Simplexa Bordetella Universal Direct

Transcription

1 REF MOL2775 Rev. A A real-time polymerase chain reaction (PCR) assay intended for the qualitative detection of Bordetella pertussis and/or Bordetella parapertussis DNA For in vitro Diagnostic Use INTENDED USE The Focus Diagnostics Simplexa TM Bordetella Universal Direct assay is intended for use on the 3M Integrated Cycler instrument for the in vitro qualitative detection and discrimination of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of bacterial respiratory tract infection. This test is an aid in the differential diagnosis of Bordetella pertussis and Bordetella parapertussis infections. Testing with the Simplexa Bordetella Universal Direct assay should not be performed unless the patient meets clinical and epidemiologic criteria for testing suspect specimens. The identification of Bordetella pertussis and Bordetella parapertussis should be performed in conjunction with clinical and epidemiological assessment. Negative results do not preclude Bordetella pertussis and Bordetella parapertussis infection and should not be used as the sole basis for treatment or other patient management decisions. The assay is not intended for use as a screening test for the presence of the analytes in blood or blood products. The assay is for professional use only. SUMMARY AND EXPLANATION Pertussis, commonly called whooping cough, is a highly contagious disease of the respiratory system caused by small gramnegative bacteria; Bordetella pertussis and Bordetella parapertussis. Clinically, it presents with a prolonged cough and patients with classic disease often have episodes of violent cough that may be followed by an inspiratory whoop and vomiting. In severe cases, seen most commonly in young infants, these symptoms can lead to hypoxia, permanent brain damage, or death. In older children and adults, particularly those who have been vaccinated or previously had disease, the illness can be milder and present as a prolonged cough. During the first half of the 20 th century, whooping cough was highly prevalent and likely affected approximately 1% of the population annually 1, with most reported cases occurring in young children. The introduction of whole-cell pertussis vaccines in the 1940s resulted in dramatic declines in the disease, however, a low level of disease incidence persisted. Since the late 1990s, there has been a marked increase in the number of reported pertussis cases in developed countries with high vaccination rates. In 2004, 25,827 cases were reported in the United States, making it the year with the highest number of reported cases since Similar increases have been seen in Canada and Europe 3. The World Health Organization estimates that there are 50 million whooping cough cases worldwide each year, resulting in 350,000 deaths 4. Pertussis can be detected among all age groups (e.g., neonates, children, adolescents and adults). A great majority of these cases is caused by Bordetella pertussis; however, a percentage of all whooping cough cases, approximately 3-35%, are caused by Bordetella parapertussis, a milder pertussis-like disease 5, 6. While the exact cause of the resurgence in whooping cough cases caused by Bordetella pertussis remains unclear, it is widely believed that vaccine-conferred immunity wanes after 7 to 10 years. Thus, immunized children may become susceptible in their teenage years, contract the disease and transmit the bacteria to very young infants in the household 1, 4. Several laboratory methods, including culture, serology and PCR, are available for diagnosis of pertussis. Culture is the reference method and almost 100% specific; however, its sensitivity is much lower. Culture sensitivity can be as high as 56% early in the disease course, but decreases over time and is lower in patients who have received antimicrobial treatment or previous vaccination. Isolating B. pertussis in culture requires special media and can take 7 to 14 days; therefore, it might not be timely for acute case management. Serology, using paired acute- and convalescent-phase sera, requires at least a 4-week interval between specimen collection and is not useful for immediate diagnosis. Single-sample serology tests for antipertussis toxin IgG have been developed for research purposes, but must be performed at least 2 weeks after symptom onset. Pertussis serology assays, using commercially available reagents, are also available, but are not clinically validated and might not differentiate between recent and

