Simplexa Flu A/B & RSV Direct

Transcription

1 Simplexa /B & Direct REF MOL2650 Rev. B A real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B & viral RNA. For in vitro Diagnostic Use INTENDED USE The Focus Diagnostics Simplexa TM /B & Direct assay is intended for use on the 3M Integrated Cycler instrument for the in vitro qualitative detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus () RNA in nasopharyngeal swabs (NPS) from human patients signs and symptoms of respiratory tract infection in conjunction clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza A, influenza B, and viral infections in humans and is not intended to detect influenza C. Negative results do not preclude influenza virus or infection and should not be used as the sole basis for treatment or other patient management decisions. Performance characteristics for influenza A were established clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary. If infection a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected appropriate infection control precautions for novel virulent Influenza viruses and sent to the state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. SUMMARY AND EXPLANATION Influenza is caused by three immunologic types (A, B, and C) of RNA viruses in the Orthomyxoviridae family. Influenza A is classified further by describing two viral proteins expressed on its surface, hemagglutinin and neuraminidase. Hemagglutinin facilitates binding of the virus to respiratory epithelial cells, whereas neuraminidase functions to break those bonds the host cell so that new virions can be released. Seasonal influenza is typically caused by viruses that contain one of three major subtypes of hemagglutinin (H1, H2, or H3) and one of two subtypes of neuraminidase (N1 or N2). Influenza B is not classified into subtypes. 1 Influenza classically presents a combination of upper and lower respiratory signs and symptoms, fever, headache, myalgia, and general malaise. Illness can take on a variety of appearances, ranging from isolated respiratory findings that resemble the common cold, to severe pneumonia requiring hospitalization. Persons at higher risk for hospitalization from seasonal influenza include children <2 years of age, adults >65 years of age, and those significant comorbidities. Flu may cause exacerbation of underlying medical conditions, such as asthma or congestive heart failure. The duration of illness is typically 2-5 days, but symptoms may last for a week or longer. infection is more prevalent in infants and toddlers and is a leading cause of hospitalization in this age group, but also causes disease in adults that can be severe in certain populations. 1 In infants and young children, disease can range from a cold-like illness, bronchitis, or croup, to lower respiratory infections such as bronchiolitis and pneumonia. In adults, symptomatic infection usually presents as an upper respiratory tract illness runny nose (rhinorrhea), sore throat (pharyngitis), and cough, some patients also complaining of headache, fatigue, and fever. High-risk adults, such as those certain chronic illnesses or immunosuppression, may have more severe disease, such as pneumonia. 2 PRINCIPLES OF THE PROCEDURE The Simplexa /B & Direct assay system is a real-time RT-PCR system that enables the direct amplification, detection and differentiation of human influenza A () virus RNA, human influenza B () virus RNA and RNA from unprocessed nasopharyngeal swabs that have not undergone nucleic acid extraction. The system consists of the Simplexa /B & Direct assay, the 3M Integrated Cycler ( Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories. In the Simplexa /B & Direct assay, bi-functional fluorescent probe-primers are used together corresponding reverse primers to amplify,, and internal control RNA. The assay provides three results; conserved regions of influenza A viruses (matrix gene), influenza B viruses (matrix gene) and (M gene) are targeted to identify these viruses in the specimen. An RNA internal control is used to detect RT-PCR failure and/or inhibition.

