Dx CT/NG/MG Assay. 1 x Direct detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium by Real-Time PCR

Transcription

1 Dx CT/NG/MG Assay 1 x Direct detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium by Real-Time PCR * /12

2 TABLE OF CONTENTS 1. INTENDED USE SUMMARY AND EXPLANATION OF THE TEST PRINCIPLES OF THE PROCEDURE REAGENTS WARNINGS AND PRECAUTIONS SPECIMENS PROCEDURE TEST LIMITATIONS EXPECTED RESULTS PERFORMANCE CHARACTERISTICS BIBLIOGRAPHY REFERENCES [EN]

3 1- INTENDED USE The Dx CT/NG/MG 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 Assay is intended for use with clinician-collected endocervical, vaginal and anorectal (male and female) swab specimens 1, patientcollected vaginal swab specimens 1, and female and male urine specimens. 1 Collected with the Dx Collection System 50-F kit (Bio-Rad cat. #37012), the Dx Collection System-F50 kit (Bio-Rad cat. #37007) or ESwab (COPAN cat. #480 CE). 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. 5 [EN]

4 3. PRINCIPLES OF THE PROCEDURE The Dx CT/NG/MG 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 kit contains all reagents necessary to perform both sample preparation and amplification/detection for 96 tests. Sample Preparation DNA extraction is performed on all sample types listed in the chapter «Specimen», using the Lysis buffer (L1) provided in the kit (Bio-Rad Chelex method). 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 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 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. 6 [EN]

5 4. REAGENTS 4.1. Description There are sufficient reagents and consumables provided in the kit to perform 96 tests. All reagents are for in vitro diagnostic use only. Label Description Presentation L1 Lysis Buffer Lysis buffer: Chelex resin in a lysis buffer. Preservative: 0.03 % ProClin 300. Each bottle contains one magnetic stir bar. R1 Amplification Mix Concentrated amplification mix, 10X: DNA Polymerase in a PCR buffer containing primers, specific fluorescent oligonucleotidic probes, dntps, UDG and MgCl 2. Preservative: 0.05 % sodium azide. 2 x 21 ml Ready to use 2 x 150 µl To be diluted by adding R2 R2 Amplification Mix Diluent Amplification mix diluent: PCR buffer containing MgCl 2. Preservative: 0.05 % sodium azide. 2 x 900 µl Ready to use C1 C3 Internal Control Positive Control Dx 24-Well PCR Strips Dx 8-Cap Strips Internal control: Non-infectious DNA in a buffered solution. Preservative: 0.03 % ProClin 300. CT,NG,MG Positive control: Non-infectious DNA from CT, NG and MG in a buffered solution. Yellow-coloured. Preservative: 0.03 % ProClin 300. Thermoplates: 24-wells white microplates, barcoded for unique identification. 2 x 1050 µl Ready to use 1 x 600 µl Ready to use Strip Caps: 8-caps strips for thermoplates NOTE: Negative Control: the negative control is an aliquot of L1 Lysis Buffer. An empty 2 ml screw-cap microtube will therefore be labeled Negative Control and processed from step 5 of DNA extraction (see chapter 7.2). 7 [EN]

6 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 Assay can be used until the expiration date given on the reagent label. Identification L1 C1 C3 R1+R2 (reconstituted mix) Conservation after opening 6 months at 2-8 C. If contamination occurs in one of these reagents, discard it 2-8 C until expiration date 4 hours at 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 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 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. 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), the Dx Collection System-F50 (Bio-Rad, cat.# 37007) or ESwab (COPAN, cat.# 480 CE). These systems are not for home use. For collection of first-void urine samples, use clean polypropylene, preservative-free specimen collection cups. Do not use the Dx Collection System 50-F (Bio-Rad, cat.# 37012), the Dx Collection System-F50 (Bio-Rad, cat.# 37007) or ESwab (COPAN, cat.# 480 CE), 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. 8 [EN]

