Dx CT/NG/MG AUTO ASSAY

5 x 96 37015 1 x 96 37029

Direct detection of , and genitalium by Real-Time PCR

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881119 - 2013/12

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Table of Content

1. INTENDED USE

2. SUMMARY AND EXPLANATION OF THE TEST

3. PRINCIPLES OF THE PROCEDURE

4. REAGENTS

5. WARNINGS AND PRECAUTIONS

6. SPECIMENS

7. PROCEDURE

8. TEST LIMITATIONS

9. EXPECTED VALUES

10. PERFORMANCE CHARACTERISTICS

11. BIBLIOGRAPHY REFERENCES

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1. INTENDED USE The Dx CT/NG/MG Auto Assay is a qualitative multiplex nucleic acid in vitro amplification testing kit allowing in a single tube the direct detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in clinical samples of symptomatic and asymptomatic individuals. The Dx CT/NG/MG Auto Assay is intended for use with clinician-collected 1 1 endocervical, vaginal and anorectal (male and female) swab specimens , patient-collected vaginal swab specimens , and female and male urine specimens. 1 Collected with the Bio-Rad Dx Collection System 50-F kit (cat. # 37012).

2. SUMMARY AND EXPLANATION OF THE TEST Chlamydia trachomatis is an obligate intracellular parasite bacterium, which causes the most common sexually transmitted in industrialized countries, with approximately 89 million new cases each year according to WHO (1). Chlamydia trachomatis are often asymptomatic in both women and men and if untreated can entail huge clinical consequences such as pelvic inflammatory disease (PID), ectopic pregnancy, tubal (2) in women, or non gonococcal and epididymitis in men (3). Molecular diagnostic tests, having superior sensitivity and specificity compared to other existing technologies, are now highly recommended for the detection of Chlamydia trachomatis infections (4). Neisseria gonorrhoeae is a nonmotile Gram-negative diplococcus bacterium which causes approximately 62 million new cases of gonorrhea worldwide each year (1). Clinical consequences are similar to those caused by Chlamydia trachomatis, with often more severe symptoms. Cell culture, commonly used for the detection of N. gonorrhoeae, is highly dependent on proper specimen handling and on viability of the in the specimens. Molecular diagnostic tests can therefore be advantageously combined to conventional techniques for the detection of N. gonorrhoeae. Mycoplasma genitalium is a small bacterium recently recognized as a frequent causative agent of urethritis in men and lower genital tract infections in women, with symptoms and microscopic signs similar to those observed in Chlamydia trachomatis. M. genitalium infections are also suspected to cause tubal infertility in women (5-13). Molecular diagnostic tests are the method of choice for the detection of Mycoplasma genitalium as its culture is extremely difficult to perform.

3. PRINCIPLES OF THE PROCEDURE The Dx CT/NG/MG Auto Assay involves two main steps: sample preparation and amplification/detection of target DNA by real- time PCR. Amplification products are detected via fluorescent dyes during the PCR. With each sample, an internal control is systematically extracted, amplified and detected in the same way as target DNA, allowing the control of the extraction procedure and the detection of potential PCR inhibition. The sample preparation (DNA extraction) is fully automated by using either the Bio-Rad Dx Prep System (cat.# 94500) and the Dx Automated Extraction Assay (cat. # 37016) or NucliSens® easyMAG® system with the corresponding reagents (bioMérieux). The Dx CT/NG/MG Auto Assay kit contains all reagents necessary to perform amplification/detection, for 96 tests (cat.# 37029) or 480 tests (cat.# 37015)

Dx Prep System Sample Preparation Automated DNA extraction is performed on all sample types listed in the chapter "Specimens", using the Bio-Rad Dx Prep System and the Dx Automated Extraction Assay (cat. # 37016) Nucleic acids are captured by magnetic beads, then washed, eluted and automatically transferred to a 96-well Dx PCR plate. A negative control is taken through the entire sample preparation procedure along with the specimens. The Internal Control (C1) is added to each specimen and to the Negative Control at the beginning of sample preparation procedure.

NucliSENS® easyMAG® Sample Preparation Automated DNA extraction is performed on all sample types listed in the chapter "Specimens", using bioMérieux NucliSENS® easyMAG® system according to the Instruction Manual. A negative control is taken through the entire sample preparation procedure with the specimens. The internal Control (C1) is added to each specimen and to the Negative Control at the beginning of the sample preparation procedure.

Real-time PCR Amplification/Detection Using the Bio-Rad Dx Prep System, the amplification master mix is added automatically into the 96-well Dx PCR plate, together with the extracted DNA. After the extraction on NucliSENS® easyMAG® system, extracted DNA are dispensed manually in 24-well Dx PCR Strips as well as the amplification master mix. In real-time PCR, specific fluorescent oligonucleotide probes are used to detect the DNA during amplification, by hybridizing to the amplicons. In the Dx CT/NG/MG Auto Assay, four different oligonucleotide probes are used: • C. trachomatis probe, targeting a sequence of the cryptic plasmid; this targeted sequence is located outside the region deleted in the new variant strain of C. trachomatis (nvCT) recently characterized (14).

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• N. gonorrhoeae probe, targeting a sequence in the pilE ; • M. genitalium probe, targeting a sequence in the MgPa gene. • Internal Control detection probe In the absence of target DNA for a given oligonucleotide probe, no corresponding fluorescence will be emitted thus no signal will be detected. When the target DNA is present, fluorescence intensity increases as the amount of amplicons increase with each round of amplification. During each PCR cycle, at the annealing step, the Dx Real-Time System optical module measures the fluorescence obtained from each fluorophore, and the associated Dx Real-Time Software plots the fluorescence intensity versus the number of cycles. At the end of the experiment, the Dx Real-Time Software automatically analyzes results for all samples and controls.

4. REAGENTS

4.1. Description There are sufficient reagents provided in the kit to perform 480 tests (cat # 37015) or 96 tests (cat.# 37029). All reagents are for in vitro diagnostic use only.

Packaging Packaging Label Reagent 37015 (480 tests) 37029 (96 tests) R1 Amplification Concentrated amplification mix, 10X: DNA 10 x 150 μl 2 x 150 μl Mix Polymerase in a PCR buffer containing primers, To be diluted To be diluted specific fluorescent oligonucleotidic probes, dNTPs by adding R2 by adding R2 and MgCl2. Preservative: 0,05 % sodium azide. R2 Amplification Amplification mix diluent: PCR buffer containing 10 x 900 μl 2 x 900 μl Mix Diluent MgCl2. Preservative: 0,05 % sodium azide. Ready to use Ready to use C1 Internal Control Internal control: Non-infectious DNA in a buffered 5 x 1300 μl 1 x 1300 μl solution. Preservative: 0,03 % ProClin™ 300. Ready to use Ready to use C2 Negative Negative control: TE buffer. 10 x 1100 μl 2 x 1100 μl Control Preservative: 0,03 % ProClin™ 300. Ready to use Ready to use C3 Positive Control CT, NG, MG Positive control: Non-infectious DNA 5 x 100 μl 1 x 100 μl from CT, NG and MG in a buffered solution. Yellow- Ready to use Ready to use coloured. Preservative: 0,03 % ProClin™ 300.

4.2. Storage and handling requirements The kits must be stored at 2-8°C. When stored at this temperature, each reagent contained in the Dx CT/NG/MG Auto Assay can be used until the expiration date given on the reagent label.

Identification Conservation after opening 15 days at 2-8°C. Each vial can be used twice. C2 (Negative Control) If contamination occurs in one of these reagents, discard it. 1 month at 2-8°C. Each vial can be used up to 4 times. C3 (Positive Control) If contamination occurs in one of these reagents, discard it. 4 hours at 18-30°C or 16 hours at 2-8°C or 2 weeks at - 20°C. If frozen, the reconstituted R1+R2 (reconstituted mix) mix can be thawed only once. Dx Prep System extraction 1 month at 2-8°C. If contamination occurs in one of these reagents, discard it. C1 + Binding Buffer The Binding Buffer is part of the Dx Automated Extraction Assay (ref 37016). NucliSENS® easyMAG® system extraction 14 days at 2-8°C. If contamination occurs in one of these reagents, discard it. Premix C1 + Silica The silica is part of the NucliSENS® easyMAG® reagents.

5. WARNINGS AND PRECAUTIONS For in vitro diagnostic use only.

5.1. Health and Safety Precautions • Wear disposable gloves, laboratory coats and protective eyewear when handling reagents and samples. • Thoroughly wash your hands after handling reagents and samples. Do not eat, drink or smoke in designated work areas. • Handle all samples as potentially infectious, in accordance with Good Laboratory Practices. • Chemicals must be handled and disposed of in accordance with Good Laboratory Practices. • The Safety Data Sheet is available upon request to your local Bio-Rad agent.