2 Simplexa /B & Direct Page 2 MATERIALS PROVIDED The Focus Diagnostics Simplexa /B & Direct kit contains sufficient reagents for 24 reactions. Upon receipt, store at -10 to -30 ºC (do not use a frost-free freezer). Each vial contains sufficient material for one use. Use in 30 minutes of removing from the freezer. Kit Description Component Name REF EC SYMBOL ON LABEL Abbreviated Name Cap Color Number of Vials Reactions per Vial/Kit Volume per Vial Simplexa /B & Direct Reaction Mix MOL2651 REAG A RM Brown 24 1/24 50 µl Kit Component Component Description Contents DNA polymerase, Reverse Transcriptase, RNase inhibitor, buffer and dntps, encapsulated RNA Template, Dye-labeled fluorescent primers specific for detection of Influenza A, Influenza B and and for the Internal Control Probe Target Fluorophore (Dye) Excitation Emission Targeted Gene Simplexa /B & Direct Reaction Mix (RM) FAM matrix JOE matrix CFR M gene Internal Control RNA IC Q N/A Simplexa /B & Direct Barcode Card Assay specific parameters, lot number, expiration date MATERIALS SUPPLIED SEPARATELY 1. Direct Amplification Disc Kit (REF MOL1455) a) Direct Amplification Discs for use on the Integrated Cycler MATERIALS REQUIRED BUT NOT SUPPLIED 1. 3M Integrated Cycler Integrated Cycler Studio Software version 4.2 or higher 2. Simplexa /B & Positive Control Pack (REF MOL2660) µl fixed volume pipette (VWR Signature Fixed Volume Ergonomic High-Performance Pipettor Model VWR FE50 or equivalent) 4. Sterile, nuclease-free disposable pipette tips filters (Extra Long tips 91 mm are recommended for pipetting directly from primary collection tubes for NPS). 5. Freezer (manual defrost) at -10 to -30 C (for kit component and specimen frozen storage) 6. Refrigerator at 2 to 8 C (for specimens) 7. Disposable, powder-free gloves RECOMMENDED MATERIALS 1. Universal Transport Media (UTM) to be used as a No Template Control (NTC). 2. Replacement Foil Wedges (REF MOL1550) REAGENT HANDLING AND STORAGE 1. Store reagents at -10 to -30 C (do not use a frost-free freezer). 2. Allow reagents to thaw at room temperature (approximate range 18 to 25 C) before use. 3. Do not use kits or reagents beyond their expiration dates. 4. After removing Reaction Mix from freezer storage, initiate the test in 30 minutes. 5. Do not vortex the Reaction Mix. 6. Do not refreeze the Reaction Mix.

3 Simplexa /B & Direct Page 3 WARNINGS AND PRECAUTIONS 1. 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. 2. Do not smoke, drink, eat, handle contact lenses or apply make-up in areas where kit reagents and/or human specimens are being used. 3. Dispose of unused kit reagents and human specimens according to local, state and federal regulations. 4. Contamination of patient specimens or reagents can produce erroneous results. Use aseptic techniques. 5. 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. 6. Assay setup should be performed at room temperature (approximate range 18 to 25 C). 7. Use fixed volume pipettes or equivalent to transfer sample and reaction mix. 8. Avoid touching the under side of the foil that will be in contact the wells and disc surface. 9. To prevent potentially erroneous results, make sure that the sample and reagent are added to the appropriate input wells. 10. Finish loading and applying adhesive foil cover to one wedge before opening the foil of adjacent wedge. 11. Initiate the run in 30 minutes of removing the Reaction-mix vial from the freezer. 12. Do not attempt to re-use a wedge that has been used in previous runs or remove adhesive foil cover from a wedge that has been used. 13. Discs may be reused until all 8 wedges have been used. Dispose of used discs out detaching foil cover. 14. Reaction Mix contains > 1% glycerol, which may cause irritation upon inhalation or skin contact. Upon inhalation or skin contact, first aid measures should be taken. 15. If kit packaging or contents appear to be broken or damaged do not use and contact Focus Diagnostics. Contact information is on the last page of this document. INSTRUCTIONS FOR USE A. SPECIMEN COLLECTION AND HANDLING Acceptable specimen types include nasopharyngeal swabs (NPS) in approximately 3mL of Universal Transport Media (UTM). Use only swabs 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. Specimens should be transported on ice and stored at 2 to 8 C, if there is a long delay before processing specimens may be frozen at -70 C in accordance the UTM instructions for use. 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. DIRECT AMPLIFICATION DISC LOADING AND REAL-TIME PCR AMPLIFICATION NOTE: No sample extraction is needed prior to PCR amplification step. 1. Select samples that need to be tested. 2. Thaw reaction-mix vials at room temperature (approximate range 18 to 25 C). Thaw one reaction-mix vial for each sample or control to be tested. 3. Scan the barcode on the Simplexa /B & Direct Reaction Mix vial or barcode card. 4. Scan the disc barcode on the Direct Amplification Disc (DAD). 5. Scan or type in each sample identifier. 6. For one wedge at a time, peel the adhesive foil back to expose the Sample (SAMPLE) and Reaction (R) wells out completely removing the adhesive foil cover.(figure 1 & 2) Avoid touching the under side of the foil that will be in contact the wells and disc surface. 7. Ensure that the reaction mix is completely thawed. Briefly spin down the tubes as needed. (Do not vortex the reaction mix) 8. Use the fixed volume pipette to transfer 50 µl of the reaction-mix into Reaction (R) well. 9. Use the fixed volume pipette to transfer 50 µl of samples or control; pipette sample or control into Sample well (SAMPLE). 10. Cover the wedge sealing the wells the peeled adhesive foil, pressing down firmly near the edge of the wedge. If the original foil is torn it should be replaced an extra Replacement Foil Wedge. 11. Tear off the tab portion of the foil cover along the perforation. 12. Repeat steps 6 to 11 for the next sample(s). 13. Load the sealed Direct Amplification Disc into the Integrated Cycler and start the run.