7 If the laboratory receives a swab specimen not collected and transported with the Dx Collection System 50-F (Bio-Rad, cat.# 37012), the Dx Collection System-F50 (Bio-Rad, cat.# 37007) or ESwab (COPAN, cat.# 480 CE) 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. Use a new filter tip for each sample. Use DNase- and RNase-free microtubes and filter tips. 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%. Work areas: within the laboratory, two dedicated areas must be unidirectionnally used to perform sample preparation and amplification/ detection steps: 1. The Pre-amplification area is dedicated to: (a) the preparation of samples and controls and (b) the PCR setup (pipetting of Amplification mix, controls and samples in the Dx 24-Well PCR Strips). These two steps must be done in two distinct zones of the Pre-amplification area. 2. The Amplification area is dedicated to the real-time PCR reaction. All reagents, equipment and laboratory coats used in one area must remain in this area and never be moved in the other area. Never transfer amplification products nor Amplification area equipment in the Preamplification area. 6. SPECIMENS 6.1. Swab specimens The Dx CT/NG/MG Assay is intended for use with clinician-collected endocervical, vaginal and anorectal (male and female) swab specimens, and with patient-collected vaginal swab specimens. 9 [EN]

8 Specimens must be collected and transported only with the Dx Collection System 50-F (Bio-Rad, cat.# 37012), the Dx Collection System-F50 (Bio-Rad, cat.# 37007) or ESwab (COPAN, cat.# 480 CE). For more information on specimen collection and transport, please refer to the Dx Collection System 50-F (Bio-Rad, cat.# 37012), the Dx Collection System-F50 (Bio-Rad, cat.# 37007) or ESwab (COPAN, cat.# 480 CE) package insert. Swab specimens collected with Bio-Rad Dx Collection Systems can be kept up to 28 days at C. Swab specimens collected with COPAN ESwab should be processed before 5 days when stored at room temperature ( C), 7 days at +2-8 C, and 2 months when stored at - 20 C. A transport tube containing a swab not supplied or validated by Bio-Rad, or multiple swabs, or no swab cannot be used with the Dx CT/NG/MG 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 Performances characteristics chapter Urine specimens The Dx CT/NG/MG 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 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 7 days. It is also posible to freeze urine specimens but in this case do not freeze and thaw urine specimens more than one time. 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. 7. PROCEDURE 7.1. Materials required Materials provided Dx CT/NG/MG Assay (Bio-Rad, cat. # 37005) 10 [EN]

9 Materials required provided separately Dx Real-Time System, Dx Real-Time Software, PC 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), Dx Collection System-F50 (Bio-Rad, cat.# 37007) or ESwab (COPAN, cat.# 480 CE) for collection and transport of female and anorectal swab specimens Materials required but not provided Clean polypropylene, preservative-free urine collection cups Desktop centrifuge for PCR thermoplates Desktop centrifuge for 1.5 ml and 2 ml microtubes Heating block able to reach 105 C Vortex mixer Magnetic stirrer Calibrated pipettes capable of delivering 10 to µl Sterile pipette tips with filters Positive displacement repeat pipettor with sterile individually wrapped 1.25 ml tips 2 ml hermetic screw-cap microtubes with round bottom shape (Simport cat.# T335-6S or equivalent) Disposable powder-free gloves 7.2. Assay Procedure Follow strictly these instructions and apply Good Laboratory Practices DNA extraction 1. Determine the number of samples to be tested and take one screw-cap microtube per sample plus one more for the Negative Control. Identify each microtube. 2. Add 1 ml of each sample (for urine sample or sample collected with Dx Collection System 50-F / Dx Collection System-F50) or 400µl for a sample collected with ESwab into its corresponding microtube. Leave one microtube empty for the Negative Control at this step. 3. Centrifuge for 10 minutes at 5,000 g, and then discard the supernatant by turning each microtube upside down. 4. Shake the bottle of Lysis Buffer (L1) by turning it upside down and then place it on a magnetic stirrer to allow the Chelex resin to stay in suspension. 5. Dispense 400 µl of Lysis Buffer (L1) into each microtube, including the Negative Control microtube. 6. Add 10 µl of Internal Control (C1) into each microtube. Screw caps tightly. 11 [EN]