2 Page 2 remote infection or vaccination 10. PRINCIPLES OF THE PROCEDURE The test is a real-time polymerase chain reaction (PCR) amplification and detection system that utilizes a bi-functional fluorescent probe-primer for the detection and differentiation of Bordetella pertussis and Bordetella parapertussis DNA in nasopharyngeal swabs (NPS). The assay has been validated with two different assay protocols. The EXTRACTION protocol uses extracted DNA from patient specimens, the DIRECT protocol uses unprocessed specimens. The DNA is amplified using a bi-functional fluorescent probe-primer together with a reverse primer for each analyte and the DNA internal control. The assay provides two results; Bordetella pertussis (Bp) is identified in the specimen by targeting IS481 while Bordetella parapertussis (Bpp) is identified in the specimen by targeting IS1001. A DNA internal control is used to monitor the extraction process (when applicable) and to detect PCR inhibition. MATERIALS PROVIDED The Focus Diagnostics Simplexa Bordetella Universal Direct assay contains sufficient material for 1000 reactions (including controls). Upon receipt, store all reagents at -10 to -30 ºC (do not use a frost-free freezer). Reagents are stable through the end of the expiration month indicated on the kit packaging when stored at -10 to -30 ºC. After initial use, store thawed reagents at 2 to 8 C for no more than 30 days. Kit Description of the Kit Labeling and Kit Components Label Focus Diagnostics Simplexa Bordetella Universal Direct (REF MOL2775) ENGLISH REF EC SYMBOL Simplexa Bordetella Primer Mix MOL2776 REAG A Simplexa Master Mix MOL2007 REAG B Simplexa Extraction and Amplification Control DNA MOL9001 CONTROL IC Simplexa Bordetella Positive Control MOL0221 CONTROL + Components Number of tubes per Kit Color Code Label Simplexa Bordetella Primer Mix 10 Brown REF MOL2776 Lot Expires Simplexa Master Mix 10 Green REF MOL2007 Lot Expires Simplexa Extraction and Amplification Control DNA (SEAC) 2 Blue REF MOL9001 Lot Expires Simplexa Bordetella Positive Control (PC) 2 Red REF MOL0221 Lot Expires Kit Component Simplexa Bordetella Primer Mix Reactions per Kit / Vial Volume (µl) per Vial 1000/ Description of the Kit Components Component Description Dye-labeled fluorescent primers specific for detection of Bordetella pertussis, Bordetella parapertussis and for the DNA Internal Control. Probe Excitation Emission Targeted Target Fluorophore (nm) (nm) Gene Bp FAM IS481 Bpp CFR IS1001 SEAC Q N/A Simplexa Master Mix 1000/ DNA polymerase, buffer and dntps Simplexa Extraction and Amplification Control (SEAC) Simplexa Bordetella Positive Control (PC) Simplexa Bordetella Universal Direct Barcode Card variable 250 Internal control DNA amplicon variable 50 IS481 and IS1001 DNA amplicon n/a n/a Assay specific parameters. MATERIALS REQUIRED BUT NOT SUPPLIED 1. Integrated Cycler with Integrated Cycler Studio Software version 3.0 or higher 2. Universal Discs for use on the Integrated Cycler

3 Page 3 3. Universal Disc Sealer 4. Single, multi-channel and/or repeater micropipette(s) with an accuracy range between 1-10 µl, µl and µl 5. Freezer (manual defrost) at -10 to -30 C (for kit component frozen storage) 6. Freezer (manual defrost) at -10 to -30 C (for specimen frozen storage) 7. Refrigerator at 2 to 8 C (for thawed kit components) 8. Biosafety cabinet (laminar flow hood) for extractions and sample handling 9. Microcentrifuge 10. Vortex mixer 11. Sterile, RNase/DNase-free disposable aerosol-barrier micropipettor tips ml polypropylene microcentrifuge tubes and racks (RNase/DNase-free tubes are recommended but not required) ml polypropylene conical tubes and racks 14. Nuclease-Free water, (RNase/DNase free) to be used as the No Template Control (NTC). 15. Disposable, powder-free gloves 16. Cooler racks for 1.5 ml microcentrifuge tubes 17. Universal Disc Cold Block MATERIALS REQUIRED BUT NOT SUPPLIED For use with the EXTRACTION protocol 1. 1X TE (ph 8.0), RNase/DNase free (for Molecular Control preparation for use with EXTRACTION protocol only) 2. Roche MagNA Pure LC System and associated consumables. 3. MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Cat. No ) 4. Simplexa Extraction and Amplification Control Set (REF MOL9000) Note: This is needed if more than 80 reactions will be run using the EXTRACTION protocol SHELF LIFE AND HANDLING 1. Store reagents at -10 to -30 C (do not use a frost-free freezer). 2. Do not use kits or reagents beyond their expiration dates. 3. Allow reagents to thaw at room temperature (approximate range 18 to 25 C) before use. 4. If PCR Setup will not be performed immediately after the Reaction Mix is prepared, store Reaction Mix at 2 to 8 C until ready to proceed with PCR Setup. Use reaction mix within one hour. 5. Once thawed, store the Primer Mix, Master Mix, Positive Control, and SEAC at 2 to 8 C for no more than 30 days. 6. Do not refreeze Primer Mix, Master Mix, SEAC or Positive Control. 7. Do not combine reagents from different kit lots. WARNINGS AND PRECAUTIONS 1. Follow standard precautions. All patient specimens and positive controls should be considered potentially infectious and handled accordingly. 2. Wear personal protective equipment, such as (but not limited to) gloves and lab coats when handling kit reagents. Wash hands thoroughly when finished performing the test. 3. Do not pipette by mouth. 