4 Simplexa /B & Direct Page 4 Figure 1 - Disc pre-use foil lifted from Sample and Reaction Wells for wedge #3 Figure 2 Sample [Sample] and Reaction [R] Wells NOTES (for informational purposes - no user action/interpretation required): 1. Focus Diagnostics kits may contain version numbers for Assay Definitions. If the version number exists, it will be appended to the Assay Definition i.e. Sample IVD Assay.2. When multiple versions exist, the software automatically uses the assay definition associated the scanned lot number. QUALITY CONTROL Simplexa TM /B & Positive Control Pack (MOL2660) may be used as an external control for QC testing, training or proficiency testing. Universal Transport Media is recommended as a No Template Control (NTC). Quality control ranges have been established as indicated in the table below. If the controls are not in these parameters, patient results should be considered invalid and the assay repeated. Focus recommends testing controls once per day. Each laboratory should establish its own Quality Control ranges and frequency of QC testing based on applicable local laws, regulations and standard good laboratory practice. Refer to the Simplexa TM /B & Positive Control package insert (PI.MOL2660) for instructions on running the positive control. Control Control Type RNA Internal Control (RNA IC) Simplexa /B & 1 Positive Control Detected Detected Detected Not applicable2 No Template Control (NTC) Not Detected Not Detected Not Detected Valid 1 Typical Ct values for the Positive Control range between 25 to Detection of the Simplexa RNA Internal Control (RNA IC) is not required for a valid result. RESULTS Upon completion of the run, the software automatically interprets and displays results. 1. For each accession ID (Sample ID) entered, software displays a result ( Detected, Not Detected or Invalid ) for and. a. Detected result indicates the presence of and/or and/or RNA in the patient sample. b. Not Detected result indicates the absence of and/or and/or RNA in the patient sample. c. Invalid result indicates inability to conclusively determine presence or absence of and/or and/or RNA in the patient sample. This result maybe due to 1) Internal Control (RNA IC) failure, or 2) failure to detect sufficient specimen. The sample needs to be retested. See Invalid section below. d. EC500 result points to a data quality error for the particular viral analyte(s). The software was unable to determine a valid amplification for that analyte(s). The sample should be re-tested if that particular virus(s) was requisitioned for testing. 2. Print the report as needed. a. Export the results as needed. INVALID RESULTS In case of an Invalid result or an error code, retest the sample a new reaction-mix vial from the same kit or a new kit. If the problem is unresolved, contact Focus Diagnostics Technical Services department. LIMITATIONS 1. For in vitro diagnostic use. 2. All results from this and other tests must be considered in conjunction the clinical history, epidemiological data and other data available to the clinician evaluating the patient. 3. The prevalence of viral infections may affect the test s predictive value.