10 Note: Alternatively, it is possible to prepare a L1+C1 premix then dispense 410 µl of this premix in each microtube. The premix tube must then be vortexed before each pipetting in order to allow Chelex resin to stay in suspension. This premix is stable for 5 days at 2-8 C. Examples of premix : 12 tests 24 tests 48 tests L1 5 ml 10 ml C1 125 µl 250 µl Add 525 µl of C1 directly in 1 bottle of L1 12 [EN] 7. Vortex for seconds. 8. Incubate for 10 minutes ± 2 minutes at 105 C. 9. Cool down for 2 minutes at room temperature (18-30 C). 10. Centrifuge for 2 minutes at 4,000 g. The supernatant contains the extracted DNA. This extracted DNA can be kept at room temperature for 2 hours. If the amplification/detection step is not performed within 2 hours, the extracted DNA can be stored at 2-8 C for up to 4 days; for longer storage, extracted DNA must be stored at -20 C for up to 6 months 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 exactly 850 µl of Amplification Mix Diluent (R2) into one vial of concentrated Amplification Mix (R1). Vortex for 10 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 Real-time PCR Amplification/Detection Please refer to the Dx Real-Time System User Manual for more detailed instructions. Each assay run must include one negative control and one positive control. In the Pre-amplification area : 1. Reconstitute as many Amplification Mix vials as necessary following the procedure on chapter (test number = n samples + 1 positive control + 1 negative control).

11 2. Take the required number of Dx 24-Well PCR Strips, place them on a holder and dispense carefully, with the repeat pipettor, 20 µl of reconstituted Amplification mix (R1+R2) into each well. 3. Add 5 µl of extracted DNA or extracted Negative Control or non-extracted Positive Control (C3) into the corresponding wells, following the established plate map. Note: Be careful not to pipette Chelex beads when adding the samples, as Chelex beads can inhibit the PCR reaction. 4. Place the Dx 8-Cap Strips on the Dx 24-Well PCR Strips. 5. Centrifuge the Dx 24-Well PCR Strips for 30 seconds at 400 g in order to avoid any bubble in the wells. In the Amplification area : 6. Switch on, in the following order, the computer then the Dx Real-Time System. Open the Dx Real-Time Software, enter your user name and Login Click on «Set up». Click on «create a new plate» and select in the PCR kit window, the PCR kit Name. Please refer to your local support for more information about the PCR kit name apropriate to Dx RTS Software version. 7. Click on «Run» button and then click on «Open Lid», follow the Dx Real-Time Software instructions to establish the plate map. 8. Place the Dx 24-Well PCR Strips in the Dx Real-Time System. 9. Use the Dx Strip Cap Tool for seating the Dx 8-caps Strips on the loaded Dx 24-Well PCR Strips. 10. Click <Close Lid>, then <Start Run> to start the real-time PCR run. 11. After completion of the real-time PCR reaction, remove the Dx 24-Well PCR Strips from the Dx Real-Time System, place them in a sealable plastic bag and dispose according to Good Laboratory Practices. 12. «Click on Results» to have a view on the results 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 F0) for each PCR curve, which are then displayed in the results spreadsheet. The estimation of these parameters 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 a sample to the other. 13 [EN]

12 14 [EN] The Ceep (Cycle of end of exponential phase) value corresponds to the cycle for which the PCR efficiency starts to decrease. The Ceep value is usually close to the cycle where the fluorescent signal starts to grow above the background. In ideal PCR conditions (no inhibitors of the reaction, high efficiency), the Ceep value is correlated to 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. The F0 value corresponds to the - non measurable - target-specific fluorescent signal before amplification starts. F0 is proportional to the copy number of the target sequence in the sample before amplification. As the estimated F0 value is usually <1, the Dx Real-Time Software displays for convenience the -log 10 (F0) value. Thus the higher the -log10 (F0) value is, the lower the initial copy number is. In the Dx CT/NG/MG Assay, analysis of results is based on Ceep values 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. 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

13 - 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 invalid for this given target. All samples showing an invalid result for a target must be reprocessed starting from the DNA extraction step 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 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. 15 [EN]