4. Do not smoke, drink, eat, handle contact lenses or apply make-up in areas where kit reagents and/or human specimens are being used. 5. Dispose of unused kit reagents and human specimens according to local, state and federal regulations. 6. Workflow in the laboratory should proceed in a uni-directional manner, beginning in the Pre-Amplification areas and moving to the Amplification/Detection area: Below is the sequence of events that takes place from specimen extraction to Real-Time PCR amplification: Begin with specimen extraction, followed by Real-Time PCR instrument set-up, reagent preparation, and finally Real-Time PCR amplification. Do not use supplies and equipment across the dedicated areas of specimen extraction and sample preparation. No crossmovement is recommended between the different areas. Supplies and equipment used for specimen preparation should not be used for reagent preparation activities or for processing amplified DNA or other sources of target nucleic acid. All amplification supplies and equipment should be kept in the Real-Time PCR Instrument Area at all times. Personal Protective Equipment, such as laboratory coats and disposable gloves, should be area-specific. 7. Contamination of patient specimens or reagents can produce erroneous results. Use aseptic techniques. 8. Pipette and handle reagents carefully to avoid mixing of samples from adjacent wells. 9. Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values. 10. Do not substitute or mix reagent from different kit lots or from other manufacturers. 11. Do not interchange the reagent tube caps. This may cause contamination and compromise the test results. 12. Only use the protocol described in this insert. Deviations from the protocol or the use of times or temperatures other than those specified may give erroneous results.

4 Page Assay setup should be performed on a universal disc cold block (approximate range 2 to 8 C). While mixing the reagents, keep the enzymes cold by utilizing a cooler block. 14. Do not re-use Universal Discs that have already been exposed to patient samples or reagents. 15. Dispose of used disc without detaching or removing cover tape. 16. If different Simplexa kits or lots are set up on the same disc, Positive Control from each kit and No Template Control (Nuclease-Free water) need to be tested. 17. Master Mix contains 1-10% glycerol, which may cause irritation upon inhalation or skin contact. Upon inhalation or skin contact, first aid measures should be taken. 18. Extended storage of extracted specimens at 2 to 8 C is not recommended; performance has not been established. INSTRUCTIONS FOR USE ASSAY PROTOCOL FOR DIRECT SPECIMEN TESTING (DIRECT PROTOCOL) A. SPECIMEN COLLECTION Acceptable specimen types include nasopharyngeal swabs (NPS) in liquid viral transport media (e.g. UTM, VCM, M4). Only use swabs with a synthetic tip (e.g. Dacron, nylon, or rayon) and an aluminum or plastic shaft. Do not use calcium alginate swabs, as they may contain substances that inhibit PCR testing. Aimes liquid transport media is not compatible with the Direct Protocol. B. REAL-TIME PCR INSTRUMENT SETUP 1. Refer to the Integrated Cycler Operator Manual for details on how to configure the Integrated Cycler Studio Software to add an assay definition, set up and analyze runs on the Integrated Cycler. C. REAGENT PREPARATION AREA Dedicated area for preparation of Simplexa TM Bordetella Universal Direct assay reaction mix. 1. Thaw the Primer Mix and the Master Mix at room temperature (approximate range 18 to 25 C). Each kit component vial contains sufficient reagents for 100 reactions. Prior to each use, gently mix the Primer Mix and Master Mix kit components by inverting 6 to 8 times and briefly centrifuge to pull contents down to bottom of tube. 2. Prepare the required volume of the Reaction Mix in an appropriately sized polypropylene microcentrifuge tube by pipetting the volume of each component as indicated in the table below. Reaction Mix Volumes for use with DIRECT Protocol Reagent Reaction Mix Reaction Mix Volume / 1 reaction Volume / 10 reactions Simplexa Master Mix 4 40 Simplexa Bordetella Primer Mix 1 10 Nuclease Free Water SEAC Total Volume Gently mix the Reaction Mix by inversion or by pipetting 8 to 10 times. 4. Briefly centrifuge to pull contents down to bottom of tube. 5. Proceed to PCR Setup. 6. Use the Reaction Mix within one hour of preparation. If PCR Setup will not be performed immediately after the Reaction Mix is prepared, store Reaction Mix at 2 to 8 C until ready to proceed with PCR Setup (within one hour). D. REAL-TIME PCR AMPLIFICATION AREA Perform in a dedicated area for preparation of the 96-well Universal Disc for Simplexa Bordetella Universal Direct assay. Use the universal disc cold block during sample and reaction mix loading. 1. Thaw Positive Control at room temperature (approximate range18 to 25 C). 2. Add 7.0 µl of the reaction mix to each well. 3. Add 3.0 µl of the Positive Control to the PC disc well. 4. Add 3.0 µl of the patient sample to the appropriate S disc well. 5. Add 3.0 µl of No Template Control (Nuclease-Free water) to the NTC disc well. 6. Cover the disc with the Universal Disc Cover Tape. 7. Load the sealed Universal Disc in the Integrated Cycler and start the run. ASSAY PROTOCOL FOR EXTRACTED SPECIMEN TESTING (EXTRACTION PROTOCOL) A. SPECIMEN COLLECTION