5 Simplexa /B & Direct Page 5 4. As other tests, negative results do not rule out, or infections and should not be used as the sole basis for treatment or other patient management decisions. 5. False-negative results may occur if the virus has genomic mutations, insertions, deletions, or rearrangements or if performed very early in the course of illness. 6. The detection of viral nucleic acid is dependent upon proper sample collection, transport, handling, storage and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results. 7. False-negative results may occur if the viruses are present at a level that is below the analytical sensitivity of the assay. 8. As other tests, false-positive results may occur. Repeat testing or testing a different device may be indicated in some settings. 9. Viral nucleic acids may persist in vivo independent of virus viability. Detection of target analyte(s) does not imply that the corresponding viruses are infectious or are the causative agent for clinical symptoms. 10. This test is a qualitative test and does not provide the quantitative value of detected organisms present. 11. This test cannot rule out infections caused by other viral or bacterial pathogens. 12. Information on the kit barcode can only be transferred into the Integrated Cycler Studio through a bar-code scanner. If the scanner is not working, or if you are unable to transfer the information for any reason, contact Focus Diagnostics Technical Services. 13. The performance of this test has not been established for screening of blood or blood products for the presence of influenza A, influenza B, or. 14. The performance of this test has been evaluated for use human specimen material only. 15. The performance of this test has not been established for immunocompromised individuals. 16. The performance of this test has not been established for patients out symptoms of viral respiratory tract infection. 17. The performance of this test has not been established for monitoring treatment of influenza A, influenza B, or infection. 18. The performance of this test has not been established for individuals who have received an inhaled influenza vaccine. 19. The performance of this test has not been evaluated for sample types other than nasopharyngeal swabs. 20. The performance of this test has not been established for individuals using throat lozenges or products containing menthol. 21. When very high levels of influenza A are present very low levels of or influenza B, the signal from the or influenza B reaction may not be adequate to be detected, due to competitive interference. PERFORMANCE CHARACTERISTICS CLINICAL AGREEMENT PROSPECTIVE STUDY Three external testing sites and one internal site participated in a prospective clinical study. Reference results for influenza A, influenza B viruses and respiratory syncytial virus were generated using culture. Culture results were carried forward from the results obtained at the time of sample collection. A total of 722 nasopharyngeal swabs specimens were obtained from prospectively collected specimens from patients signs and symptoms of viral respiratory tract infection. Prospective samples were collected in Eastern United States from 10-November-2010 to 11-March-2011, the Mid-Western United States from 08- January-2011 to 02-February 2011 and in Australia from 17-August-2010 to 20-October Of the 722 specimens 325 specimens were collected from female patients and 397 specimens were collected from male patents. A total of 327 specimens were from patients <5 years of age, 223 specimens were from patients between 5-22 years of age, 158 specimens were from patients between 22 to 60 years of age and 14 specimens were from patients >60 years of age, One (1) sample was excluded from the prospective analysis due to Invalid result for, three (3) samples were excluded due to an invalid result for and one (1) sample were excluded due to an invalid result for. During the study the percentage of specimens invalid results was 0.4% (3/722) a of 0.1% to 1.2%. Clinical (Prospective All sites combined) Simplexa - Sensitivity/Specificity n Detected Not Detected Detected Not Detected Sensitivity: 97.1%(66/68) : 89.9 to 99.2% Specificity: 97.9%(639/653) : 96.4 to 98.7% Clinical (Prospective All sites combined) Simplexa - FluB Sensitivity/Specificity N Detected Not Detected Detected Not Detected Sensitivity: 100.0%(21/21) : 84.5 to 100.0% Specificity: 99.9%(697/698) : 99.2 to 100.0%