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5.2. Precautions Related to the Procedure • This kit is validated for use with the Dx Real-Time System (Bio-Rad, cat. # 94000) and its Dx Real-Time Software only. • Automated DNA extraction must be done either with the Dx Automated Extraction Assay kit (Bio-Rad, cat. # 37016) on the Dx Prep System (Bio-Rad) or on the NucliSENS® easyMAG® system with corresponding reagents. • For collection of clinician-collected endocervical, vaginal and anorectal swab specimens and of patient-collected vaginal swab specimens, use only the Dx Collection System 50-F (Bio-Rad, cat. # 37012). This system is not for home use. • For collection of first-catch urine samples, use clean polypropylene, preservative-free specimen collection cups. First-catch Urine samples must be transferred into a Dx Transfer System tube (Bio-Rad, cat. # 37014) if the samples are loaded on the Dx Prep System.

In case the DNA extraction is performed on the NucliSENS® easyMAG® system, samples (urine or Dx Collection System 50- F) are directly loaded on the “shuttle”.

• Do not use the Dx Collection System 50-F if the packaging is damaged or if transport medium has leaked from the tube. Discard unused, damaged or leaking systems in accordance with Good Laboratory Practices. • If the laboratory receives a swab specimen not collected and transported with the Dx Collection System 50-F or a swab specimen transport tube containing no swab or two swabs, the specimen must be rejected. • Do not use expired reagents. • Do not interchange, mix or combine reagents from kits with different lot numbers. • Do not change the assay procedure. • Carefully avoid cross-contamination during specimen handling steps: ensure that specimen containers do not contact one another; if gloves come in contact with specimen, change them immediately. • Carefully reconstitute the Amplification Mix avoiding any contamination. • Check the pipettes and other equipment for accuracy and correct operation. • Avoid spilling samples or solutions containing samples. Spills must be rinsed with bleach diluted at 10%. The material used for cleaning must be discarded in a contaminated residue container. • Work surfaces, pipettes and other equipment must be decontaminated on a regular basis with bleach diluted at 1%.

6. SPECIMENS

6.1. Swab specimens collected with Bio-Rad Dx Collection System 50-F The Dx CT/NG/MG Auto Assay is intended for use with clinician-collected endocervical, vaginal and anorectal (male and female) swab specimens, and with patient-collected vaginal swab specimens. Specimens must be collected and transported only with the Dx Collection System 50-F. For more information on specimen collection and transport, please refer to the Dx Collection System 50-F (Bio-Rad, cat. # 37012) package insert. A transport tube containing a swab not supplied by Bio-Rad, or multiple swabs, or no swab cannot be used with the Dx CT/NG/MG Auto Assay. The presence of blood, of some spermicidal agents and treatments for vaginal conditions may interfere with the real-time PCR reaction. For more information on interfering substances, please refer to the “Performance characteristics” chapter. Once collected, swab specimens can be kept up to 28 days at 18-30°C.

6.2. Urine specimens The Dx CT/NG/MG Auto Assay is intended for use with female and male urine specimens from symptomatic and asymptomatic patients. NOTE: the patient should not have urinated for at least 1 hr prior to specimen collection. 1. Collect 10-20 ml from the first part of urine stream in a clean polypropylene, preservative-free specimen collection cup. 2. Close the cup and label with patient identification and date/time collected. 3. Transport the specimens to the test site at room temperature (18-30°C). Urine samples are stable during 24 hrs at room temperature. If analysis cannot be done within 24 hrs of collection, urine specimens must be stored at 2-8°C and analyzed within 14 days. 4. Urine specimens analyzed at external test sites must be transported to the test site within 24 hrs of collection. If room temperature shipment is chosen, specimens must be stored at 2-8°C prior to shipment and upon arrival in order to ensure that room temperature storage does not exceed 24 hrs of collection. 5. If the DNA extraction step is performed on the Dx Prep System, transfer 3 ml of each urine sample to a Dx Transfer System (Bio-Rad cat. # 37014) tube. This tube will be loaded on the Dx Prep System. In case the extraction is performed on the NucliSENS® easyMAG® system, load the sample directly from collection cup on the "shuttle”.

Once collected, urine specimens can be kept up to 14 days at 2-8°C or up to 60 days at -20°C. If frozen, urine specimens can be thawed only twice.

7. PROCEDURE Follow strictly these instructions and apply Good Laboratory Practices.

7.1. Materials required

7.1.1. Materials provided • Dx CT/NG/MG Auto Assay 480 tests (Bio-Rad, cat. # 37015 only for use on the Dx Prep System extraction) ) or 96 tests (Bio-Rad, cat # 37029 use on both Dx Prep System extraction and NucliSENS® easyMAG® system extraction) 5 [EN]

7.1.2. Materials required provided separately For Dx Prep System extraction • Dx Prep System and accessories (Bio-Rad, cat.# 94500) • Dx Automated Extraction Assay (Bio-Rad, cat.# 37016) • Dx Real-Time System and accessories (Bio-Rad, package cat.# 94000) • Dx Strip Cap Tool (provided in the Dx Real-Time System package Bio-Rad cat.#94000) • Dx Collection System 50-F (Bio-Rad, cat.# 37012) for collection and transport of female and anorectal swab specimens • Dx Transfer System (Bio-Rad, cat. # 37014) for transferring first-catch urine samples. • Capture Cap Set (Bio-Rad, cat. # 37017) for recapping samples tubes after use. • Dx PCR Plates & caps (Bio-Rad, cat.# 94023) • Dx Processing Plate (cat.# 94534) • Dx Elution Plate (cat.# 94022) • Dx Prep filter tips 1100 µl (cat.# 94531) • Dx Prep Filter tips 300 µl (cat.# 94532) • Dx Prep Filter tips 50µl (cat.# 94533)

For NucliSENS® easyMAG® system extraction • Dx Real-Time System and accessories (Bio-Rad, package cat.# 94000) • Dx Strip Cap Tool (provided in the Dx Real-Time System package Bio-Rad cat.#94000) • Dx Collection System 50-F (Bio-Rad, cat.# 37012) for collection and transport of female and anorectal swab specimens • Capture Cap Set (Bio-Rad, cat. # 37017) for recapping samples tubes after use • Dx 24-well PCR strips & caps ( cat # 94020)

7.1.3. Materials required but not supplied For Dx Prep System extraction • Clean polypropylene, preservative-free urine collection cups • Vortex mixer • Desktop centrifuge • Calibrated pipettes capable of delivering 1,000 to 1,200 µl • Sterile pipette tips with filters • Disposable powder-free gloves

For NucliSENS® easyMAG® system extraction • Clean polypropylene, preservative-free urine collection cups • NucliSENS® easyMAG®, reagents and accessories from bioMérieux: EasyMAG Extraction Buffers 1, 2 and 3 (cat. # 280130, 280131, 280132), EasyMAG Magnetic Silica (cat. # 280133), EasyMAG Lysis Buffer (cat. # 280134) and EasyMAG disposables (cat. # 280135).

• Vortex mixer • Desktop centrifuge • 1.5-2 ml tubes (for C1+ silica mixing) • Adjustable micropipettes (P 200 µl and 1000 µl), calibrated, • Multichannel pipette (P 10 µl) calibrated, • Sterile pipette tips with filters • Positive displacement repeat pipettor with sterile individually wrapped 1.25 ml tips • Disposable powder free gloves

For Dx Prep System extraction: • Add the Internal Control (C1) into the Binding Buffer (BB): • Take one bottle of Binding Buffer (BB) from the Dx Automated Extraction Assay and one vial of Internal Control (C1) from the Dx CT/NG/MG Auto Assay. • Add 1200 µl of Internal Control (C1) into the Binding Buffer (BB) bottle, and gently mix by inversion. • Keep the empty vial of Internal Control (C1): it will be loaded on the Dx Prep System.

This mix “Binding Buffer + Internal Control” is stable for one month at 2-8°C.

For NucliSENS® easyMAG® system extraction During the sample lysis phase on NucliSENS® easyMAG® system, mix Internal Control (C1) with Silica according to the following volumes. Once prepared, vortex briefly.

1 test 12 tests 23 tests Silica Volume 50 µl 600 µl 1200 µl C1 Volume 10 µl 120 µl 240 µl

Note: Alternatively, it is possible to add 50µl of Silica and 10µl of C1 into each individual extraction well of the NucliSENS® easyMAG® shuttle. 6 [EN]

Reconstitution of Amplification Mix Before reconstitution, vials R1 (Concentrated amplification mix, 10X) and R2 (Amplification mix diluent) must be vortexed for 5 seconds then centrifugated for 15 seconds. Add 867 µl of Amplification Mix Diluent (R2) into one vial of concentrated Amplification Mix (R1). Vortex for 15 seconds then centrifuge briefly. One vial of reconstituted Amplification mix (R1+R2) is sufficient for 48 real-time PCR reactions. Prepare as many vials of reconstituted Amplification mix (R1+R2) as necessary (for example, 2 vials if 96 tests are to be processed). The reconstituted Amplification Mix (R1+R2) can be used immediately or aliquoted and stored at –20°C for up to 2 weeks.