5 Page 5 Acceptable specimen types include nasopharyngeal swabs (NPS) in Amies transport media or other liquid viral transport media (e.g. UTM, VCM, M4). Only use swabs with a synthetic tip (e.g. Dacron, nylon, or rayon) and an aluminum or plastic shaft. Do not use calcium alginate swabs, as they may contain substances that inhibit PCR testing. B. SPECIMEN EXTRACTION AREA Perform in a dedicated area for specimen extraction. Sample preparation for extraction should be performed in a biosafety cabinet. POSITIVE CONTROL PREPARATION Prepare Positive Control by adding 5 µl Positive Control to 195 µl 1X TE (ph 8.0). Extraction using Roche MagNA Pure LC extraction method 1. Nucleic acids are extracted from patient specimens and No Template Control using the Roche MagNA Pure Total Nucleic Acid kit and the Roche MagNA Pure LC Automated Nucleic Acid Extractor instrument. Refer to the manufacturer s Instructions for Use for nucleic acid extraction using this kit. 2. Under the Protocol drop-down menu on the MagNA Pure LC System, select Total NA, and then Total NA Variable_elution_volume.blk from the list. This will load the appropriate settings for the run. 3. The Sample Protocol should be Total NA Variable_elution_volume µl should be set for the Sample Volume, and the elution volume should be set at 50 µl. 5. The dilution volume should be set at zero for all samples. 6. Ensure that the Post Elution Protocol is set to None. 7. Ensure that specimens and controls are in the correct position on the Sample Cartridge. 8. Vortex each sample for 2 to 4 seconds and briefly centrifuge to pull contents down to bottom of tube. 9. For each set of 16 samples (1-16 samples), pipette 100 µl of the SEAC DNA into 6 ml MagNA Pure lysis buffer in a conical tube. Mix by vortexing briefly. Add to the appropriate tray on the MagNA Pure extraction instrument. For example, if 32 samples are extracted, pipette 200 µl of SEAC DNA into 12 ml MagNA Pure lysis buffer in a conical tube. Mix by vortexing briefly. Add to the appropriate tray on the MagNA Pure extraction instrument. 10. Pipette 200 µl of each specimen, No Template Control (Nuclease-Free water) and Positive Control into the corresponding position in the sample cartridge. 11. Visually check the level of samples and controls in the Sample Cartridge to ensure sample(s) were added. 12. Transfer the Sample Cartridge containing the samples to the MagNA Pure LC Automated Nucleic Acid extractor and begin the extraction run. 13. After nucleic acid extraction is complete, the cartridge containing the extracted specimens can be removed from the MagNA Pure and sealed. Store the extracted DNA at 2 to 8 C prior to use. Long-term storage of extracted samples at this temperature is not recommended. Keep extracted DNA samples on a cooler block while loading disc. C. REAL-TIME PCR INSTRUMENT SETUP 1. Refer to the Integrated Cycler Operator Manual for details on how to configure the Integrated Cycler Studio Software to add an assay definition, set up and analyze runs on the Integrated Cycler. D. REAGENT PREPARATION AREA Dedicated area for preparation of Simplexa TM Bordetella assay reaction mix. 1. Thaw the Primer Mix and the Master Mix at room temperature (approximate range 18 to 25 C). Each kit component vial contains sufficient reagents for 100 reactions. Prior to each use, gently mix the Primer Mix and Master Mix kit components by inverting 6 to 8 times and briefly centrifuge to pull contents down to bottom of tube. 2. Prepare the required volume of the Reaction Mix in an appropriately sized polypropylene microcentrifuge tube by pipetting the volume of each component as indicated in the table below. Reaction Mix Volumes for use with EXTRACTION Protocol Reagent Reaction Mix Reaction Mix Volume / 1 reaction Volume / 10 reactions Simplexa Master Mix 4.0 µl 40 µl Simplexa Bordetella Primer Mix 1.0 µl 10 µl Total Volume 5.0 µl 50 µl 3. Gently mix the Reaction Mix by inversion or by pipetting 8 to 10 times. 4. Briefly centrifuge to pull contents down to bottom of tube. 5. Proceed to PCR Setup. 6. Use the Reaction Mix within one hour of preparation. If PCR Setup will not be performed immediately after the Reaction Mix is prepared, store Reaction Mix at 2 to 8 C until ready to proceed with PCR Setup (within one hour). E. REAL-TIME PCR AMPLIFICATION AREA Perform in a dedicated area for preparation of the 96-well Universal Disc for Simplexa Bordetella Universal Direct assay.