14 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 invalid 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. However do not freeze and thaw urine specimens more than one time. 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 performances of this test depend directly on the quality of specimens. It is therefore important to comply with indications given in the chapter Collection and handling of 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 Performances 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 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. The targeted sequence in pile gene is shared with some of the Neisseria meningitidis strains leading to potential false positive NG results in rare circumtances. 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 Assay is not intended for use in suspected sexual abuse evaluation nor other medico-legal indications. 16 [EN]

15 Therapeutic success or failure cannot be determined by the Dx CT/NG/MG Assay since pathogen DNA can persist after appropriate antimicrobial therapy. As with any diagnostic test, results from the Dx CT/NG/MG Assay should be interpreted in conjunction with other laboratory and clinical findings. 9. EXPECTED RESULTS Prevalence 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 Assay during clinical studies in the 2 sites are shown in the table below: Site Population Sex 1 2 Asymptomatic Symptomatic Asymptomatic Symptomatic Asymptomatic Symptomatic Asymptomatic Symptomatic N C. trachomatis N. gonorrhoeae M. genitalium patient positive positive positive % 16.0% 6.9% 3.4% 7.6% 8.3% 10.2% 14.8% % 16.0% 0.0% 0.0% 0.8% 16.7% 0.6% 7.4% % 2.7% 1.5% 1.1% 1.3% 12.5% 1.8% 7.4% Total in the 2 sites: Population Asymptomatic Symptomatic Sex N patient C. trachomatis positive % 14.9% N. gonorrhoeae positive % 16.1% M. genitalium positive % 4.0% Asymptomatic Symptomatic % 6.0% % 1.7% % 2.6% 17 [EN]

16 10. PERFORMANCE CHARACTERISTICS Analytical Performance Analytical Sensitivity/Limit of detection The limit of detection of the Dx CT/NG/MG Assay was determined for each target using: - for C. trachomatis: the international panel QCMD06 CTDNA06B in urine and in transport medium; - for N. gonorrhoeae: the international panel QCMD06 NgDNA06 in urine; - for M. genitalium: the ATCC strain 33530D at a concentration of 1 ng DNA/µL in urine. A dilution series was prepared for each analyte and tested 24 to 36 times. The limit of detection was determined using the Probit analysis retaining the dilution corresponding to a positive level of 95% or higher: - The limit of detection for C.trachomatis was estimated at: 73 copies of DNA/ml in transport medium, with a 95% confidence interval of 45 to 182 copies of DNA/ml; 158 copies of DNA/ml in urine, with a 95% confidence interval of 106 to 315 copies of DNA/ml. - The limit of detection for N. gonorrhoeae was estimated at 1559 CFU/ml in urine, with a 95% confidence interval of 1066 to 3283 CFU/ml. - The limit of detection for M. genitalium was estimated at 450 equivalentgenome/ml in urine, with a 95% confidence interval of 250 to 1246 CFU/ml. These limits of detection were confirmed by testing 16 serovars of C. trachomatis (A, B, Ba, C, D, E, F, G, H, I, J, K, LGV1, LGV2, LGV3, nv CT) and 19 N. gonorrhoeae strains isolated from patients. 18 [EN]