6 Simplexa /B & Direct Page 6 Clinical (Prospective Site 1) Simplexa N Detected Not Detected Sensitivity/Specificity Detected Not Detected a 323 Sensitivity: 100.0%(1/1) : 20.7 to 100.0% Specificity: 98.2%(323/329) : 96.1 to 99.2% a) 4/6 samples were confirmed as positive by an FDA cleared NAT Clinical (Prospective Site 2) Simplexa N Detected Not Detected Sensitivity/Specificity a Sensitivity: 98.6%(72/73) Detected : 92.6 to 99.8% Not Detected b 154 Specificity: 89.5%(154/172) : 84.1 to 93.3% a) 1/1 sample confirmed as positive by an FDA cleared DFA b) 11/18 samples confirmed as positive by an FDA cleared DFA Clinical (Prospective Site 3) Simplexa N Detected Not Detected Sensitivity/Specificity Detected Not Detected a 115 Sensitivity: 90.0%(9/10) : 59.6 to 98.2% Specificity: 84.6%(115/136) : 77.5 to 89.7% a) 20/21 samples confirmed as positive by an FDA cleared NAT CLINICAL AGREEMENT RETROSPECTIVE STUDY Three external testing sites participated in a retrospective clinical study. Reference results for influenza A, influenza B viruses and respiratory syncytial virus were generated using culture. Culture results were carried forward from the results obtained at the time of sample banking. A total of 223 nasopharyngeal swabs specimens were obtained from retrospectively banked specimens from patients signs and symptoms of viral respiratory tract infection. Clinical (Retrospective All sites combined) Simplexa PPA/NPA* n Detected Not Detected Detected Not Detected PPA: 96.2% (76/79) : 89.4 to 98.7% NPA: 99.3% (143/144) : 96.2 to 99.9% * PPA = Positive Percent, NPA = Negative Percent Clinical (Retrospective All sites combined) Simplexa PPA/NPA* N Detected Not Detected Detected Not Detected PPA: 97.6% (40/41) : 87.4 to 99.6% NPA: 100.0% (182/182) : 97.9 to 100.0% * PPA = Positive Percent, NPA = Negative Percent

7 Simplexa /B & Direct Page 7 Clinical (Retrospective All sites combined) Simplexa PPA/NPA* N Detected Not Detected Detected Not Detected PPA: 100.0%(12/12) : 75.7 to 100.0% NPA: 98.6%(208/211) : 95.9 to 99.5% * PPA = Positive Percent, NPA = Negative Percent REPRODUCIBILITY Three investigative sites assessed the device's inter-site, inter-day and inter/intra-assay reproducibility. Each of the laboratories tested a panel of seven contrived sample pools including a low (approximately 2-4 times LoD) and medium positive (approximately 20 times LoD) for each analyte and a high negative. The high negative sample contained a small amount of influenza A, influenza B and, it was designed to be negative approximately 95% of the time. The assays were performed in triplicate on five different days. Each site had two operators who each assayed the entire sample panel once per day, for a total of two sets of data per day. Combined results for all sites are presented in the tables below. Sample Site 1 Site 3 Site 4 Mean Ct* Mean Ct* Mean Ct* Total Low Positive (90/90) 95.9 to Medium Positive (90/90) 95.9 to Low Positive (90/90) 95.9 to Medium Positive (90/90) 95.9 to Low Positive (90/90) 95.9 to Medium Positive (90/90) 95.9 to High Negative 90% (27/30) 96.7% (87/90) 90.7 to 98.9% Total (210/210) 98.6% (207/210) (210/210) 99.5% (627/630) 98.6 to 99.8% Samples that are negative for an analyte are assigned a value of 40.1 for the purposes of statistical analysis only. Samples that are negative have a Ct value of zero (0). Sample Low Positive Medium Positive Low Positive Medium Positive Site 1 Site 3 Site 4 Mean Ct Mean Ct 96.7% (29/30) Mean Ct Total (90/90) 95.9 to (90/90) 95.9 to % (89/90) 94 to 99.8% (90/90) 95.9 to