7.3. Assay Procedure with Dx Prep System 7.3.1. Automated DNA extraction and PCR Setup Switch on the Dx Prep System then the computer. Ensure that the Dx Prep System front door is closed. 1. The Dx Prep Software starts automatically. Enter the user name and password in the “Login” window. 2. Open the Dx Prep System metal flap to load the instrument. 3. Load the samples: Note: Before loading the samples, make sure that the volume of specimen in the transport tube is at least 1 ml. a. Vortex each sample tube. b. Gently tap the sample tubes on the bench to bring down the drops. c. Open the sample tubes, discard the caps and swabs and place the tubes on the Dx Prep Sample Racks. d. Load the Dx Prep Sample Racks on the Dx Prep System following the instrument’s indications (flashing lights). e. If a Dx Prep Sample Rack is not full, confirm it by selecting it on the Dx Prep Software then click on the “Confirm rack” button. Note: As the Dx CT/NG/MG Auto Assay requires the presence of one Negative Control and one Positive Control per run, never load more than 94 samples per run. 4. Click on the “Extraction” then “Defining test orders” buttons on the Dx Prep Software. 5. Select “STD” on the “Analyte group(s)” list. 6. Ensure that the test assignment is correct for all samples. 7. Click on the “Defining the Run” button. 8. Select “STD” on the “Extraction” and “PCR setup” lists. Note: It is also possible to perform only the extraction step without PCR setup. If extraction only is done, the eluted samples in elution plate can be stored for up to 16 hours at room temperature or up to 4 days at 2-8°C. Please refer to the Dx Prep System User Manual for more information. 9. Click on the “Loading Plates” button. 10. Pull the Plates drawer and load the Dx Processing Plate, the Dx Elution Plate and the Dx PCR Plate on the Dx Prep System following Dx Prep Software’s indications. 11. Close the Plates drawer. The Dx Prep System automatically reads the plates barcodes. 12. Click on the “Loading Reagents” button. 13. Take one set of Dx Automated Extraction Assay reagents (Magnetic Beads, Binding Buffer, Proteinase K, Wash Buffer and Elution Buffer). Note: It is also possible to load two sets of reagents (for example a previously opened set + a new one). Please refer to the Dx Prep System User Manual for more information. 14. Vortex the MB bottle for 5 seconds and gently mix the PK, WB and EB vials by inversion. 15. Place the set of Dx Automated Extraction Assay reagents and the empty Internal Control (C1) vial on the Dx Prep Extraction Rack of the Dx Prep System, following Dx Prep System User Manual indications. 16. Load the Dx Prep Extraction Rack on the Dx Prep System following the instrument’s indications (flashing lights). 17. If the rack is not full, confirm it by selecting it on the Dx Prep Software then click on the “Confirm rack” button. 18. If the Dx Automated Extraction Assay or Dx CT/NG/MG Auto Assay kit lot is used for the first time, click on the “Kit Lot Input” button and enter the lot information by reading the corresponding lot card with the barcode reader. Please refer to the Dx Prep System User Manual for more information. 19. Vortex the Negative Control (C2) and Positive Control (C3) vials then centrifuge briefly. 20. Place the Negative Control (C2), the Positive Control (C3) and the reconstituted Amplification Mix (R1+R2) vials on the Dx Prep PCR rack of the Dx Prep System, following Dx Prep System User Manual indications. 21. Load the Dx Prep PCR rack on the Dx Prep System following the instrument’s indications (flashing lights). 22. If the rack is not full, confirm it by selecting it on the Dx Prep Software then click on the “Confirm rack” button. 23. Click on the “Loading Tips” button. 24. Pull the Tips drawer and if needed load the tips trays: if the quantity of tips already loaded is not sufficient, click on “Show loading selection”, load tips according to the Dx Prep Software suggestion, then click on “Accept suggestion”. Close the Tips drawer. Note: There are 3 tip sizes: 50 µl (yellow box), 300 µl (brown box) and 1100 µl (grey box). Ensure to load the box types as suggested by the Dx Prep software. Any modification from the suggestion must be validated by clicking on the corresponding location on the Dx Prep Software then enter the tip size and number of tips in the box.

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25. The Dx Prep Software then indicates the level of filling of the Solid and Liquid Wastes: a. If the level of filling of the Solid Waste is too high, a “Failed” message appears; replace it with a new one then click on the “Change bin” button. b. If the level of filling of the Liquid Waste is too high, a “Failed” message appears; empty it according to Good Laboratory Practices then put it back in its place and click on the “Empty waste” button. 26. Click on the “Final Check” button. 27. If the loading is correct, the Dx Prep Software then displays the word “Passed” in the “Run Status” window. 28. Close the metal flap and click on the “Run Status” button. 29. Click on the “Start run” button. Note: Never open the Dx Prep System front door during the run. The metal flap can be opened ONLY WHEN the “Unload samples” button becomes active on the Dx Prep Software and for a limited time. For more information, please refer to the Dx Prep System User Manual. 30. At the end of the run, open the Dx Prep System metal flap, then pull the Plates drawer. 31. Take the Dx PCR Plate from the Dx Prep System, put it on the bench and place the Dx 8-Cap Strips on the Dx PCR Plate. Note: If the last column of the Dx PCR Plate is empty, put the caps anyway in order to equilibrate the Dx Real-Time System heated lid. 32. Proceed to Real-time PCR Amplification/Detection (see 7.3.2) within 30 minutes following Dx PCR Plate preparation. 33. Unload Samples from the Dx Prep System: unload the sample racks, close the samples tubes with new caps (Capture Cap Set, Bio-Rad,cat. # 37017) and store or discard them according to Good Laboratory Practices. 34. Unload the Dx Prep Extraction and PCR racks, close each reagent with its corresponding cap and store them at 2-8°C, except the reconstituted amplification mix which must be stored at -20°C. Empty bottles and vials must be discarded according to Good Laboratory Practices. Unload and discard the Dx Processing Plate according to Good Laboratory Practices. If needed, the Dx Elution Plate can be stored for up to 16 hours at room temperature or up to 4 days at 2-8°C. If not, discard it according to Good Laboratory Practices.

7.3.2 Real-time PCR Amplification/Detection Please refer to the Dx Real-Time System User Manual for more detailed instructions 1. Switch on the computer then the Dx Real-Time System. Open the Dx Real-Time Software. Enter your user name and login 2. Click on the “Run” and click on “Open Lid”. 3. Load the Dx PCR Plate in the Dx Real-Time System and use the Dx Strip Cap Tool for seating the Dx 8-Cap Strips on the loaded Dx PCR Plate. The Dx Real-Time Software automatically recognizes the Dx PCR Plate and imports the plate map from the Dx Prep System.The corresponding plate map is displayed on the screen. 4. Click on the “Close lid” button then on the “Start run” button. 5. After completion of the real-time PCR reaction, remove the Dx PCR Plate from the Dx Real-Time System, place it in a sealable plastic bag and dispose according to Good Laboratory Practices. 6. Click on “Results” to have a view on the results

7.4 Assay procedure with NucliSENS® easyMAG® system 7.4.1 NucliSENS® easyMAG® extraction

System preparation - Switch on first the NucliSENS® easyMAG® system, and then the computer. - When the NucliSENS® easyMAG® system’s green light is on, enter loggin and password. - In the main Menu click on “Instrument » and the sub menu will appear automatically. Scan first the label barcode of one position and then the reagent barcode. Repeat the same for all reagents. NB: check that reagent caps vials are clean (without crystals). If not, clean with water or ethanol.

Define extraction orders - In the main menu click on "Routine" and the sub menu "define extraction order" will appear automatically - Create a list of specimens selecting the following settings: -Protocole: Generic 2.0 -Matrix: other -Sample volume: 0,500 ml for collection medium and 1 ml for urine -Elution volume: 60 µl -Type: primary -Enter sample ID for each sample

-Click on “ENTER “on the keyboard

Create an extraction working list - In the main menu "Routine", click on “doublele green arrow” button and then on the “sun” icon to create a run. - Enter the name of the extraction working list and validate the workflow proposed by default - Click on “OK” - Transfer samples from the extraction orders to the working list clicking on “>>>”.

