When added as a high target to the mixture, CT was added at 10° IFU/ml, NG was added at 10° CFU/ml and TV was added at 106 trophozoites/ml. No interference was observed when either CT, NG or TV were tested in the presence of exceedingly high concentrations of the other two targets.

H. ASSAY CUT-OFF

The Encompass MDx workstation's optical system and software evaluates the intensity of the end point PCR hybridization spots on the DNA array contained within the CARD cartridge devices used to perform the assay. Based on predetermined intensity values, the software reports the results as either Positive, Negative, Indeterminate or an Error Code for each of the three targets. Intensity values equal to or less than the lower cut off value generate a negative result, values between the lower and the upper cut offs generate an indeterminate result, and values equal to or greater than the upper cut off generate a possitive result. If the workstation encounters any unacceptable assay parameters, appropriate Error code messages appear on the final report.

2. COMPARISON STUDIES

A. METHOD COMPARISON WITH PREDICATE DEVICE: Not Applicable.

B. MATRIX COMPARISON

Not Applicable.

3. CLINICAL STUDIES

A multi-site and geographically diverse study was conducted to evaluate the Rheonix STT TriPlex Assay as performed on the Rheonix Encompass MDx Workstation using male urine specimens collected in the Rheonix Urine Specimen Collection Kit.

During the clinical study to evaluate the performance of the Rheonix STI TriPlex Assay, specimens from a total of 1627 male subjects (aged 14 years and older) were collected at 8 geographically distinct sites in US. The study enrolled both symptomatic and asymptomatic subjects. One firstcatch urine specimen was collected from each subject. The collected specimen from each subject was subsequently aliquoted and transferred into a total of four different manufacturers' transport tubes (one from Rheonix Urine Specimen Collection kit and three others for reference methods). All processed urine specimens were shipped via overnight courier to a central laboratory for Patient

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Infection Status (PIS) testing and then distributed to one of three separate testing laboratories for test by Rheonix STI TriPlex assay.

Out of the total of 1627 subjects, 14 were excluded from test for reasons including incligibility issues (N = 1), Patient Withdrawal (N=3), sample mishandling/shipping issues (N=10). Of the remaining 1613 evaluable subjects, 1606 were evaluated for CT and NG (7 subjects were excluded from performance analysis due to testing noncompliance or unevaluable PIS testing results), while 1586 subjects were evaluated for TV (27 were excluded from performance analysis due to testing noncompliance or unevaluable PIS testing results). Of the 1586 subjects evaluated for TV, results from one subject were not included in the calculation of Sensitivity and Specificity for TV because of invalid test results in Rheonix STI TriPlex Assay. Therefore, results from a total of 1585 subjects were used to calculate the Sensitivity and Specificity of the Rheonix STI TriPlex Assay for the TV target while results from a total of 1606 subjects were used to calculate the Sensitivity and Specificity for the CT and NG targets.

The performance characteristics of the Rheonix STI TriPlex Assay was then compared to the PIS results determined from analysis of the comparator test results. For males the PIS testing for CT, NG and TV consisted of up to three different FDA cleared Nucleic Acid Amplification Tests (NAATs). When the first two NAATs yielded either two positive or two negative results, the PIS was defined as either PIS Positive or PIS Negatively. If the first two PIS tests were not concordant, then a third "tie-breaker" test was used to establish the PIS of the specimen whereby the PIS was considered either Positive or Negative based on the results of 2 out of the 3 results being either positive or negative, respectively.

The prevalence of each pathogen at each clinical study site and all sites combined based on the comparator results is shown below.

Collection Site	CT	NG	TV
1	20.4%	18.6%	7.1%
2	4.6%	7.2%	0.0%
3	9.7%	4.7%	1.7%
4	13.5%	5.0%	1.6%
5	12.2%	2.0%	2.0%
6	9.1%	10.6%	5.1%
7	1.9%	4.8%	0.0%
8	23.1%	23.1%	0.0%
All Sites	10.1%	7.1%	2.2%

Table 14 Prevalence of each pathogen at each clinical study site and all sites combined based on the comparator results

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2) Clinical Sensitivity and Specificity

Based upon comparison against the PIS of each enrollee, the following performance estimates were calculated for each of the analytes:

| Gender | Specimen | Symptom | N | TP | FP | TN | FN | CT
%Sens
(95% CI) | CT
%Spec
(95% CI) |
|--------|----------|---------|------|-----|----|------|----|-------------------------|-------------------------|
| Male | Urine | A | 1202 | 96 | 0 | 1104 | 2 | 98% (92.9% - 99.4%) | 100.0% (99.7% - 100.0%) |
| | | S | 404 | 62 | 0 | 339 | 3 | 95.4% (87.3% - 98.4%) | 100.0% (98.9% - 100.0%) |
| | | ALL | 1606 | 158 | 0 | 1443 | 5 | 96.9% (93.0% - 98.7%) | 100.0% (99.7% - 100.0%) |

Table 15 Clinical Performance for CT Detection

Table 16 Clinical Performance for NG Detection

| Gender | Specimen | Symptom | N | TP | FP | TN | FN | NG
%Sens
(95% CI) | %Spec
(95% CI) |
|--------|----------|---------|------|-----|----|------|----|-------------------------------------|-------------------------------------|
| Male | Urine | A | 1201 | 14 | 0 | 1187 | 0 | 100.0% (78.5% - 100.0%) | 100.0% (99.7% - 100.0%) |
| | | S | 405 | 99 | 0 | 305 | 1 | 99% (94.6% - 99.8%) | 100.0% (98.8% - 100.0%) |
| | | ALL | 1606 | 113 | 0 | 1492 | 1 | 99.1% (95.2% - 99.8%) | 100.0% (99.7% - 100.0%) |