17 Analytical Specificity (Cross-reactivity) The analytical specificity of the Dx CT/NG/MG Assay was evaluated on a total of 112 bacteria, viruses, yeast and molds strains. This panel includes strains that may be isolated in the urogenital tract and strains representing a phylogenetic cross-section with Chlamydia trachomatis, Neisseria gonorrhoeae or Mycoplasma genitalium: Achromobacter xerosis Haemophilus influenzae Neisseria perflava Acinetobacter calcoaceticus Hepatitis B virus (HBV) Neisseria polysaccharea Acinetobacter lwoffii Herpes simplex virus 1 Neisseria sicca Actinomyces isrealii Herpes simplex virus 2 Neisseria subflava flava Aerococcus viridans Human Immunodeficiency Virus (HIV) Neisseria subflava perflava Agrobacterium radiobacter Human Papilloma Virus HPV 16 Paracoccus denetrificans Alcaligenes faecalis Human Papilloma Virus HPV 18 Peptostreptococcus anaerobius Bacillus subtilis Kingella dentrificans Plesiomonas shigelloides Bacteriodes fragilis Kingella kingae Propionibacterium acnes Bacteroides ureolyticus Klebsiella oxytoca Proteus mirabilis Bifidobacterium adolescentis Klebsiella pneumoniae Providencia stuartii Bifidobacterium breve Lactobacillus acidophilus Pseudomonas aeruginosa Brevibacterium linens Lactobacillus brevis Pseudomonas putida Campylobacter jejuni Lactobacillus jensenii Rahnella aquatilis Candida albicans Lactobacillus vaginalis Rhodospirillum rubrum Candida glabrata Legionella pneumophila Salmonella minnesota Candida tropicalis Leuconostoc mensenteroides Salmonella typhimurium Chlamydia pneumoniae Micrococcus luteus Serracia marscence Chlamydia psittaci Moraxella (Branhamella) catarrhalis Staphylococcus aureus Chromobacterium violaceum Moraxella lacunata Staphylococcus epidermidis Citrobacter freundii Moraxella osloensis Staphylococcus saprophyticus Clostridium perfringes Morganella morganii Streptococcus agalactiae Corynebacterium genitalium Mycobacterium gordonae Streptococcus bovis Corynebacterium xerosis Mycobacterium smegmatis Streptococcus griseinus Cryptococcus neoformans Mycoplasma fermentans Streptococcus mitis Cytomegalovirus Mycoplasma hominis Streptococcus mutans Derxia gummosa Mycoplasma orale Streptococcus pneumoniae Edwardsiella tarda Mycoplasma penetrans Streptococcus pyogenes Eikenella corrodens Mycoplasma pneumoniae Streptococcus salivarus Enterobacter cloacae Neisseria cinerea Streptococcus sanguis Enterococcus avium Neisseria elongata Treponema pallidium Enterococcus faecalis Neisseria flavescens Trichomonas vaginalis Enterococcus faecium Neisseria lactamina Ureaplasma urealyticum Escherichia coli Neisseria meningitidis 135 Veillonella parvula Flavobacterium meningosepticum Neisseria meningitidis serogroupe A Vibrio parahaemolyticus Fusobacterium nucleatum Neisseria meningitidis serogroupe B Yersinia enterocolitica Gardnerella vaginalis Neisseria meningitidis serogroupe C Gemella haemolysans Neisseria mucosa 19 [EN]

18 All the strains except 22 were cultivated, calibrated at 10 8 CFU/ml in transport medium, extracted and tested with the Dx CT/NG/MG Assay. For the 22 noncultivated strains, commercial DNA at 40 µg/ml to 200 µg/ml was directly tested with the Dx CT/NG/MG Assay. One strain, Neisseria meningitidis serogroup B, gave a positive result with the Neisseria gonorrhoeae target. This cross- reactivity was also observed during clinical evaluations. Interferences Potential interferences on the Dx CT/NG/MG Assay were evaluated with endogenous or exogenous substances that may be present in swab and/or urine samples. Fourteen substances were diluted to various final concentrations and added to a swab pool in 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 10 replicates. The following substances were tested: Potentially interfering substance Concentration Matrix Bilirubin 1 mg/ml Urine Blood 5% v/v Urine 5% v/v Swab Glucose 1 mg/ml Urine ph 4 Urine ph 9 Urine Proteins (Human Serum Albumin) 5% Urine Delfen (Contraceptive foam) 5% Swab Betadine 5% Swab Miconazole (anti-bactericide and antifungal) 5% Swab Isoconazole (anti-bactericide and antifungal) 5% Swab Trophicrem (vaginal cream) 5% Swab Acyclovir (Antiherpetic) 5% Swab Lubricant KY Gel 5% Swab Progesterone 5% Swab No interference on the Dx CT/NG/MG Assay was observed with the substances indicated above, except for: - Delfen 5%: inhibitor effect on the detection of Neisseria gonorrohoeae and Mycoplasma genitalium. - Blood at 5%: inhibitor effect on the detection of Neisseria gonorrohoeae. 20 [EN]