6 Page 6 Use the universal disc cold block during sample and reaction mix loading. For use with the EXTRACTION Protocol 1. Add 5.0 µl of the reaction mix to each well. 2. Add 5.0 µl of the extracted Positive Control to the PC disc well. 3. Add 5.0 µl of extracted patient sample to the appropriate S disc well. 4. Add 5.0 µl of extracted No Template Control (Nuclease-Free water) to the NTC disc well. 5. Cover the disc with the Universal Disc Cover Tape. 6. Load the sealed Universal Disc in the Integrated Cycler and start the run. REPORTING RESULTS Reporting results is a three step process. 1. Examination of controls to determine if the run is valid. The Integrated Cycler Studio Software will suppress interpretation of patient results if any of the samples programmed as controls are invalid. 2. Examination of validity of patient specimen results. 3. Interpretation of patient results. If the controls are not valid, the patient results cannot be interpreted. 1. Determine if the run is valid by examining the No Template Control, Bordetella Positive Control, and DNA Internal Control. Criteria for a Valid Control (simplified)* Control Bp Ct Bpp Ct DNA IC Ct No Template Control (NTC) , but 0 Positive Control 40, but 0 40, but 0 Not Applicable (N/A) * See notes below for full description. a. If the No Template Control is: i. Positive (Ct value 40, but 0 for Bp or Bpp), then this indicates possible contamination of prepared samples. The control is invalid and all patient specimens must be re-assayed. ii. Negative for Bp and Bpp detector (Ct = 0), then this control is valid and acceptable. iii. If the DNA IC is not detected in the No Template Control, the assay run is invalid and all patient specimens must be re-assayed. iv. If the DNA IC is detected for the No Template Control, the assay run is considered valid and acceptable. b. Positive Control i. If the Positive Control result is a Ct = 0 for Bp or Bpp, the assay run is considered invalid and unacceptable. All patient specimens must be re-assayed. ii. If the Ct values for Bp and Bpp are 40, but 0 the assay run is considered valid and acceptable. 2. Examination of Patient Specimen Results Examination of clinical specimen results should be performed after the Positive and No Template Controls have been examined and determined to be valid and acceptable. Bp, Bpp and DNA IC results must be examined for each patient specimen. a. Amplification plots should be examined for every result with a Data Quality message. A valid amplification curve shows a smooth, exponential increase. An invalid amplification curve may be a non-exponential or linear curve or a curve with data spikes where the curve may cross the threshold. If the curve is valid after examination, the Ct value reported may be used to determine if Bp or Bpp targets are detected as indicated in section 3 below. Examples of valid and invalid curves are shown below.