Table 17 Clinical Performance for TV Detection

| Gender | Specimen | Symptom | N | TP | FP | TN | FN | %Sens
(95% CI) | %Spec
(95% CI) |
|--------|----------|---------|-------|----|----|------|----|--------------------------|---------------------------|
| Male | Urine | A | 1187 | 22 | 1 | 1163 | 1 | 95.7%
(79.0% - 99.2%) | 99.9%
(99.5% - 100.0%) |
| | | S | 398 | 12 | 0 | 386 | 0 | 100%
(75.8% - 100.0%) | 100%
(99.0% - 100.0%) |
| | | ALL | 1585* | 34 | 1 | 1549 | 1 | 97.1% (85.5% -
99.5%) | 99.9%
(99.6% - 100.0%) |

One of the tested 1586 subjects was excluded from the above performance analysis due to the lack of valid result for Rheonix STI TriPlex assay.

3) Rate of non-reportable results

Indeterminate or Error Code Results

The Rheonix Encompass MDx workstation reports results as Positive, Negative or Indeterminate for each of the three target microorganisms. In addition, the workstation also reports a variety of

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error codes (ERR) that would require testing. On a target-by-target basis, the combined IND and ERR code rates are reported (Table 18). The values reported as "Initial" represent samples that displayed either an IND or ERR code that required a reanalysis. The "Unresolved" values represent those samples that, upon repeat, did not yield valid results. As noted, only one urine specimen vielded an unresolved final result, also representing 0.06% of the male population evaluated. All other initially failed runs yielded valid, interpretable results upon repeat testing.

		Chlamydia trachomatis			Neisseria gonorrhoeae			Trichomonas vaginalis
Subject	Total N	Initial	Unresolved	Total N	Initial	Unresolved	Total N	Initial	Unresolved
Male	1606	20
(1.3%)	0
(0.0%)	1606	16
(1.0%)	0
(0.0%)	1586	24
(1.5%)	1
(0.1%)
		95% CI			95% CI			95% CI
		(0.8% -
1.9%)	(0.0% -
0.2%)		(0.6% -
1.6%)	(0.0% -
0.2%)		(1.0% -
2.2%)	(0.0% -
0.04%)

Table 18 Rates of Initial and Final Unresolved Results
--------------------------------------------------------

4) Hypothetical Positive and Negative Predictive Values

The hypothetical Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were calculated for urine specimens evaluated from male subjects. The calculations are based on the observed clinical sensitivity and clinical specificity for each specimen type, as compared to the Patient Infection Status (Table 19).

Table 19 Hypothetical Positive and Negative Predictive Values of the Rheonix STI TriPlex Assay

| Specimen
Type | Hypothetical
Prevalence | Chlamydia trachomatis | | | | Neisseria gonorrhoeae | | | | Trichomonas vaginalis | | | |
|------------------|----------------------------|-----------------------|-----------|-------|-------|-----------------------|-----------|-------|-------|-----------------------|-----------|-------|-------|
| | | %
Sen | %
Spec | % PPV | % NPV | %
Sen | %
Spec | % PPV | % NPV | %
Sen | %
Spec | % PPV | % NPV |
| Male
Urine | 1% | | | 100% | 100% | | | 100 % | 100% | | | 90.8% | 100% |
| | 2% | | | 100% | 99.9% | | | 100 % | 100% | | | 95.2% | 99.9% |
| | 5% | | | 100% | 99.8% | | | 100% | 100% | | | 98.1% | 99.9% |
| | 10% | 96.9% | 100% | 100% | 99.7% | 99.1 | 100 | 100 % | 99.9% | 97.1 | 99.9 | 99.1% | 99.7% |
| | 15% | | | 100% | 99.5% | | | 100 % | 99.8% | | | 99.4% | 99.5% |
| | 20% | | | 100% | 99.2% | | | 100 % | 99.8% | | | 99.6% | 99.3% |
| | 25% | | | 100% | 99.0% | | | 100 % | 99.7% | | | 99.7% | 99.0% |

4. CLINICAL CUT-OFF

Not Applicable

5. EXPECTED VALUES/REFERENCE RANGE

The prevalence of infection with CT and/or NG and or TV in patient populations is dependent upon multiple risk factors including age, gender, type of collection site clinic, and the sensitivity of

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the test used to detect the three target microorganisms. The observed positivity rates observed using the Rheonix STI TriPlex Assay in male (Table 20) subjects enrolled in the studies are provided.

| Collection Site | Positivity Rate as Determined by Rheonix STI
TriPlex™ Assay by Clinical Site | | |
|-----------------|---------------------------------------------------------------------------------|-------|------|
| | CT | NG | TV |
| 1 | 19.5% | 17.7% | 7.1% |
| 2 | 4.6% | 7.2% | 0.0% |
| 3 | 9.7% | 4.7% | 1.7% |
| 4 | 13.2% | 5.0% | 1.3% |
| 5 | 10.9% | 2.0% | 2.0% |
| 6 | 8.7% | 10.6% | 5.9% |
| 7 | 1.9% | 4.8% | 0.0% |
| 8 | 23.1% | 23.1% | 0.0% |
| All Sites | 9.8% | 7.0% | 2.2% |

Table 20. Rheonix STI TriPlex Observed Positivity Rates in Male Subjects

N. INSTRUMENT NAME

Rheonix Encompass MDx® Workstation.