Sample loading Dispense in the shuttle 500 µl of Negative Control (C2) and samples (500 µl for collection medium and 1 ml for urine) 8 [EN]

- Click on “shuttle” button. - Load the shuttle and scan first the barcode position and then the shuttle barcode - Insert the pipetting comb and check on the screen that comb positionning is Ok (green light)

Note: for the option of traceability, in the sub menu “barcode registration” (left bottom side of the screen): 1. click on “tube” button and enter the Internal Control barcode, 2. Then click on the “bead” button and scan the Magnetic Silica barcode.

Lysis and extraction process - Click on the icon “lysis buffer distribution” in the right side of the screen (loaded lysis mode) - During the lysis, prepare the premix Silica + Internal Control by adding 120 µl of Internal Control (C1) in the 600 µl of silica tube - Once the lysis is finished, add 60µl of the premix in each well (re-suspend the premix before pipetting) - Mix with the BIOHIT multipipette - Click on “the green arrow” button to start the extraction phase - When the run is finished transfer the extracted samples (eluate) from the shuttle to tubes or plate. Extracted samples can be stored for up to 16 hours at room temperature or 4 days at 2-8°C.

7.4.2 PCR Plate preparation 1. Reconstitute Amplification Mix vials as necessary following the procedure on chapter 7-2 (test number = n samples + 1 positive control + 1 negative control). 2. Take the required number of Dx 24-Well PCR Strips, place them on a holder and dispense carefully, with the repeat pipettor, 20µl of reconstituted Amplification mix (R1+R2) into each well. 3. Add 5µl of extracted DNA or extracted Negative Control or non-extracted Positive Control (C3) into the corresponding wells, following the established plate map. 4. Place the Dx 8-Cap Strips on the Dx 24-Well PCR Strips and close them firmly 5. Centrifuge the Dx 24-Well PCR Strips for 30 seconds at 400 g in order to avoid any bubble in the wells

7.4.3 Real-time PCR Amplification/Detection Please refer to the Dx Real-Time System User Manual for more detailed instructions 1. Switch on the computer then the Dx Real-Time System. Open the Dx Real-Time Software. Enter your user name and login Click on “Setup” Click on “create a new plate” and select the PCR kit name in “PCR kit”. Please refer to your local support for more information about the PCR kit name. Note: It is also possible to import test orders into the Dx Real-Time Software. Please refer to your local support for more information 2. Click on the “Run” button then click on “Open Lid”. 3. Load the 24-well Dx PCR Strips in the Dx Real-Time System and use the Dx Strip Cap Tool for seating the Dx 8-Cap Strips on the loaded 24-well Dx PCR Strips. 4. Click on the “Close lid” button then on the “Start run” button. 5. After completion of the real-time PCR reaction, remove the 24-well Dx PCR Strips from the Dx Real-Time System, place it in a sealable plastic bag and dispose according to Good Laboratory Practices. 6. Click on “Results” to have a view on the results

7.5. Calibration During each PCR cycle, at the annealing step, the Dx Real-Time System optical module measures the fluorescence obtained from each fluorophore, and the associated Dx Real-Time Software plots the fluorescence intensity versus number of cycles. At the end of the experiment, the software automatically analyzes results for all samples and controls. The Dx Real-Time Software uses a proprietary mathematical algorithm to determine the values of a set of mathematical parameters (among them Ceep and Cmin) for each PCR curve, which are then displayed in the results spreadsheet. Cmin is determined from a statistical method and corresponds to the last (integer) cycle of the background phase. The trend of the background phase is then subtracted from the whole amplification curve before proceeding to the Ceep determination. The estimation of Ceep is based on the assumption that the efficiency rate of the PCR reaction is maximal at the beginning of the reaction and decreases after a number of cycles which varies from one sample to another. The Ceep (Cycle of end of exponential phase) value corresponds to the cycle for which the PCR efficiency starts to decrease. Ceep is usually a few cycles after Cmin. The Ceep value depends on both the initial copy number and the efficiency rate of the PCR reaction. In ideal PCR conditions (no inhibitors of the reaction, high efficiency), the Ceep value is inversely proportional to the logarithm of the copy number of the target sequence in the sample before amplification: the higher the Ceep value is, the lower the initial copy number is. In the Dx CT/NG/MG Auto Assay, analysis of results is based on Ceep values. 9 [EN]

7.6. Quality Control

Positive and Negative Controls A Negative and aPositive controls must be included in each assay run in order to detect potential failure in specimen processing, amplification or detection steps. If Ceep values of the controls are out of their expected range, the Dx Real-Time Software invalidates the whole assay run and displays one of the following flags:

Negative Control: Flag Signification - Invalid_IC : Invalid internal control - CT_conta : contamination by CT DNA - NG_conta : contamination by NG DNA - MG_conta : contamination by MG DNA - CTNG_conta : contamination by CT and NG DNA - CTMG_conta : contamination by CT and MG DNA - NGMG_conta : contamination by NG and MG DNA - CTNGMG_conta : contamination by CT, NG and MG DNA

Positive Control: Flag Signification - CT_out : CT value out of range - NG_out : NG value out of range - MG_out : MG value out of range - CTNG_out : CT and NG values out of range - CTMG_out : CT and MG values out of range - NGMG_out : NG and MG values out of range - CTNGMG_out : CT, NG and MG values out of range

All samples and controls from the assay run must then be reprocessed starting from DNA extraction step.

Detection of Inhibition: Internal Control The Internal Control is added to each sample and to the Negative Control at the beginning of DNA extraction step, and is detected with a specific probe during the real-time PCR reaction. Specimens whose Internal Control’s Ceep value is above the expected value (meaning possibly inhibited) are interpreted by the Dx Real-Time Software as follows: • Any sample with a Ceep value ≤ 48 for a given target (CT, NG or MG) is reported as “positive” for this given target. • Any sample with no Ceep value or a Ceep value > 48 for a given target is reported as “failed” for this given target. All samples showing a failed result for a target must be reprocessed starting from the DNA extraction step.

7.7. Test Validation Criteria The assay run is valid if: • The Positive Control’s Ceep value is within the expected range for the three targets CT, NG and MG, and • The Negative Control has no Ceep value or a Ceep value > 48 for the three targets CT, NG and MG If the assay run is valid, the Dx Real-Time Software reports the run status as “Passed”. If not, the Dx Real-Time Software reports the run status as “Failed” If the run status is “Failed”, all test results in that run are reported as “invalid” and all corresponding samples and controls must be reprocessed starting from the DNA extraction step.

7.8. Calculation / Interpretation of the results Results calculation is performed by the Dx Real-Time Software, which automatically determines Ceep values for CT, NG and MG and for the Internal Control in samples and controls. If the test is valid, any sample (inhibited or not) with a Ceep value ≤ 48 for a given target (CT, NG or MG) is reported as “positive” for this given target by the Dx Real-Time Software. As an example, if a sample is positive for CT, the reported result is “CT_POS”. If the test is valid, any non-inhibited sample with no Ceep value or a Ceep value > 48 for a given target (CT, NG or MG) is reported as “negative” for this given target by the Dx Real-Time Software. As an example, if a sample is negative for CT, the reported result is “CT_neg”. If the test is valid, any inhibited sample with no Ceep value or a Ceep value > 48 for a given target (CT, NG or MG) is interpreted as “failed” for this given target by the Dx Real-Time Software, which then reports the result as “Failed” with the flag “Invalid_IC”. The corresponding sample must therefore be reprocessed starting from the DNA extraction step. NOTE: If the inhibited sample is a urine sample, it is recommended to freeze it one night at -20°C before reprocessing, as freezing can eliminate inhibitors in urine. If after reprocess the sample is still inhibited, it is recommended to perform a new test with a newly collected sample.

8. TEST LIMITATIONS • Optimal performance of this test depends directly on the quality of specimens. It is therefore important to comply with indications given in the chapter “Specimens”. 10 [EN]

• The assay should be performed only on indicated sample types. Other sample types have not been validated. • The presence of blood, mucus, some spermicidal agents and treatments for vaginal conditions may interfere with the real- time PCR reaction. For more information on interfering substances, please refer to the “Performance characteristics” chapter. • The potential effects of other factors, such as vaginal discharge, use of tampons, vaginal douching, have not been determined. • Use of this assay is limited to personnel who have been trained on the use of the Dx CT/NG/MG Auto Assay, Dx Prep System, Dx Automated Extraction Assay, Dx Real-Time System , Dx Real-Time Software and NucliSENS® easyMAG® system. • A negative result does not exclude the possibility of infection because results are dependent on several variables. An improper specimen collection or handling, the presence of inhibitors or a technical error can lead to a false result. • The targeted sequence in pile gene is shared with some of the Neisseria meningitidis strains leading to potential false positive NG results in rare circumstances. • The collection of vaginal, anorectal or urine samples is not intended to replace cervical exams and endocervical samples for diagnosis of female urogenital infections. Patients may have , urethritis, urinary tract or vaginal infections due to other causes or concurrent infections with other . • The Dx CT/NG/MG Auto Assay is not intended for use in suspected sexual abuse evaluation nor other medico-legal indications. • Therapeutic success or failure cannot be determined by the Dx CT/NG/MG Auto Assay since DNA can persist after appropriate therapy. • As with any diagnostic test, results from the Dx CT/NG/MG Auto Assay should be interpreted in conjunction with other laboratory and clinical findings.