8 Simplexa /B & Direct Page 8 Sample Low Positive Medium Positive High Negative Site 1 Site 3 Site 4 Mean Ct 96.7% (29/30) Mean Ct 93.3% (28/30) Mean Ct Total (90/90) 95.9 to (90/90) 95.9 to 96.7% (29/30) 95.6% (86/90) 89.1 to 98.3% Total 99.5% (209/210) 98.6% (207/210) 99.5% (209/210) 99.2% (625/630) 98.2 to 99.7% Samples that are negative for an analyte are assigned a value of 40.1 for the purposes of statistical analysis only. Samples that are negative have a Ct value of zero (0). Site 1 Site 3 Site 4 Total Sample Mean Mean Mean Ct Ct Ct Low Positive Medium Positive Low Positive Medium Positive Low Positive Medium Positive High Negative 90% (27/30) Total (210/210) 98.6% (207/210) 99.5% (209/210) 99.4% (626/630) 98.4 to 99.8% (90/90) 95.9 to (90/90) 95.9 to (90/90) 95.9 to (90/90) 95.9 to (90/90) 95.9 to (90/90) 95.9 to 96.7% (29/30) % (86/90) 89.1 to 98.3% Samples that are negative for an analyte are assigned a value of 40.1 for the purposes of statistical analysis only. Samples that are negative have a Ct value of zero (0). ANALYTICAL SENSITIVITY/LIMIT OF DETECTION The Limit of Detection (LoD) was determined for the Simplexa /B & Direct assay using quantified stocks of influenza A, influenza B and virus strains serially diluted in negative swab matrix. The lowest concentration 95% detection (at least 19 out of 20 replicates) was determined to be the limit of detection for each assay. Simplexa /B & Direct Limit of Detection Viral Strain LoD (TCID 50 /ml) Influenza A/PR/8/34 (H1N1) Influenza A/Hong Kong/8/68 (H3N2) 10 Influenza A/Swine NY/02/2009 (H1N1) 0.1 Influenza B/Great Lakes/1739/54 2 Influenza B/Malaysia/2506/ A2 1 B CH93-18(18) 3

9 Simplexa /B & Direct Page 9 ANALYTICAL REACTIVITY / CROSS REACTIVITY Analytical Reactivity Different strains of influenza A including H1, H3 and H5 subtypes, influenza B and including A and B subtypes were evaluated. The most recent strains and geographically diverse strains were chosen. Quantified viral material was spiked into negative swab matrix at a single dilution a concentration of approximately 1.0 x10 2 or 1.0 x10 3 TCID 50 /ml and assayed in triplicate. Ct values obtained during testing indicate all viral strains were tested near the LoD. All strains tested were appropriately detected. Analytical Reactivity Additional Viral Strains Viral Strain Concentration Tested (TCID 50 /ml) Result Influenza A Brisbane/10/07 H Detected Influenza A Brisbane/59/07 H Detected Influenza A New Caledonia/20/99 H1N Detected Influenza A Port Chalmers/1/73 H3N Detected Influenza A Solomon Island/03/06 H Detected Influenza A Taiwan/42/06 H1N Detected Influenza A Wisconsin/67/05 H Detected Influenza A WS/33 H1N Detected Influenza A/California/7/2009 NYMC x-179-a Detected Tissue Culture Adapted Influenza A/Swine H1N1/Iowa/15/ Detected Tissue Culture Adapted Influenza A Swine H1N1/USA/1976/ Detected Influenza A PR8 Vietnam/1203/2004 (H5N1 - inactivated virus) unknown Detected Influenza B Allen/ Detected Influenza B Florida/02/ Detected Influenza B Florida/04/ Detected Influenza B Florida/07/ Detected Influenza B Hong Kong/5/ Detected Influenza B Lee/ Detected Influenza B Maryland/1/ Detected Influenza B Panama/45/ Detected Influenza B Taiwan/2/ Detected A Long Detected B Detected B Wash/18537/ Detected B WV/14617/ Detected Cross Reactivity (Analytical Specificity) The Simplexa assay s analytical specificity was evaluated by testing the ability to exclusively identify influenza A virus and/or influenza B virus and/or no cross reactivity to organisms that are closely related, or cause similar clinical symptoms, or present as normal flora in the specimen types of interest. The panel of thirty-two (32) potential cross reactants were individually spiked into a swab matrix at clinically relevant concentrations. The unspiked matrix was also tested to serve as a baseline. Samples were tested in triplicate to screen for cross reactivity. If signal was detected in any detection channel (,, ) in any of the three replicates, an additional 5 replicates were tested for confirmation. No cross reactivity was detected for, or.

10 Simplexa /B & Direct Page 10 Cross Reactant Simplexa TM /B & Direct Cross Reactivity Testing * Concentration Units Adenovirus TCID 50 /ml Adenovirus 7A TCID 50 /ml Bordetella pertussis A cfu/ml Chlamydia pneumoniae copies/ml 1 Cytomegalovirus (CMV) TCID 50 /ml Coronavirus 229E TCID 50 /ml Coronavirus OC TCID 50 /ml Corynebacterium diphtheriae cfu/ml Enterovirus TCID 50 /ml Epstein-Barr Virus (EBV) copies/ml 2 Escherichia coli O cfu/ml Haemophilus influenzae cfu/ml Lactobacillus plantarum, cfu/ml Legionella longbeachae cfu/ml Measles virus TCID 50 /ml Metapneumovirus TCID 50 /ml Moraxella catarrhalis Ne cfu/ml Mumps virus TCID 50 /ml Mycobacterium tuberculosis cfu/ml Mycoplasma pneumoniae M TCID 50 /ml Neisseria elongata cfu/ml Neisseria meningitidis cfu/ml Parainfluenza type TCID 50 /ml Parainfluenza type TCID 50 /ml Parainfluenza type TCID 50 /ml Pseudomonas aeruginosa cfu/ml Rhinovirus 1A TCID 50 /ml Staphylococcus aureus, COL cfu/ml Staphylococcus epidermidis cfu/ml Streptococcus pneumoniae cfu/ml Streptococcus pyogenes cfu/ml Streptococcus salivarius cfu/ml * The minus symbol ( ) indicates Not Detected 1) C. pnuemoniae material used had a titer of 1.52 x 10 5 IFU/mL, it was further quantified using a real-time qpcr assay. 