DX CT/NG/MG AUTO ASSAY

DX CT/NG/MG AUTO ASSAY 5 x 96 37015 Direct detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium by Real-Time PCR * 881106-2013/02

Table of Content 1- INTENDED USE...3 2- SUMMARY AND EXPLANATION OF THE TEST...3 3- PRINCIPLES OF THE PROCEDURE...3 4- REAGENTS...5 5- WARNINGS AND PRECAUTIONS...6 6- SPECIMENS...7 7- PROCEDURE...8 8- TEST LIMITATIONS...16 9- EXPECTED VALUES...16 10- PERFORMANCE CHARACTERISTICS...18 11- BIBLIOGRAPHY REFERENCES...33 2 [EN]

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 endocervical, vaginal and anorectal (male and female) swab specimens 1, patient-collected vaginal swab specimens 1, 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 infection in industrialized countries, with approximately 89 million new cases each year according to WHO (1). Chlamydia trachomatis infections 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 infertility (2) in women, or non gonococcal urethritis 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 bacteria 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. 3 [EN]

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 the Bio- Rad Dx Prep System and the Dx Automated Extraction Assay (cat. # 37016). The Dx CT/NG/MG Auto Assay kit contains all reagents necessary to perform amplification/detection, for 480 tests. 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. Real-time PCR Amplification/Detection The Bio-Rad Dx Prep System automatically adds the amplification master mix into the 96-well Dx PCR plate, together with the extracted DNA. 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). N. gonorrhoeae probe, targeting a sequence in the pile gene; 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 [EN]

4. REAGENTS 4.1. Description There are sufficient reagents provided in the kit to perform 480 tests. All reagents are for in vitro diagnostics use only. R1 R2 C1 C2 C3 Label Reagent Packaging Amplification Mix Amplification Mix Diluent Internal Control Negative Control Positive Control Concentrated amplification mix, 10X: DNA Polymerase in a PCR buffer containing primers, specific fluorescent oligonucleotidic probes, dntps and MgCl 2. Preservative: 0,05 % sodium azide. Amplification mix diluent: PCR buffer containing MgCl 2. Preservative: 0,05 % sodium azide. Internal control: Non-infectious DNA in a buffered solution. Preservative: 0,03 % ProClin 300. Negative control: TE buffer. Preservative: 0,03 % ProClin 300. 10 x 150 μl To be diluted by adding R2 10 x 900 μl Ready to use 5 x 1300 μl Ready for use 10 x 1100 μl Ready for use CT, NG, MG Positive control: 5 x 100 μl Non-infectious DNA from CT, NG and MG Ready for use in a buffered solution. Yellow-coloured. Preservative: 0,03 % ProClin 300. 5 [EN]

4.2. Storage and handling requirements The kit 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. C2 C3 Identification C1 + Binding Buffer R1+R2 (reconstituted mix) Conservation after opening 15 days at 2-8 C. Each vial can be used twice. 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. If contamination occurs in one of these reagents, discard it. 1 month at 2-8 C. If contamination occurs in one of these reagents, discard it. The Binding Buffer is part of the Dx Automated Extraction Assay (Cat. # 37016). 4 hours at 18-30 C or 16 hours at 2-8 C or 2 weeks at - 20 C. If frozen, the reconstituted mix can be thawed only once. 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. 5.2. Precautions related to the procedure This kit is validated for use with the Dx Real-Time System (Bio-Rad) and its Dx Real-Time Software only. Automated DNA extraction must be done with the Dx Automated Extraction Assay kit (Bio-Rad, cat. # 37016) on the Dx Prep System (Bio-Rad). 6 [EN]

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) in order to be loaded on the Dx Prep System. 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. 7 [EN]

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. Transfer 3 ml of each urine sample to a Dx Transfer System (Bio-Rad cat. # 37014) tube. This tube is to be loaded on the Dx Prep System. 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 (Bio-Rad, cat. # 37015) 7.1.2. Materials required provided separately 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) 8 [EN]

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) 7.1.3. Materials required but not provided Clean polypropylene, preservative-free urine collection cups Vortex mixer Calibrated pipettes capable of delivering 1,000 to 1,200 µl Sterile pipette tips with filters Disposable powder-free gloves 7.2. Reagents Preparation Adding the Internal Control (C1) into the Binding Buffer (BB): 1. 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. 2. Add 1200 µl of Internal Control (C1) into the Binding Buffer (BB) bottle, and gently mix by inversion. 3. 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. 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 realtime 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. 9 [EN]

10 [EN] 7.3. Assay Procedure 7.3.1. Automated DNA extraction and PCR Setup 1. Switch on the Dx Prep System then the computer. Ensure that the Dx Prep System front door is closed. 2. The Dx Prep Software starts automatically. Enter the user name and password in the Login window. 3. Open the Dx Prep System metal flap to load the instrument. 4. 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 System Sample Racks. d. Load the Sample Racks on the Dx Prep System following the instrument s indications (flashing lights). e. If a 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. 5. Click on the Extraction then Defining test orders buttons on the Dx Prep Software. 6. Select STD on the Analyte group(s) list. 7. Ensure that the test assignment is correct for all samples. 8. Click on the Defining the Run button. 9. 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. 10. Click on the Loading Plates button. 11. 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. 12. Close the Plates drawer. The Dx Prep System automatically reads the plates barcodes. 13. Click on the Loading Reagents button.