9. EXPECTED VALUES The prevalence of positive samples for C. trachomatis, N. gonorrhoeae and M. genitalium in the populations studied depends on the clinical picture, age, risk factors, sex and test method. The prevalences observed with the Dx CT/NG/MG Auto Assay during clinical studies in the two sites are shown in the tables below:

Table 1: CT/NG/MG prevalence Site 1

Chlamydia Neisseria Mycoplasma Population trachomatis gonorrhoeae genitalium Gender N site 1 CT + NG + MG + N % N % N % Asymptomatic ♀ 141 17 12.1% 1 0.7% 6 4.3% Symptomatic ♀ 8 2 25.0% 0 0.0% 1 12.5% Symptomatic ♂ 37 8 21.6% 2 5.4% 2 5.4% Asymptomatic or ♀ 308* 16 5.2% 1 0.3% 4 1.3% Symptomatic Asymptomatic or ♂ 162 19 11.7% 15 9.3% 6 3.7% Symptomatic Total ♀+ ♂ 656 62 9.5% 19 2.9% 19 2.9% * 1 patient positive for CT and failed for NG and MG

Table 2: CT/NG/MG prevalence Site 2

Chlamydia Neisseria Mycoplasma trachomatis gonorrhoeae genitalium Population Site 2 Gender N CT + NG + MG + N % N % N %

Sampling ♀ 75 2 2.7% 0 0.0% 1 1.3% center 1 ♂ 6 1 16.7% 1 16.7% 0 0.0% Prospective Sampling ♀ 36 1 2.8% 0 0.0% 0 0.0% Asymptomatic center 2 or Symptomatic ♂ 1 0 0.0% 0 0.0% 0 0.0% sampling ♀ 30 1 3.3% 1 3.3% 0 0.0% center 3 ♂ 4 2 50.0% 1 25.0% 0 0.0% Total Prospective ♀+ ♂ 152 7 4.6% 3 2.0% 1 0.7%

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Table 3: CT/NG/MG prevalence Sites 1 + 2

Chlamydia Neisseria Mycoplasma POPULATION trachomatis gonorrhoeae genitalium Sex N Site 1 & Site 2 CT + NG + MG + N % N % N % Asymptomatic ♀ 141 17 12.1% 1 0.7% 6 4.3% Symptomatic ♀ 8 2 25.0% 0 0.0% 1 12.5% Symptomatic ♂ 37 8 21.6% 2 5.4% 2 5.4% Asymptomatic or ♀ 449 20 4.5% 2 0.4% 5 1.1% Symptomatic Asymptomatic or ♂ 173 22 12.7% 17 9.8% 6 3.5% Symptomatic

Total ♀+ ♂ 808 69 8.5% 22 2.7% 20 2.5%

10. PERFORMANCE CHARACTERISTICS * * performances have been obtained initially with automated extraction performed on Dx Prep System. However equivalent performances have been obtained with extraction on NucliSENS® easyMAG® system

10.1. Precision measurement

10.1.1 Repeatability and Intermediate Precision According to the CLSI instructions EP5A2, two sample panels (one in Urine and one in Dx Collection System transport medium) constituted of negative samples and CT/NG/MG positive samples at different amounts (low, medium and high) were tested for intermediate precision study in 4 extraction replicates by 1 operator. This study was done on 2 Dx Automated Extraction Assay lots with 2 Dx Prep Systems on a period of 5 days with 1 run per day. The assay continued for 15 additional days with 2 Dx Automated Extraction Assay lots and with 1 Dx Prep System and 1 operator. The Nested ANOVA function was used to measure the variance components for each condition (batch - instrument - day -replicate).

The results are recorded in tables 4, 5, and 6. Table 4: Chlamydia trachomatis: Total, between run, between day precision results per panel member- Ceep value of positive samples

CT Within Run Between Day Between Lot TOTAL Extraction Panel Sample N Mean SD CV% SD CV% SD CV% SD CV% Lot A 80 34.4 0.46 1.3% 0.33 0.9% Low 0.52 1.5% 0.63 1.8% B 80 34.9 0.18 0.5% 0.40 1.1% Urine A 80 31.4 0.69 2.2% 0.49 1.6% Medium 0.54 1.7% 0.75 2.4% B 80 31.7 0.23 0.7% 0.44 1.4% A 80 27.3 0.16 0.6% 0.21 0.8% High 0.38 1.4% 0.43 1.6% B 80 27.5 0.22 0.8% 0.38 1.4% A 80 33.6 0.45 1.3% 0.54 1.6% Low 0.36 1.1% 0.69 2.1% B 80 33.7 0.47 1.4% 0.50 1.5% Transport A 80 30.0 0.31 1.0% 0.35 1.2% Medium 0.32 1.1% 0.42 1.4% Medium B 80 30.1 0.16 0.5% 0.33 1.1% A 80 26.7 0.17 0.6% 0.41 1.5% High 0.36 1.3% 0.39 1.5% B 80 26.7 0.14 0.5% 0.25 0.9%

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Table 5: Neisseria gonorrhoeae: Total, between run, between day precision results per panel member- Ceep value of positive samples

NG Within Run Between Day Between Lot TOTAL Extraction Panel Sample N Mean SD CV% SD CV% SD CV% SD CV% Lot A 80 35.3 0.43 1.2% 0.93 2.6% Low 0.99 2.8% 1.07 3.0% B 80 36.0 0.38 1.1% 0.49 1.4% Urine A 80 32.2 0.67 2.1% 0.65 2.0% Medium 0.79 2.4% 0.93 2.9% B 80 32.6 0.23 0.7% 0.60 1.9% A 80 27.1 0.16 0.6% 0.52 1.9% High 0.74 2.7% 0.77 2.8% B 80 27.6 0.23 0.8% 0.53 1.9% A 80 30.6 0.22 0.7% 0.19 0.6% Low 0.20 0.6% 0.33 1.1% B 80 30.8 0.23 0.8% 0.27 0.9% Transport A 80 27.1 0.42 1.5% 0.58 2.2% Medium 0.38 1.4% 0.54 2.0% Medium B 80 27.1 0.20 0.7% 0.18 0.7% A 80 23.4 0.17 0.7% 0.63 2.7% High 0.48 2.0% 0.55 2.3% B 80 23.3 0.34 1.4% 0.21 0.9%

Table 6: Mycoplasma genitalium: Total, between run, between day precision results per panel member- Ceep value of positive samples

MG Within Run Between day Between Lot TOTAL Extraction Panel Sample N Mean SD CV% SD CV% SD CV% SD CV% Lot A 80 34.8 0.57 1.6% 1.16 3.3% Low 1.33 3.8% 1.4 4.1% B 80 35.9 0.60 1.7% 0.71 2.0% Urine A 80 31.3 0.96 3.1% 0.71 2.3% Medium 0.94 3.0% 1.21 3.8% B 80 32.0 0.46 1.4% 0.71 2.2% A 80 26.3 0.31 1.2% 0.35 1.3% High 0.64 2.4% 0.74 2.8% B 80 26.9 0.43 1.6% 0.45 1.7% A 80 30.8 0.66 2.1% 0.28 0.9% Low 0.00 N/A 0.69 2.2% B 80 30.9 0.56 1.8% 0.34 1.1% Transport A 80 26.7 0.63 2.3% 0.15 0.6% Medium 0.28 1.0% 0.63 2.3% Medium B 80 26.9 0.49 1.8% 0.23 0.8% A 80 23.2 0.28 1.2% 0.20 0.9% High 0.19 0.8% 0.36 1.5% B 80 23.2 0.33 1.4% 0.15 0.6%

10.1.2 Inter-Systems Precision An inter-systems precision study was assessed within 3 to 5 days on up to 4 Dx Prep Systems with 2 to 3 Dx Real-Time Systems (RTS) using 3 Dx Automated Extraction Assay lots and 1 Dx CT/NG/MG Auto Assay batch. For the inter-Dx Prep System precision study, the one way ANOVA method was used. All acceptance criteria were correct: negative sample was negative, positive samples were positive; CVs (calculated on Ceep value) on positive samples were <= 3% for repeatability, and intermediate precision, and <= 5% for total precision, inter-lot and inter-systems precision.