7 Page 7 Valid Amplification Curve Valid Amplification Curve Invalid Amplification Curve b. If the amplification curve is valid for Bp or Bpp, the DNA IC is not required to be detected to report a positive result. 3. Interpretation of Results Interpretation of Results Bp Bpp DNA IC Example Interpretation Ct value Ct value Ct value , 0 Bp and Bpp Not Detected 2 40, 0 0 N/A Bp Detected , 0 N/A Bpp Detected 4 40, 0 40, 0 N/A Bp and Bpp Detected Ct = cycle threshold. Detected is a Ct 40, 0. Not Detected is a Ct = 0, Detection of the Simplexa DNA Internal Control is not required to be detected to report a positive result. LIMITATIONS 1. For in vitro diagnostic use. 2. For export only. 3. Analysts should be trained and familiar with testing procedures and interpretation of results prior to performing the assay. 4. All results from this and other tests must be used in conjunction with the clinical history, epidemiological data and other data available to the clinician evaluating the patient. 5. The prevalence of infection will affect the test s predictive value. 6. As with other tests, negative results do not rule out Bp or Bpp infections. 7. False-negative results may occur when the infecting organism has genomic mutations, insertions, deletions, or rearrangements or when performed very early in the course of illness. 8. False-negative results may occur if inadequate numbers of organisms are present in the specimen due to improper collection, transport or handling. 9. As with other tests, false-positive results may occur. Repeat testing or testing with a different device may be indicated in some settings. 10. This test is a qualitative test and does not provide the quantitative value of detected organism present. 11. The performance of this test has not been established for patients without symptoms of Bordetella pertussis or Bordetella parapertussis infection. 12. The performance of this test has not been established for monitoring treatment of Bordetella pertussis or Bordetella parapertussis infection. 13. The performance of this test has not been established for screening of blood or blood product for the presence of Bordetella pertussis or Bordetella parapertussis. 14. This test cannot rule out diseases caused by other bacterial or viral pathogens. 15. Dual-infection with both Bordetella pertussis and Bordetella parapertussis is possible 6,7, This test may not distinguish between infection by Bordetella pertussis and Bordetella holmesii. Both organisms contain the IS481 gene. The incidence of Bordetella holmesii in clinical specimens is reported to be very low (<0.5%) and it remains unclear whether Bordetella holmesii can cause respiratory illness in otherwise healthy individuals This test may not distinguish between infection by Bordetella pertussis and Bordetella bronchiseptica. B. pertussis and some strains of B. bronchiseptica contain the IS481 gene. Although rare, B. bronchiseptica infections have been documented in infants and immunocompromised individuals 11. PERFORMANCE CHARACTERISTICS ANALYTICAL SENSITIVITY/LIMIT OF DETECTION Limit of Detection (LoD) is the concentration that results in 95% detection rate. LoD of the Simplexa assay was evaluated using Bordetella pertussis A639, Bordetella pertussis ATCC 8467, Bordetella parapertussis A747, and Bordetella parapertussis E595. Bordetella pertussis A639 LoD was confirmed at 2.5 cfu/ml for the EXTRACTED protocol (20/20 detected) and 250 cfu/ml for the DIRECT protocol (20/20 detected). Bordetella parapertussis A747 LoD was confirmed at 10 cfu/ml for the EXTRACTED protocol (19/20 detected) and 1000 cfu/ml for the DIRECT protocol (19/20 detected). Both Bordetella pertussis ATCC 8467 and Bordetella parapertussis E595 were detected with both protocols. REPRODUCIBILITY Two separate reproducibility studies were conducted. The reproducibility study for the EXTRACTION protocol was performed by two operators, each using a different instrument. Each operator performed a single extraction on each reproducibility panel sample and performed two PCR runs a day for a total of 5 days (n=20 total runs). Each run tested three replicates of each sample and positive control.

8 Page 8 EXTRACTION Protocol Reproducibility - Bp Intra- and Inter-assay Inter-instrument Sample ID n Mean Ct Intraassay SD Intra-assay %CV Interassay SD Inter-assay %CV Mean Ct SD %CV BP-Med BP-Low BPP-Med BPP-Low Negative EXTRACTION Protocol Reproducibility - Bpp Intra- and Inter-assay Inter-instrument Sample ID n Mean Ct Intraassay SD Intra-assay %CV Interassay SD Inter- assay %CV Mean Ct SD %CV BPP-Med BPP-Low BP-Med BP-Low Negative The reproducibility study for the DIRECT protocol was performed by two operators,. Each operator performed 15 runs, one run on each of three instruments per day for a total of 5 days (n=30 total runs). Each run tested three replicates of each sample and positive control. DIRECT Protocol Reproducibility - Bp Inter-Instrument Inter-Operator Inter-Run/Day Intra-Run Total Sample N Mean Ct SD %CV SD %CV SD %CV SD %CV SD %CV Positive Control (PC) BP Medium Positive 89* BP Low Positive DIRECT Protocol Reproducibility - Bpp Inter-Instrument Inter-Operator Inter-Run/Day Intra-Run Total Sample N Mean Ct SD %CV SD %CV SD %CV SD %CV SD %CV Positive Control (PC) BPP Medium Positive 89* BPP Low Positive * One sample replicate was Invalid.