14. Take one set of Dx Automated Extraction Assay reagents (Magnetic Beads, 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. 15. Vortex the MB bottle for 5 seconds and gently mix the PK, WB and EB vials by inversion. 16. 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. 17. Load the Dx Prep Extraction Rack on the Dx Prep System following the instrument s indications (flashing lights). 18. If the rack is not full, confirm it by selecting it on the Dx Prep Software then click on the Confirm rack button. 19. 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. 20. Vortex the Negative Control (C2) and Positive Control (C3) vials then centrifuge briefly. 21. 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. 22. Load the Dx Prep PCR Rack on the Dx Prep System following the instrument s indications (flashing lights). 23. If the rack is not full, confirm it by selecting it on the Dx Prep Software then click on the Confirm rack button. 24. Click on the Loading Tips button. 25. 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. 26. Click on the Checking Waste button. 27. The Dx Prep Software then indicates the level of filling of the Solid and Liquid Wastes: 11 [EN]

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. 28. Click on the Final Check button. 29. If the loading is correct, the Dx Prep Software then displays the word Passed in the Run Status window. 30. Close the metal flap and click on the Run Status button. 31. 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. 32. At the end of the run, open the Dx Prep System metal flap, then pull the Plates drawer. 33. 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. 34. Proceed to Real-time PCR Amplification/Detection (see 7.3.2) within 30 minutes following Dx PCR Plate preparation. 35. 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. 36. 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. 37. 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. 12 [EN]

2. Click on the Run button on the left, then in the Run window, 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 recognises the Dx PCR Plate and imports the plate map from the Dx Prep System. 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. 7.4. 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. 7.5. Quality Control Positive and Negative Controls A Negative and a Positive controls must be included in each assay run in order to detect potential failure in specimen processing, amplification or detection steps. 13 [EN]

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. 14 [EN]

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. 7.6. 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. 7.7. 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. 15 [EN]

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. 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 and Dx Real-Time Software. 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. In rare circumstances, the Dx CT/NG/MG Auto Assay can lead to false NG positive results when the sample contains Neisseria meningitidis serogroup C or D. 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 cervicitis, urethritis, urinary tract or vaginal infections due to other causes or concurrent infections with other pathogens. 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 pathogen DNA can persist after appropriate antimicrobial 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. 16 [EN]

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 Population site 1 Gender N Chlamydia trachomatis CT + Neisseria gonorrhoeae NG + Mycoplasma genitalium MG + N % N % N % Asymptomatic 141 17 12.2% 1 2.6% 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 Symptomatic 308* 16 5.2% 1 0.3% 4 1.3% Asymptomatic or Symptomatic 162 19 11.8% 15 9.3% 6 3.7% 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 Population Site 2 Gender N Chlamydia trachomatis CT + Neisseria gonorrhoeae NG + Mycoplasma genitalium MG + Prospective Asymptomatic or Symptomatic Sampling center 1 Sampling center 2 sampling center 3 N % N % N % 75 2 3% 0 0% 1 1% 6 1 17% 1 17% 0 0% 36 1 3% 0 0% 0 0% 1 0 0% 0 0% 0 0% 30 1 3% 1 3% 0 0% 4 2 50% 1 25% 0 0% Total Prospective + 152 7 5% 3 2% 1 1% 17 [EN]

Table 3: CT/NG/MG prevalence Sites 1 + 2 POPULATION Site 1 & Site 2 Sex N Chlamydia trachomatis CT + Neisseria gonorrhoeae NG + Mycoplasma genitalium MG + N % N % N % Asymptomatic 141 17 12% 1 3% 6 4% Symptomatic 8 2 25% 0 0% 1 13% Symptomatic 37 8 22% 2 5% 2 5% Asymptomatic or Symptomatic 449 20 4% 2 0.4% 5 1% Asymptomatic or Symptomatic 173 22 13% 17 10% 6 3% Total + 808 69 8.5% 22 2.7% 20 2.5% 10. PERFORMANCE CHARACTERISTICS 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. 18 [EN]