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Table 7: CT, NG and MG: Inter systems precision results per panel member. Sample ID Replicates Mean Ceep SD CV CT Negative 22 Neg NA NA CT low 22 32.6 0.25 0.78% CT Medium 22 30.0 0.24 0.79% CT High 22 24.8 0.35 1.40% NG Negative 22 Neg NA NA NG low 22 31.3 0.17 0.55% NG Medium 22 28.4 0.25 0.89% NG High 22 22.6 0.43 1.92% MG Negative 22 Neg NA NA MG low 22 31.7 0.68 2.14% MG Medium 22 28.4 0.54 1.89% MG High 22 23.0 0.46 2.02%

10.2. Clinical performance Clinical performance characteristics of the Dx CT/NG/MG Auto Assay were established during a study performed in two clinical sites in France. 1102 symptomatic and asymptomatic patients consulting in STI (Sexually Transmitted Infection) prevention centers and in anonymous free screening centers were recruited for this study. 16 patients did not meet the previously defined inclusion criteria and were excluded. The 1086 other patients were included in the study (723 women and 363 men). A total of 1086 patients with 1255 different specimens were analyzed: Retrospective study: 278 samples • 125 female first-catch urine specimens • 153 male first-catch urine specimens Prospective study: 977 samples • 141 female self vaginal samples, • 364 female endocervical specimens, • 49 female vaginal / endocervical samples • 32 female vaginal samples, • 130 female first-catch urine samples, • 1 female anorectal sample, • 9 female unknown swab origin samples • 158 male first-catch urine samples, • 36 male first-catch urine with prostatic massage, • 6 male urethral samples, • 51 male anorectal samples. Among these specimens: • Women patients with multiple sites of sampling: 457 women, of whom 121 provided endocervical swab and urine specimens, and 7 provided vaginal and urine samples. • Men patients with multiple sites of sampling: 199 men, of whom 36 provided both first-catch urine (FCU) and post-prostatic massage first-catch urine specimens, 4 provided FCU and anorectal swab specimens and 1 provided FCU and urethral swab samples.

All the specimens were collected using the Bio-Rad’s Dx Collection Systems (Male and female collection system kits ref 37013 and 37012). The performance of the Dx CT/NG/MG Auto Assay for the target C. trachomatis was established by comparison with three EC registered real time PCR tests (1 PCR test on the site 1 and 2 others tests on the site 2). The performance of the Dx CT/NG/MG Auto Assay for the target N. gonorrhoeae was established by comparison with three EC registered real time PCR tests (1 PCR test on the site 1 and 2 others tests on the site 2) or with culture. The performance of the Dx CT/NG/MG Auto Assay for the target M. genitalium was established by comparison with an in-house PCR test using the TaqMan technique according to the protocol described by Jensen. Only the specimens that tested positive with the Dx CT/NG/MG Auto Assay were tested with the in-house PCR test.

10.2.1 Chlamydia trachomatis performance 1255 different specimens from the two sites were analyzed using the Dx CT/NG/MG Auto Assay. Among them, 825 samples were tested with Site 1 PCR CT/NG test assay, 311 with the first Site 2 PCR CT/NG test assay and 119 with the second Site 2 PCR CT/NG test assay. The three comparative assay tests were declared Reference assays. 7 specimens (1 self vaginal, 3 endocervical swabs, 1 vaginal swab, 1 female first-catch urine, and 1 male anorectal) were excluded from the calculation because of empty tube or insufficient volume. 14 [EN]

Table 8: CT comparative results

Reference CT Reference CT Positive or Equivocal* or Negative Failed Beyond cutoff** Total Inv IC Dx CT Auto Dx CT Auto Dx CT Auto Dx CT Auto specimens Negative Positive Negative Positive ♀ self vaginal 121 1(a) 0 15+1* 2 140 ♀ endocervical swab 345 0 0 15 1 361 ♀ vaginal 27 1(b) 0 3 0 31 ♀ vaginal / endocervical swab 47 0 0 2 0 49 ♀ first-catch urine 195 1(c) 1(d) 57 0 254 ♀ anorectal 1 0 0 0 0 1 ♀ not communicated 9 0 0 0 0 9 ♂ first-catch urine 229 0 0 82 0 311 ♂ FCU after Prostatic massage 35 0 0 1 0 36 ♂ urethral 4 0 0 1+1** 0 13 ♂ anorectal 36 0 0 13 1 43 total 1049 3 1 191 4 1248 *Equivocal: The Site 1 PCR CT/NG test equivocal sample was considered as positive: this sample from self vaginal population was found equivocal for CT with the Site 1 PCR CT/NG test and positive for CT with the Bio-Rad Dx CT/NG/MG Auto Assay. **Beyond cutoff: The Site 2 PCR CT/NG test beyond cutoff sample was considered as low positive: this sample from urethral population was found beyond cutoff for CT with the Site 2 PCR CT/NG test and positive for CT with the Bio-Rad Dx CT/NG/MG Auto Assay.

Diagnostic Specificity The samples a, b and c were excluded from the specificity calculation because these specimens were true positive samples. Specimens a and b were found true CT positive with Site 1 PCR CT confirmatory assay and the sample c was confirmed CT positive with the associated endocervical swab samples. The relative specificity of the Dx CT/NG/MG Auto Assay with respect to the 3 reference PCR assays is 1049/1049 = 100% CI 95% [99.7% – 100%].

Diagnostic Sensitivity The sample d was reproducible and confirmed discrepant with the Site 1 PCR CT/NG test. Moreover this sample was associated to an endocervical swab specimen that was tested positive with the Bio-Rad Dx CT/NG/MG Auto Assay and the Site 1 PCR CT confirmatory assay. This patient was declared real positive. The relative sensitivity of the Dx CT/NG/MG Auto Assay with respect to the 3 reference PCR assays is 191/192 = 99.5% CI 95% [97.1% – 100%].

10.2.2 Neisseria gonorrhoeae performance 1255 different specimens from the two sites were analyzed using the Dx CT/NG/MG Auto Assay. Among them, 825 samples were tested with Site 1 PCR CT/NG test assay, 311 with the first Site 2 PCR CT/NG test assay and 119 with the second Site 2 PCR CT/NG test assay. The three comparative assay tests were declared Reference assays. 6 specimens (3 endocervical, 1 vaginal, 1 female first-catch urine, and 1 male anorectal) were excluded from the calculation because of empty tube or insufficient volume.

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Table 9: NG comparative results

Reference NG PCR test Reference NG PCR test Failed Negative Positive or Equivocal Total Inv IC Dx NG Auto Dx NG Auto Dx NG Auto Dx NG Auto specimens negative Positive negative Positive ♀ self vaginal 138 0 0 1 2 141 ♀ endocervical swab 358 0 0 2 1 361 ♀ vaginal 31 0 0 0 0 31 ♀ vaginal / endocervical swab 49 0 0 0 0 49 ♀ first-catch urine 245 0 1(d) 7 1 254 ♀ anorectal 1 0 0 0 0 1 ♀ not communicated 9 0 0 0 0 9 ♂ first-catch urine 278 1(a) + 1(e) 0 31 0 311 ♂ FCU after prostatic massage 35 0 0 1 0 36 ♂ urethral 4 0 0 2 0 6 ♂ anorectal 38 1(b) 1*(c) 9 1 50 total 1186 3 2 53 5 1249 *Equivocal: The equivocal sample was considered as positive. This sample (c) from anorectal population was found equivocal for NG with Site 1 PCR CT/NG test and negative for NG with the Bio-Rad Dx CT/NG/MG Auto Assay.

Diagnostic Specificity 3 samples were found discrepant between the Dx CT/NG/MG Auto Assay and the reference assay: • Sample (a) was not reproducible with the Dx CT/NG/MG Auto Assay and was re-tested negative using the Site 1 PCR NG confirmatory assay. Moreover, the culture was found gonococci negative. • Sample (b) was reproducibly positive when retested with the Dx CT/NG/MG Auto Assay and was confirmed negative after re-test with the Site 1 PCR NG confirmatory assay. The result of the culture was gonococci negative. No sample was found positive for meningococcal bacteria during the evaluation. • Sample (e) was not reproducible with the Dx CT/NG/MG Auto Assay and was re-tested positive with the Site 2 PCR NG assay. This sample with low rate of N. gonorrhoeae was at the limit of detection for the Site 2 PCR NG and the Dx CT/NG/MG Auto Assay. The relative specificity of the Dx CT/NG/MG Auto Assay with respect to the 3 reference PCR assays is 1186/1189 = 99.8% CI 95% [99.3% – 100%].