19 Competition study between the various Multiplex analytes In order to validate that the presence of the 3 micro-organisms in the same sample does not result in a competition effect between the targets that could lead to a detection defect of one of them, urine and transport medium specimens were spiked with low and high concentrations of each of the three microorganisms, in order to prepare all the combinations (1 low/2 high or 2 low/1 high). These specimens were tested with the Dx CT/NG/MG Assay in parallel with specimens spiked with a low concentration of only one of each micro-organism in order to detect any impact on each of the targets. The percentage of positive responses was analyzed on tests performed in triplicate over 3 days. Irrespective of the microorganism studied, none of the combinations tested had an impact on the expected result. Therefore, there is no competition between the targets and the presence of one of the micro-organisms in high concentration does not result in any detection defect of the other two. However, the presence in high concentration of the 3 micro-organisms in the same sample results in an inhibition of the internal control. Precision Study A study was performed in order to determine the intra-assay, inter-day, interinstrument and inter-batch precision. Three urine samples (U2, U3, U4) and 3 transport medium samples (S2, S3, S4) were spiked with respectively low, medium and high concentrations of C. trachomatis, N. gonorrohoeae or M. genitalium. According to the CLSI instructions EP5A2, this panel was tested in 3 replicates per day over 21 days on 2 different Dx Real-Time System instruments and on 2 different batches of Dx CT/NG/MG Assay. The analysis on positive samples was performed using the Ceep values. The Nested ANOVA function was used to measure the variance components for each condition (batch - instrument - day - replicate). All the tests gave the expected qualitative result. 21 [EN]

20 The results are as follows: Chlamydia trachomatis CT Intra Assay Inter Day * The variance value is negative and estimated at 0. Neisseria gonorrhoeae * The variance value is negative and estimated at 0. Mycoplasma genitalium Inter Instrument * The variance value is negative and estimated at 0. Inter Batch Total Precision PANEL N Mean SD CV% SD CV% SD CV% SD CV% SD CV% U2 (low) % % 0* NA % % U3 (medium) % % 0* NA % % U4 (high) % % 0* NA % % S2 (low) % % 0* NA % % S3 (medium) % % 0* NA % % S4 (high) % % 0* NA % % NG Intra Assay Inter Day Inter Instrument Inter Batch Totale Precision PANEL N Mean SD CV% SD CV% SD CV% SD CV% SD CV% U2 (low) % % 0* NA % % U3 (medium) % % 0* NA % % U4 (high) % % % % % S2 (low) % % % 0* NA % S3 (medium) % % % 0* NA % S4 (high) % % 0* NA 0* NA % MG Intra Assay Inter Day Inter Instrument Inter Batch Totale Precision PANEL N Mean SD CV% SD CV% SD CV% SD CV% SD CV% U2 (low) % % 0* NA % % U3 (medium) % % 0* NA % % U4 (high) % % 0* NA 0* NA % S2 (low) % % 0* NA % % S3 (medium) % % 0* NA % % S4 (high) % % 0* NA % % 22 [EN]

21 10.2. Clinical performance Clinical performance characteristics of the Dx CT/NG/MG Assay were established during a study performed in 2 clinical sites in France symptomatic and asymptomatic patients consulting in STI prevention centers and in anonymous free screening centers were recruited for this study. 12 patients did not meet the previously defined inclusion criteria and were excluded. The 1408 other patients were included in the study (821 women and 587 men). A total of 2361 specimens were analyzed: 430 patient-collected vaginal specimens; 407 endocervical specimens; 386 clinician-collected vaginal specimens; 540 female first-void urine specimens; 532 male first-void urine specimens; 57 anorectal specimens; 9 urethral specimens; Among these specimens: 381 vaginal, endocervical and first-void urine specimens were taken from the same patients; 26 vaginal and endocervical specimens were taken from the same patients; 151 women provided a first-void urine and a self-collected vaginal specimen; A first-void urine and its associated urethral specimen were available for 6 men. All the specimens were collected using the Dx Collection System-F50 (Bio-Rad, cat.# 37007) and Dx Collection System-M15 (Bio-Rad, cat.# 37006). Furthermore, 60 frozen samples (10 first-void urine specimens, 22 anorectal specimens, 16 vaginal specimens, 1 endocervical specimen and 11 urethral specimens) kept in 2SP medium and initially found to be positive for one of the 3 micro-organisms (20 per microorganism) were tested retrospectively using the Dx CT/NG/MG Assay. The performance of the Dx CT/NG/MG Assay for the target C. trachomatis was established by comparison with two EC registered real time PCR tests. The performance of the Dx CT/NG/MG Assay for the target N. gonorrhoeae was established by comparison with a EC registered PCR test or with the culture. The performance of the Dx CT/NG/MG 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 (15). On one of the sites, only the specimens that tested positive with the Dx CTNG/MG Assay were tested with the in-house PCR test. 23 [EN]