9 Page 9 ANALYTICAL REACTIVITY / CROSS-REACTIVITY Seventeen (17) organisms were tested for potential cross-reactivity with Bordetella pertussis and parapertussis. Results of testing are summarized in the following tables: Analytical Reactivity / Cross-Reactivity - Bp Potential Cross-Reactant EXTRACTION Protocol Results Initial Run; Confirmation Run DIRECT Protocol Results Initial Run; Confirmation Run Bacillus cereus 0/3 0/3 Chlamydophila pneumoniae 0/3 0/3 Corynebacteria diptheria 2/3; 0/5 0/3 Haemophilus influenzae 1/3; 0/5 0/3 Haemophilus parainfluenzae 0/3 2/3; 1/5* Influenza A/Solomon Island/03/06 H1 0/3 1/3; 0/5 Influenza B/Florida/02/2006 0/3 0/3 Klebsiella pneumoniae 0/3 0/3 Legionella pneumophila 0/3 0/3 Moraxella catarrhalis 0/3 0/3 Mycoplasma pneumoniae 0/3 0/3 Neisseria meningitides 0/3 0/3 RSV B WV/14617/85 0/3 0/3 Staphylococcus aureus 1/3; 0/5 0/3 Staphylococcus epidermidis 0/3 0/3 Streptococcus pneumoniae 1/3; 3/5** 0/3 Streptococcus pyogenes 0/3 0/3 *Sequence analysis showed no significant homology between the BP assay design and the H. parainfluenzae genome. **Positive BP results with S. pneumoniae could not be replicated in subsequent testing. Analytical Reactivity / Cross-Reactivity - Bpp Potential Cross-Reactant EXTRACTION Protocol Results Initial Run; Confirmation Run DIRECT Protocol Results Initial Run; Confirmation Run Bacillus cereus 0/3 0/3 Chlamydophila pneumoniae 0/3 0/3 Corynebacteria diptheria 0/3 0/3 Haemophilus influenzae 0/3 0/3 Haemophilus parainfluenzae 0/3 0/3 Influenza A/Solomon Island/03/06 H1 0/3 0/3 Influenza B/Florida/02/2006 0/3 0/3 Klebsiella pneumoniae 0/3 0/3 Legionella pneumophila 0/3 0/3 Moraxella catarrhalis 0/3 0/3 Mycoplasma pneumoniae 0/3 0/3

10 Page 10 EXTRACTION Protocol Results DIRECT Protocol Results Potential Cross-Reactant Initial Run; Confirmation Run Initial Run; Confirmation Run Neisseria meningitides 0/3 0/3 RSV B WV/14617/85 0/3 0/3 Staphylococcus aureus 0/3 0/3 Staphylococcus epidermidis 0/3 0/3 Streptococcus pneumoniae 0/3 0/3 Streptococcus pyogenes 0/3 0/3 INTERFERENCE Fifteen (15) endogenous and exogenous substances were spiked into low positive Bordetella pertussis and parapertussis samples and tested for potential interference in the detection of Bp/Bpp. In the EXTRACTION Protocol, Bordetella pertusiss strain A639 was tested at a concentration of 25 cfu/ml and Bordetella parapertusiss strain A747 was tested at a concentration of 50 cfu/ml. In the DIRECT Protocol, Bordetella pertusiss strain A639 was tested at a concentration of 1250 cfu/ml and Bordetella parapertusiss strain A747 was tested at a concentration of cfu/ml. The table below summarizes the interference study results. Interference Potential Interferent Potential Interferent Concentration EXTRACTION Protocol Results DIRECT Protocol Results Bp Bpp Bp Bpp Ampicillin 25mg/mL 3/3 3/3 3/3 3/3 Azithromycin 25mg/mL 3/3 3/3 3/3 3/3 Beclomethasone 10% v/v 3/3 3/3 N/A N/A Beclomethasone 25 mg/ml N/A N/A 3/3 3/3 Blood 2% v/v 3/3 3/3 3/3 3/3 Ciprofloxacin 25 mg/ml 3/3 3/3 N/A N/A Ciprofloxacin 250 µg/ml N/A N/A 3/3 3/3 Erythromycin 25 mg/ml 3/3 3/3 3/3 3/3 Fluticasone 10% v/v 3/3 3/3 3/3 3/3 Quaifenesin/ dextromethorphan 10% v/v 3/3 3/3 3/3 3/3 Mucin 60ug/mL 3/3 3/3 3/3 3/3 Mupirocin 25mg/mL 3/3 6/8 1 3/3 3/3 Oxymetazoline 10% v/v 3/3 3/3 3/3 3/3 Phenol chloraseptic 10% v/v 3/3 3/3 3/3 3/3 Phenylephrine 10% v/v 3/3 3/3 3/3 3/3 Rifampicin 25 mg/ml 3/3 7/8 2 N/A N/A Rifampicin 250 µg/ml N/A N/A 3/3 3/3 Sodium chloride 10% v/v 3/3 7/8 2 3/3 3/3 1 One of three initial replicates