2) EBV was quantified by the vendor using a real time qpcr assay. INTERFERENCE The performance of this assay was evaluated potentially interfering substances that may be present in nasopharyngeal swabs at the concentrations indicated in the table below. The potentially interfering substances were evaluated in a contrived sample that contained influenza A (influenza A/PR/8/34 H1N1) at a concentration of 0.01 TCID 50 /ml and influenza B (influenza B/Malaysia/2506/2004) at a concentration of 40 TCID 50 /ml and A2 at a concentration of 4 TCID 50 /ml. All strains were tested at two to four times the LoD. There was no evidence of interference caused by the substances tested. Potential Interferents Active Ingredient Interferent Concentration Afrin Nasal Spray Oxymetazoline 15% (v/v) Antibacterial, systemic Tobramycin 4 µg/ml Antibiotic, nasal ointment Mupirocin 6.6 mg/ml Blood N/A 2%(v/v) Purified Mucin Protein Bovine Submaxillary Gland Type I-S 60 µg/ml Nasal Corticosteroid - Beconase AQ Beclomethasone 5% (v/v)

11 Simplexa /B & Direct Page 11 Potential Interferents Active Ingredient Interferent Concentration Nasal Corticosteroid - Fluticasone Fluticasone 5% (v/v) Relenza Antiviral Drug Zanamivir 3.3 mg/ml Tamiflu Antiviral Drug Oseltamivir 1 µm Zicam Nasal Gel Luffa Opperculata, Galphimia glauca, histaminum hydrochloricum 5% (v/v) COMPETITIVE INTERFERENCES The Competitive Interference study evaluated the effects of clinically relevant co-infections each of the analytes probed by the assay. The study assessed whether a high concentration of one virus in the specimen could potentially affect the Simplexa TM assay performance for another target present at low levels in the multiplex assay. A low sample Baseline Strain was contrived at approximately two to four times the LoD for each target (influenza A, influenza B and ), and a baseline Ct was determined for each sample. Each potential concomitant infecting virus was spiked into the low level specimen according to the table below. When two viruses were present, high levels of or influenza B did not interfere detection of influenza A; high levels of influenza A may interfere the detection of very low concentrations of influenza B or, Baseline Strain Influenza A/PR/8/34 H1N1 Baseline Concentration [TCID 50 /ml] 0.01 Competitive Interferent Strain Influenza B/GL/1739/54 Competitive Interferent Result (# Detected/# Total) Concentration [TCID 50 /ml] /3 3/3 0/ A /8 0/8 8/ B Ch93-18(18) /8 0/8 8/8 Influenza B/GL/1739/54 8 Influenza A/PR/8/34 H1N /3 3/3 0/3 8 A /3 3/3 3/3 8 B Ch93-18(18) /3 3/3 3/3 A2 B Ch93-18(18) Influenza A/PR/8/34 H1N1 Influenza B/GL/1739/54 Influenza A/PR/8/34 H1N1 Influenza B/GL/1739/ /3 0/3 3/ /3 3/3 3/ /3 0/3 3/ /8 8/8 7/8 INHIBITION BY OTHER MICROORGANISMS The Simplexa assay was evaluated by testing the ability to identify influenza A virus, influenza B virus, and when potentially inhibitory organisms are present. The panel of thirty two (32) potentially inhibitory organisms was individually spiked into a pool a low concentration (approximately two times LoD) of influenza A (Influenza A/PR/8/34 H1N1), influenza B (Influenza B/Malaysia/2506/2004) and (A2). Samples were tested in triplicate to screen for inhibition. If signal was not detected in any detection channel (,, ) in any of the three replicates, an additional five replicates were tested for confirmation. No inhibitory effects were confirmed for influenza A, influenza B, or at the concentrations tested. Microorganism Microorganism Concentration Concentration Units Adenovirus x10 5 TCID 50 /ml Adenovirus 7A 1.1 x10 5 TCID 50 /ml Bordetella pertussis 1.1 x10 6 cfu/ml Chlamydia pneumoniae 1.1 x10 6 copies/ml Cytomegalovirus (CMV) 1.1 x10 5 TCID 50 /ml + + +