22 Chlamydia trachomatis performance: 1361 specimens from the 2 sites were analyzed in parallel using the Dx CT/NG/ MG Assay and the PCR 1 test: Site PCR 1 Test Negative PCR 1 Test Positive Samples Dx CT Dx CT Dx CT Dx CT Inv IC Total Negative Positive Negative Positive self-collected vaginal endocervical vaginal first-void urine anorectal first-void urine anorectal urethral Total The relative specificity of the Dx CT/NG/MG Assay with respect to the PCR 1 test is 1223/1229 = 99.5% CI 95 [98.9% 99.8%]. The relative sensitivity of the Dx CT/NG/MG Assay with respect to the PCR 1 test is 112/114 = 98.2% CI 95 [93.8% 99.8%]. On one of the 2 sites, 1014 specimens were analyzed in parallel using the Dx CT/ NG/MG Assay and a second real time PCR test: Site 1 PCR 2 Test Negative PCR 2 Test Positive Samples Dx CT Dx CT Dx CT Dx CT Inv IC Total Negative Positive Negative Positive self-collected vaginal endocervical vaginal first-void urine first-void urine anorectal Total * * Two of these specimens (1 vaginal and 1 first-void urine) are actually true negatives, from patients for which the other specimens were negative with the Dx CT/NG/MG Assay and PCR 2 test. 24 [EN]

23 The relative specificity of the Dx CT/NG/MG Assay with respect to the PCR 2 test is 950/954 = 99.6% CI 95 [98.9% 99.9%]. The relative sensitivity of the Dx CT/NG/MG Assay with respect to the PCR 2 test is 51/57 = 89.5% CI 95 [78.5% 96.0%]. After analysis of the discordant specimens, 2 specimens were removed from the calculations; they come for the 2 patients for whom the other associated specimens were negative with the Dx CT/NG/MG Assay and PCR 2 test. The relative sensitivity after exclusion is 51/55 = 92.7% CI 95 [82.4% 98.0%]. Neisseria gonorrhoeae performance: 1012 specimens were analyzed in parallel with the Dx CT/NG/MG Assay and a real time PCR test; 712 other speøcimens were analyzed in parallel with the Dx CT/NG/MG Assay and the culture. The results are presented below: Comparison to a real time PCR test Site 1 PCR NG Test PCR NG Test Negative Positive Samples Dx NG Dx NG Dx NG Dx NG Negative Positive Negative Positive Inv IC Total self-collected vaginal endocervical vaginal first-void urine anorectal first-void urine anorectal Total The relative specificity of the Dx CT/NG/MG Assay with respect to the PCR test is 997/998 = 99.9% CI 95 [99.4% 100%]. The relative sensitivity of the Dx CT/NG/MG Assay with respect to the PCR test is 10/11 = 90.9%. 25 [EN]

24 Comparison to the culture: Site 1 NG Culture NG Culture Negative Positive Samples Dx NG Dx NG Dx NG Dx NG Negative Positive Negative Positive Inv IC Total self-collected vaginal endocervical vaginal first-void urine anorectal first-void urine anorectal urethral Total The relative specificity of the Dx CT/NG/MG Assay with respect to the culture is 675/677 = 99.8% CI 95 [98.9% 100%]. The relative sensitivity of the Dx CT/NG/MG Assay with respect to the culture is 30/30 = 100%. Mycoplasma genitalium performance: On site 2, 656 specimens were analyzed in parallel using the Dx CT/NG/MG Assay and the in-house TaqMan MG PCR test: Site 2 PCR MG Test PCR MG Test Negative Positive Samples Dx MG Dx MG Dx MG Dx MG Negative Positive Negative Positive Inv IC Total first-void urine endocervical vaginal first-void urine anorectal urethral Total The relative specificity of the Dx CT/NG/MG Assay with respect to the in-house TaqMan PCR test is 616/617 = 99.8% CI 95 [99.1% 100%]. The relative sensitivity of the Dx CT/NG/MG Assay with respect to the in-house TaqMan PCR test is 10/11 = 90.9%. 26 [EN]