and one of five additional replicates (used to confirm possible interference) did not detect Bpp. 2 One of three initial replicates did not detect Bpp; however, Bpp was detected in five additional replicates (used to confirm possible interference). CLINICAL CONCORDANCE A total of 222 samples, comprised of Bordetella pertussis positive (spiked and endogenous) and negative samples and Bordetella parapertussis positive (spiked and endogenous) and negative samples, were tested using the Focus Diagnostics Bordetella kit (MOL0200) and the Simplexa Bordetella Universal Direct kit using the EXTRACTION protocol (MOL2700 / MOL2775). The

11 Page 11 results of the method comparison are summarized below. Clinical Concordance Results for Bp Predicate (MOL0200) Simplexa Bordetella (MOL2700) n Detected Not Detected Indeterminate %Agreement(Observed/Expected) 95% CI Detected %(65/66) 95% CI: % Not Detected %(151/156) 95% CI: % Results for Bpp Predicate (MOL0200) Simplexa Bordetella (MOL2700) n Detected Not Detected Indeterminate %Agreement(Observed/Expected) 95% CI Detected %(71/71) 95% CI: % Not Detected %(148/151) 95% CI: % METHOD COMPARISON EXTRACTION Protocol vs. DIRECT Protocol In order to demonstrate the relationship between the results obtained with the EXTRACTION protocol and DIRECT protocol a comparative analysis of the Ct values obtained from a combination of spiked and endogenous positive samples was under taken. The Ct values of the samples that were positive using the EXRACTION protocol were compared to the Ct values for the DIRECT protocol for the same specimens. For Bordetella pertussis the slope of the regression line is 0.98 with an intercept of For Bordetella parapertussis the slope of the regression line is 1.04 with an intercept of In each case, the slope of the regression line indicates a good correlation between the two protocols. Figure 11: Passing-Bablok Regression - Bordetella pertussis

12 Page 12 Figure 22: Passing-Bablok Regression - Bordetella parapertussis AMPLIFICATION CARRY-OVER CONTAMINATION Amplification carry-over using the Universal Disc and Universal Disc Cover Tape has been evaluated with other Simplexa assays and is not assay dependent. REFERENCES 1. Mattoo & Cherry. Clin Microbiol Rev : Centers for Disease Control Data available at: 3. National Consensus Conference on Pertussis. Available at: 4. Kerr et al., Eur J Clin Microbiol Infect Dis : Linneman et al., Am J Dis Child : Mertsola, J. Eur J Clin Microbiol : Hoppe, J.E. Pediatr Infect Dis : Iwatta et al. Dev Biol Stand : Loeffelholz, M.J et al. J. Clin Microbiol : Centers for Disease Control and Prevention. Morbidity Mortality Weekly Report (MMWR) 2007 Aug 24; 56: Register, J.B. and Sanden, G.N., Journal of Clinical Microbiology, Dec 2006, p The use of Scorpions Probes for human in vitro diagnostic purposes is covered by a license to Focus Diagnostics, Inc. from DxS, Ltd. Black Hole Quencher, CAL Fluor, Quasar dyes are trademarks of Biosearch Technologies, Inc. ('BTI'). Black Hole Quencher, CAL Fluor and Quasar dye technology is licensed pursuant to an agreement with BTI, and these products are sold exclusively for clinical, diagnostic, or research and development purposes. AUTHORIZED REPRESENTATIVE mdi Europa GmbH, Langenhagener Str , Langenhagen-Hannover, Germany ORDERING INFORMATION Telephone: Fax: TECHNICAL ASSISTANCE (800) (U.S.A. only) (562) (562) (International) PI.MOL2775 Rev. A Date written: 08 June 2012 Telephone: (800) (U.S.A. only) Fax: (562) Visit our website at (562) (International) Cypress, California USA