12 Simplexa /B & Direct Page 12 Microorganism Microorganism Concentration Concentration Units Coronavirus 229E 1.1 x10 5 TCID 50 /ml Coronavirus OC x10 5 TCID 50 /ml Corynebacterium diphtheriae 1.1 x10 6 cfu/ml E. coli O x10 6 cfu/ml Epstein-Barr Virus (EBV) 1.1 x10 5 copies/ml Enterovirus x10 5 TCID 50 /ml Haemophilus influenzae 1.1 x10 6 cfu/ml Lactobacillus plantarum, x10 6 cfu/ml Legionella longbeachae 1.1 x10 6 cfu/ml Measles virus 1.1 x10 5 TCID 50 /ml Metapneumovirus 1.1 x10 5 TCID 50 /ml Moraxella catarrhalis Ne x10 6 cfu/ml Mumps virus 1.1 x10 5 TCID 50 /ml Mycobacterium tuberculosis 1.1 x10 6 cfu/ml Mycoplasma pneumoniae M x10 6 TCID 50 /ml Neisseria elongata 1.1 x10 6 cfu/ml Neisseria meningitidis 1.1 x10 6 cfu/ml Parainfluenza type x10 5 TCID 50 /ml Parainfluenza type x10 5 TCID 50 /ml Parainfluenza type x10 5 TCID 50 /ml Pseudomonas aeruginosa 1.1 x10 6 cfu/ml Rhinovirus 1A 1.1 x10 5 TCID 50 /ml Staphylococcus aureus, COL 1.1 x10 6 cfu/ml Staphylococcus epidermidis 1.1 x10 6 cfu/ml Streptococcus pneumoniae 1.1 x10 6 cfu/ml Streptococcus pyogenes 1.1 x10 6 cfu/ml Streptococcus salivarius 1.1 x10 6 cfu/ml ) Initial testing appeared to show possible inhibition, upon repeat testing there was no evidence of inhibition. The C. pneumoniae used had a titer of 1.52 x 10 5 IFU/mL it was further quantified using a real-time qpcr assay. 2) EBV was quantified by the vendor using a real-time qpcr assay. CARRY-OVER CONTAMINATION An internal carry-over study searched for the presence of contamination in negative samples. The study was designed by alternately placing a high positive and a negative sample on each disc. The carryover effect was evaluated by comparing the observed negative rate for the negative sample the expected rate under normal reproducibility conditions. No carry-over contamination effect was seen in the, or channels. EXPECTED VALUES The prevalence of influenza varies each year flu-season occurring during the fall and winter months in the US. Variables that affect the rate of positivity observed in respiratory testing include: the efficiency and timing of specimen collection, handling and transport of the specimen, the time of year, age of the patient, and local disease prevalence. Prospective specimens used in our clinical study were obtained from the United States and Australia. The prevalence of all influenza viruses in the US 4 during the September 2010 to March 2011 collection period ranged from 2.1 to 35.5%. During the season, among influenza positives, 72.9% were positive for Influenza A and 27.1 % for Influenza B. Outbreaks of occur each year, usually lasting 3 4 months during the fall, winter, and/or spring months. The exact timing of the season can vary by region 4. The positivity rate for in the United States during the period that included the September 2010 to March 2011 collection period was 15.9% 5. In Australia during the 2010 influenza season, 9% of specimens have tested positive for influenza 6 ; the positivity rate for was not reported. Prevalence (positives as determined by reference method) observed during our prospective clinical study is indicated in the tables below.

13 Simplexa /B & Direct Page 13 Simplexa /B & Direct - Stratified by Collection Sites Australia (n=330) Analyte Total (Prevalence) < 5 years (n=63) 5-21 years (n=106) years (n=147) > 60 years (n=14) Influenza A 2.7% (9/330) Influenza B 2.9% (7/330) Respiratory Syncytial Virus 0.7% (1/330) Ohio (n=245) Analyte Total (Prevalence) < 5 years (n=195) 5-21 years (n=48) years (n=2) > 60 years (n=0) Influenza A 11.4% (28/245) Influenza B 1.2% (3/245) Respiratory Syncytial Virus 29.8% (73/245) Virginia (n=147) Analyte Total (Prevalence) < 5 years (n=69) 5-21 years (n=69) years (n=9) > 60 years (n=0) Influenza A 21% (31/147) Influenza B 7.5% (11/147) Respiratory Syncytial Virus 7.5% (11/147) REFERENCES 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 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: (800) (U.S.A. only) (562) (562) (International) PI.MOL2650.IVD Rev. B Date written: 12 - July 2012 TECHNICAL ASSISTANCE Telephone: Fax: (800) (U.S.A. only) (562) (562) (International) Visit our website at Cypress, California USA