Diagnostic Sensitivity 2 samples were found discrepant between the Dx CT/NG/MG Auto Assay and the reference assay: • Sample (c) was reproducible and confirmed negative discrepant with the Site 1 PCR NG equivocal results (confirmed equivocal with the Site 1 PCR NG Assay). The result of the culture was gonococci negative. The Dx CT/NG/MG Auto Assay result is in agreement with the culture. • Sample (d) was reproducible and confirmed negative discrepant with the Site 2 PCR NG first result but was re-tested negative with the Site 2 PCR NG assay. This sample with low rate of N. gonorrhoeae was at the limit of detection of the Site 2 PCR NG assay. The relative sensitivity of the Dx CT/NG/MG Auto Assay with respect to the 3 reference PCR assays is 53/55 = 96.4% CI 95% [87.5% – 99.6%].

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10.2.3 Neisseria gonorrhoeae: Comparison to the culture A total of 533 samples (201 from male and 332 from female) were compared to the results obtained for the culture of Neisseria gonorrhoeae.

Table 10: NG comparative results with culture

Culture NG Culture NG

Negative Positive Failed - Total Dx NG Auto Dx NG Auto Dx NG Auto Dx NG Auto Invalid-IC Specimens negative Positive negative Positive

♀ self vaginal 0 0 0 0 2 2 ♀ endocervical swab 207 0 0 0 1 208 ♀ vaginal 1 0 0 0 0 1 ♀ first-catch urine 120 0 0 0 1 121 ♀ anorectal 0 0 0 0 0 0 ♂ first-catch urine 112 1(a) 0 7 0 120 ♂ FCU after prostatic 36 0 0 0 0 36 massage ♂ urethral 1 0 0 1 0 2 ♂ anorectal 33 1(b) 0 8 1 43 total 510 2 0 16 5 533

Diagnostic Specificity 2 samples were found discrepant with culture (samples a and b) and also found discrepant with the reference PCR assay (see specificity discrepant table above). The relative specificity of the Dx CT/NG/MG Auto Assay with respect to the culture is 510 / 512 = 99.6% CI 95% [98.6 -100%].

Diagnostic Sensitivity All the positive samples found with the Dx CT/NG/MG Auto Assay were also positive in culture. The relative sensitivity of the Dx CT/NG/MG Auto Assay with respect to the culture is 16/16 = 100 % CI 95% [79.4 - 100%].

10.2.4 Mycoplasma genitalium performance On 2 sites, 35 samples were found positive for Mycoplasma genitalium with the Bio-Rad Dx CT/NG/MG Auto Assay: 21 specimens from 19 patients on Site 1 and 14 specimens from 14 patients on Site 2. 2 specimens (1 endocervical, 1 vaginal) were excluded from the calculation because of empty tube or insufficient volume. A total of 36 specimens from these 33 patients were sent to Site 3 for analysis with the in-house TaqMan MG PCR test.

Table 11: Bio-rad Dx MG Auto results

Dx MG Auto Dx MG Auto specimens Failed Invalid IC Total negative Positive

♀ self vaginal 133 6 2 141 ♀ endocervical swab 358 4 1 363 ♀ vaginal 31 0 0 31 ♀ vaginal / endocervical swab 48 1 0 49 ♀ first-catch urine 244 10 1 255 ♀ anorectal 1 0 0 1 ♀ not communicated 9 0 0 9 ♂ first-catch urine 301 10 0 311 ♂ FCU after Prostatic massage 36 0 0 36 ♂ urethral 6 0 0 6 ♂ anorectal 46 4 1 51 total 1213 35 5 1253

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Table 12: MG comparison results

Dx MG Auto In House MG sites Specimens Total Positive Negative Positive Negative

Urine 7 0 7 0 7 Site 1 Swab 14 1* 9 6 15 Urine 13 0 12 1 13 Site 2 Swab 1 0 0 1 1 Total 35 1 28 8 36

Three patients provided 2 different samples (swab and urine) which were reproducibly MG-positive except for 1 swab which was found negative even after retest.

Comparison with the “In house” method Among the 36 specimens tested with the Dx CT/NG/MG Auto Assay, 35 specimens were found positive; 28 of them were confirmed positive with the “in house” Mycoplasma genitalium Taqman Method realized at Site 3.

The agreement between the Dx CT/NG/MG Auto Assay and the Mycoplasma genitalium Taqman Method is equal to 27/36 (75%).

The samples not found positive with the Taqman method were low concentrated MG samples; the default of detection was not due to PCR inhibitors.

The positive agreement was equal to 96.4% (=27/28) CI 95% [81.7 - 100%] with 1 specimen found negative with the Dx CT/NG/MG Auto Assay and positive close to the limit of detection with the Mycoplasma genitalium Taqman PCR assay.

Coinfection: Among these 35 specimens found MG-positive with the Dx CT/NG/MG Auto Assay, 12 samples were also tested CT positive with the Dx CT/NG/MG Auto Assay and reference CT PCR assays which leads us to think that these patients were considered as at-risk co infected patients.

10.2.5 Retrospective study (Site 2) 278 first-catch urine (FCU) frozen samples were tested retrospectively using the Dx CT/NG/MG Auto Assay.

Table 13: C. trachomatis

Reference CT PCR Reference CT PCR Negative Positive Total Dx CT Auto Dx CT Auto Dx CT Auto Dx CT Auto specimens Negative Positive Negative Positive

♀ first-catch urine 73 0 0 52 125 ♂ first-catch urine 87 0 0 66 153 TOTAL 160 0 0 118 278

Table 14: N. gonorrhoeae

Reference NG PCR Reference NG PCR

Negative Positive Total Dx NG Auto Dx NG Auto Dx NG Auto Dx NG Auto specimens Negative Positive Negative Positive

♀ first-catch urine 117 0 1 7 125 ♂ first-catch urine 128 1 0 24 153 TOTAL 245 1 1 31 278

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Table 15: M. genitalium,

Dx MG Auto Dx MG Auto specimens Total Negative Positive ♀ first-catch urine 118 7 125 ♂ first-catch urine 147 6 153 TOTAL 245 13 278

For C. trachomatis, 118/118 specimens were concordant positive with the Site 2 PCR CT/NG assay.

For N. gonorrhoeae, 31/32 specimens were concordant positive with the Site 2 PCR CT/NG assay

For M. genitalium, 13 specimens were found positive with the Dx CT/NG/MG Auto Assay and 12 samples were found in agreement with Mycoplasma genitalium Taqman PCR assay of Site 3.

10.3. Limit of detection The limit of detection (LOD) has been determined using the probit analysis retaining the dilution corresponding to a positive level of 95%. A dilution series was prepared for each analyte and tested 24 times. The limit of detection was determined using the probit analysis as the lowest dilution value leading to 95% of positive response. - The limit of detection for C.trachomatis was estimated at: • 45 copies of DNA/ml in transport medium, with a 95% confidence interval of 31 to 189 copies of DNA/ml. This corresponds to 6.4 CFU/ml. • 50 copies of DNA/ml in urine, with a 95% confidence interval of 41 to 84 copies of DNA/ml. This corresponds to 7 CFU/ml. - The limit of detection for N. gonorrhoeae was estimated at: • 26 CFU/ml in transport medium, with a 95% confidence interval of 21 to 45 CFU/mL. • 1694 CFU/ml in urine, with a 95% confidence interval of 1232 to 3191 CFU/mL. - The limit of detection for M. genitalium was estimated at: • 79 Genome equivalent/ml (Geq/ml) in transport medium, with a 95% confidence interval of 59 to 151 Geq/ml. This corresponds to 79 CFU/ml. • 615 Geq/ml in urine, with a 95% confidence interval of 436 to 1886 Geq/ml. This corresponds to 615 CFU/ml. These limits of detection were confirmed by testing 17 serovars of C. trachomatis (A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L3, LGV2, nv CT, L2a), 21 N. gonorrhoeae strains (2013, 2014, 2015, 2016, 2017, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2028, 2029, 2030, 2031, 2032, 2033, 2036, 2037, 2050) isolated from patients and 6 M. genitalium strains (M30, R32G, TW10- 5G, TW10-6G, TW48-5G, UTMB).

10.4. Analytical specificity

10.4.1 Cross-reactivity Study The analytical specificity of the Dx CT/NG/MG Auto Assay was evaluated on a panel of assumed non-chlamydial, non- gonococcal, and non-genitalium pathogens, virus and other microorganisms representing >=5 diseases and >= 120 pathogens or microorganisms. All the strains except 13 were cultivated, calibrated at 106 CFU/ml in transport medium + urine, extracted and tested with the Dx CT/NG/MG Auto Assay. For the 13 non-cultivated strains, commercial DNA at 0.636 ng/ml (equivalent to 106 copies/ml) was directly tested with the Dx CT/NG/MG Auto Assay. Two strains, Neisseria meningitidis serogroup C and serogroup D, gave a positive result with the Neisseria gonorrhoeae target and led to obtain an assay specificity for NG equal to 98.3% IC 95% [94.1% - 99.8%] and 100% for CT and MG.