25 25 specimens from 20 patients who tested positive for M. genitalium with the Dx CT/NG/MG Assay on Site 1 were sent to Site 2 for analysis with the in-house TaqMan MG PCR test. 22/25 specimens tested positive with the Dx CT/NG/MG Assay on Site 2, the remaining 3 specimens were close to the limit of detection. Out of these 22 specimens, 2 tested negative with the in-house TaqMan MG PCR test. The concordance between the Dx CT/NG/MG Assay and the in-house TaqMan PCR test is therefore 20/22 = 90.9% for the specimens coming from Site 1. However, at least one of the two discordant specimens is actually a true positive as it was obtained from a patient for whom all the associated specimens tested positive for M. genitalium with the Dx CT/NG/MG Assay. Therefore the final concordance between the Dx CT/NG/MG Assay and the the in-house TaqMan PCR test, obtained after addition of the results from Site 1 and Site 2, is 626/629 = 99,5 %. Retrospective study (Site 2) Sixty samples (10 first-void urine specimens, 22 anorectal specimens, 16 vaginal specimens, 1 endocervical specimen and 11 urethral specimens) kept at -80 C in 2SP medium and initially found to be positive for one of the 3 micro-organisms with the test routinely used in the laboratory (20 per microorganism) were tested retrospectively using the Dx CT/NG/MG Assay. For C. trachomatis, 19/19 specimens were concordant positives, the twentieth presented an invalid internal control. For N. gonorrhoeae, 19/20 specimens were concordant positives, the twentieth had been previously found positive in culture with only 1 colony. For M. genitalium, 16/20 specimens were concordant positives; out of the 4 discordant (urines), 2 tested negative after control with the routine test. 11. BIBLIOGRAPHY REFERENCES 1 WHO. Global prevalence and incidence of selected curable sexually transmitted infections. Overview and estimates. Geneva: WHO; Cates W Jr, Rolfs RT Jr, Aral SO. Sexually transmitted diseases, pelvic inflammatory disease, and infertility: an epidemiologic update. Epidemiol Rev 1990; 12: 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, Centers for Disease Control and Prevention. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections. MMWR 2002; 51 (No. RR-15) : Taylor-Robinson D. Mycoplasma genitalium: an update. Int J STD AIDS 2002; 13: [EN]

26 28 [EN] 6 Simms I, Eastick K, Mallinson et al. Associations between Chlamydia trachomatis, Mycoplasma genitalium, and pelvic inflammatory disease. Sex Transm Infect 2003; 79: Cohen CR, Manhart LE, Bukusi EA et al. Associations between Mycoplasma genitalium and acute endometritis. Lancet 2002; 359: Clausen HF, Fedder J, Drasbek M et al. Serological investigation of Mycoplasma genitalium in infertile women. Human Reprod 2001; 16: 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: Moi H, Reinton N, Moghaddam A. Mycoplasma genitalium in women with lower genital tract inflammation. Sex Transm Infect 2009; 85: Moi H, Reinton N, Moghaddam A. Mycoplasma genitalium is associated with symptomatic and asymptomatic non-gonococcal urethritis in men. Sex Transm Infect 2009; 85: Haggerty CL, Totten PA, Astete SG, Lee S, Hoferka SL, Kelsey FL, Ness RB. Failure of cefoxitin and doxycycline to eradicate endometrial Mycoplasma genitalium and the consequence for clinical cure of pelvic inflammatory disease. Sex Transm Infect 2008; 84: 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: 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: 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 : 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 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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