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 Panel Sample Extraction Lot N Mean SD CV% SD CV% SD CV% SD CV% Urine Transport Medium Low Medium High Low Medium High A 80 34.4 0.46 1.3% 0.33 0.9% 0.52 1.5% 0.63 1.8% B 80 34.9 0.18 0.5% 0.40 1.1% A 80 31.4 0.69 2.2% 0.49 1.6% 0.54 1.7% 0.75 2.4% B 80 31.7 0.23 0.7% 0.44 1.4% A 80 27.5 0.16 0.6% 0.21 0.8% 0.38 1.4% 0.43 1.6% B 80 33.6 0.22 0.8% 0.38 1.4% A 80 33.7 0.45 1.3% 0.54 1.6% 0.36 B 80 30.0 0.47 1.4% 0.50 1.5% 1.1% 0.69 2.1% A 80 30.1 0.31 1.0% 0.35 1.2% 0.32 1.1% 0.42 1.4% B 80 26.7 0.16 0.5% 0.33 1.1% A 80 26.7 0.17 0.6% 0.41 1.5% 0.36 1.3% 0.39 1.5% B 80 26.7 0.14 0.5% 0.25 0.9% 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 Panel Sample Extraction Lot N Mean SD CV% SD CV% SD CV% SD CV% Urine Transport Medium Low Medium High Low Medium High A 80 35.3 0.43 1.2% 0.93 2.6% 0.99 2.8% 1.07 3.0% B 80 36.0 0.38 1.1% 0.49 1.4% A 80 32.2 0.67 2.1% 0.65 2.0% 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% 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% 0.20 B 80 30.8 0.23 0.8% 0.27 0.9% 0.6% 0.33 1.1% A 80 27.1 0.42 1.5% 0.58 2.2% 0.38 1.4% 0.54 2.0% B 80 27.1 0.20 0.7% 0.18 0.7% A 80 23.4 0.17 0.7% 0.63 2.7% 0.48 2.0% 0.55 2.3% B 80 23.3 0.34 1.4% 0.21 0.9% 19 [EN]

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 Panel Sample Extraction Lot N Mean SD CV% SD CV% SD CV% SD CV% Urine Transport Medium Low Medium High Low Medium High A 80 34.8 0.57 1.6% 1.16 3.3% 1.33 3.8% 1.4 4.1% B 80 35.9 0.60 1.7% 0.71 2.0% A 80 31.3 0.96 3.1% 0.71 2.3% 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% 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% 0.00 B 80 30.9 0.56 1.8% 0.34 1.1% N/A 0.69 2.2% A 80 26.7 0.63 2.3% 0.15 0.6% 0.28 1.0% 0.63 2.3% B 80 26.9 0.49 1.8% 0.23 0.8% A 80 23.2 0.28 1.2% 0.20 0.9% 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. 20 [EN]

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, 21 [EN]

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 cat. # 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. 22 [EN]

Table 8: CT comparative results Specimens Reference CT Dx CT Auto Negative Negative Dx CT Auto Positive Reference CT Positive or Equivocal* or Beyond cutoff** Dx CT Auto Negative Dx CT Auto Positive Failed Inv IC self vaginal 121 1(a) 0 15+1* 2 140 endocervical swab 345 0 0 15 1 361 Total 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%]. 23 [EN]

24 [EN] 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. Table 9: NG comparative results Specimens Reference NG PCR test Dx NG Auto Negative Negative Dx NG Auto Positive Reference NG PCR test Positive or Equivocal Dx NG Auto Negative Dx NG Auto Positive Failed Inv IC self vaginal 138 0 0 1 2 141 endocervical swab 358 0 0 2 1 361 Total 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 (d) 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%]. 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. 25 [EN]

Table 10: NG comparative results with culture Specimens Dx NG Auto Negative Culture NG Negative Dx NG Auto Positive Dx NG Auto Negative Culture NG Positive Dx NG Auto Positive Failed - Invalid-IC 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 Total first catch urine 112 1(a) 0 7 0 120 FCU after Prostatic massage 36 0 0 0 0 36 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. 26 [EN]

Table 11: Bio-rad Dx MG Auto results Specimens Dx MG Auto negative Dx MG Auto Positive Failed Invalid IC self vaginal 133 6 2 141 endocervical swab 358 4 1 363 Total 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 Table 12: MG comparison results sites Specimens Dx MG Auto In House MG Positive Negative Positive Negative Total Site 1 Site 2 Urine 7 0 7 0 7 Swab 14 1* 9 6 15 Urine 13 0 12 1 13 Swab 1 0 0 1 1 Total 35 0 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. 27 [EN]

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 Specimens Table 14: N. gonorrhoeae Reference CT PCR Dx CT Auto Negative Negative Dx CT Auto Positive Reference CT PCR Dx CT Auto Negative Positive Dx CT Auto Positive First catch urine 73 0 0 52 125 First catch urine 87 0 0 66 153 Total Total 245 0 0 31 278 Specimens Reference NG PCR Dx NG Auto Negative Negative Dx NG Auto Positive Reference NG PCR Dx NG Auto Negative Positive Dx NG Auto Positive First catch urine 117 0 1 7 125 First catch urine 128 1 0 24 153 Total Total 245 1 1 31 278 28 [EN]

Table 15: M. genitalium Specimens Dx MG Auto Negative Dx MG Auto Positive Total First catch urine 118 7 125 First catch urine 147 6 153 Total 245 13 278 For C. trachomatis, 31/31 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. 29 [EN]

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 10 6 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 10 6 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. 30 [EN]