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Table 16: Cross reactivity pathogens

Achromobacter xerosis Gemella haemolysans Neisseria subflava Acinetobacter calcoaceticus Haemophilus ducreyi Paracoccus denitrificans Acinetobacter lwoffi anaerobius Actinomyces israelii Kingella kingae Plesiomonas shigelloides Actinomyces (Arcanobacterium) Klebsiella oxytoca Propionibacterium acnes pyogenes (pyogenes) Aerococcus viridans Klebsiella pneumoniae subsp. pneumoniae Proteus mirabillis Aeromonas hydrophila Lactobacillus acidophilus Proteus vulgaris Agrobacterium (rhizobium) radiobacter Lactobacillus brevis Providencia stuartii (radiobacter) Alcaligenes faecalis Lactobacillus jensenii Pseudomonas aeruginosa subtilis Lactobacillus delbrueckii subsp.lactis Pseudomonas fluorescens Bacteroides fragilis Lactococcus lactis subsp. cremoris Pseudomonas putida Bacteroides ureolyticus Legionella pneumophila Rahnella aquatilis Bifidobacterium adolescentis Leuconostoc mesenteroides Rhodospirillum rubrum Bifidobacterium brevi monocytogenes Ruminococcus productus Brevibacterium linens Micrococcus luteus Campylobacter jejuni Moraxella lacunata lacunata Salmonella enterica Candida albicans Moraxella osloensis osloensis Salmonella enterica serovar Typhimurium Candida glabrata Moraxella (Branhamella) catarrhalis Serratia marcescens (catarrhalis) Candida parapsilosis Morganella morganii Candida tropicalis Mycobacterium gordonae Staphylococcus epidermidis Chromobacterium violaceum Mycoplasma fermentans fermentans Staphylococcus saprophyticus Citrobacter freundii hominis agalactiae perfringens pneumoniae Corynebacterium genitalium Neisseria meningitidis Corynebacterium xerosis Neisseria meningitidis Serogroup A (carie) Cryptococcus neoformans Neisseria meningitidis Serogroup B Deinococcus radiodurans Neisseria meningitidis Serogroup C Derxia gummosa Neisseria meningitidis Serogroup D Streptococcus salivarius Eikenella corrodens Neisseria meningitidis Serogroup W135 Enterobacter aerogenes Neisseria meningitidis Serogroup Y Streptomyces griseinus Enterobacter cloacae Neisseria cinerea Vibrio parahaemolyticus avium Neisseria (Bergeriella) denitrificans Weissella paramesenteroides Neisseria elongata (3) Yersinia enterocolitica Neisseria flava CMV Erwinia (Pantoea) herbicola Neisseria flavescens(2) HBV (agglomerans) Erysipelothrix rhusiopathiae Neisseria lactamica(9) HIV Neisseria mucosa (3) HSV1 Flavobacterium (Elizabethkingia) Neisseria perflava HSV2 meningosepticum (meningoseptica) Fusobacterium nucleatum Neisseria polysaccharea HPV16 Gardnerella vaginalis Neisseria sicca (3) HPV18

10.4.2 Interference Study According to the CLSI instruction EP07-A2, potential interferences on the Dx CT/NG/MG Auto Assay were evaluated with endogenous or exogenous substances that may be present in swab and/or urine samples. Thirteen substances were diluted to various final concentrations and added to a swab pool in Dx Collection System transport medium or to a urine pool containing the 3 targets CT, NG and MG at a concentration of about 3 times the limit of detection, as well as to a swab pool in transport medium or to a urine pool not containing any of the 3 targets. Each condition was tested in 6 replicates. The following substances were tested:

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Table 17: Interfering substances

Interfering Substance Concentration Matrix Bilirubin 1 mg/ml Urine Blood 1% v/v Urine Blood 0.1% v/v Transport medium Glucose 1 mg/ml Urine pH 4 Urine pH 9 Urine Proteins ( serum albumin) 1% Urine Betadine 0.1% Transport medium Miconazole (anti-bactericide and antifungal) 1% Transport medium Isoconazole (anti-bactericide and antifungal) 5% Transport medium Trophicrème (vaginal cream) 5% Transport medium Aciclovir (Anti herpetic) 5% Transport medium Lubrifiant Gel KY (Sensilube) 5% Transport medium Progesterone 1% Transport medium

None of the potentially interfering compounds used at the concentration recorded in the table above with the Dx CT/NG/MG Auto Assay, show significant discordance and impact on the interpretation as well as on the Ceep results.

10.5. Competition study between targets In order to challenge the performance of the Dx CT/NG/MG Auto Assay in samples containing low amounts of CT, NG or MG in the presence of high amounts of the opposite analytes, a study using urine and transport medium specimens spiked with low and high concentrations of each of the three micro- was conducted. Samples were prepared in order to contain low level of analytes (around 3 times the LoD) or high level of analytes (CT, and/or NG and/or MG). The percentage of positive responses was analyzed on tests performed in triplicate over 3 days. No competition was found between CT and NG and MG targets for low and high rate of positive samples.

11. BIBLIOGRAPHY REFERENCES 1. WHO. Global prevalence and incidence of selected curable sexually transmitted infections. Overview and estimates. Geneva: WHO; 2001. 2. Cates W Jr, Rolfs RT Jr, Aral SO. Sexually transmitted diseases, pelvic inflammatory disease, and infertility: an epidemiologic update. Epidemiol Rev 1990; 12: 199–220. 3. Stamm WE. Chlamydia trachomatis infections of the adult. In: Holmes KK, Sparling PF, Mardh PA, eds. Sexually Transmitted Diseases, 3rd ed. New York: McGraw-Hill, 1999. 4. Centers for Disease Control and Prevention. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections. MMWR 2002; 51 (No. RR-15) : 1-27. 5. Taylor-Robinson D. Mycoplasma genitalium: an update. Int J STD AIDS 2002; 13: 145-151. 6. Simms I, Eastick K, Mallinson et al. Associations between Chlamydia trachomatis, Mycoplasma genitalium, and pelvic inflammatory disease. Sex Transm Infect 2003; 79: 154-156. 7. Cohen CR, Manhart LE, Bukusi EA et al. Associations between Mycoplasma genitalium and acute . Lancet 2002; 359: 765-766. 8. Clausen HF, Fedder J, Drasbek M et al. Serological investigation of Mycoplasma genitalium in infertile women. Human Reprod 2001; 16: 1866-1874. 9. Falk L, Fredlund H, Jensen JS. Symptomatic urethritis is more prevalent in men infected with Mycoplasma genitalium than with Chlamydia trachomatis. Sex Transm Infect 2004; 80: 289-293. 10. Moi H, Reinton N, Moghaddam A. Mycoplasma genitalium in women with lower genital tract . Sex Transm Infect 2009; 85: 10-14. 11. Moi H, Reinton N, Moghaddam A. Mycoplasma genitalium is associated with symptomatic and asymptomatic non- gonococcal urethritis in men. Sex Transm Infect 2009; 85: 15-18. 12. Haggerty CL, Totten PA, Astete SG, Lee S, Hoferka SL, Kelsey FL, Ness RB. Failure of cefoxitin and to eradicate endometrial Mycoplasma genitalium and the consequence for clinical cure of pelvic inflammatory disease. Sex Transm Infect 2008; 84: 338-342. 13. Falk L, Fredlund H, Jensen JS. Signs and symptoms of urethritis and cervicitis among women with or without Mycoplasma genitalium or Chlamydia trachomatis infection. Sex Transm Infect 2005; 81: 73-78. 14. Ripa T, Nilsson PA. A Chlamydia trachomatis strain with a 377-bp deletion in the cryptic plasmid causing false-negative nucleic acid amplification tests. Sex Transm Dis 2007:34:255-56. 15. Jensen J S, Björnelius E, Dohn B, Lidbrink P. Use of TaqMan 5’ Nuclease Real-Time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a Sexually Transmitted Disease clinic. J.Clin. Microbiol. 2004 42: 683 – 692.

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The purchase of this product allows the purchaser to use it for the performance of diagnostic services for human in vitro diagnostics. No general patent or other license of any kind other than this specific right of use from purchase is granted hereby to the purchaser. Any brand name that should appear in this product insert belongs to its respective owners.

* The Dx CT/NG/MG Auto Assay is CE-IVD marked for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium. CT parameter, as listed in Annex IIB of the 98/79/EC directive, is the only parameter evaluated by the 0459 notified body in accordance with Annex IV.3 about it.

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