Verigene® Respiratory Virus Plus Nucleic Acid Test
Contents
1. 7 ey A Port Chalmers 1 73 1X10 y 7 R i i i _ Influenza A Hong Kong 8 68 H3N2 1X10 7 Influenza A Aichi2 68 H3N2 1X10 Z Influenza A Victoria 3 75 H3N2 1X10 Page 17 of 27 027 00024 02 Rev B A Cust Servi Technical Service Nanosphere wo one ee E Mail productsupport nanosphere us Influenza A Wisconsin 67 05 H3N2 1X10 Influenza A Hiroshima 52 05 H3N2 1x10 Influenza A NY 55 04 H3N2 1x10 Influenza A California 04 2009 H1N1 1X10 Influenza A 2009H1N1 PSF1 Clinical 1X102 E z E Isolate 09 H1N1 Influenza A Wisconsin 629 D01606 2 miu an 1x10 P e Influenza A 2009H1N1 Clinical Isolate 1X10 Pi _ i H1N1 Influenza A Swine lowa 15 30 1X10 Influenza A Swine 1976 31 1X10 gt T Influenza A Duck Hunan 795 02 2 E So E E EE Influenza A Chicken Korea 1S 2006 3 E LAA Influenza A Scaly Breasted 4X10 F a 7 k Munia Hong Kong 2006 H5N1 Influenza A Netherlands 219 2003 4 a w E Influenza A New York 107 2003 0 So Influenza B Wisconsin 2 2006 1X10 A A Florida 02 2006 1x10 i i Influenza B Malaysia 2506 2004 1X10 Influenza B Ohio 1 2005 1x10 Influenza B Lee 40 1X10 Influenza B GL 1739 54 1x10 E Influenza B Taiwan 2 62 1X10 Influenza B Hong Kong 5 72 1X10 Influenza B Maryland 1 59 1x10 E EE EEE EE2. B Verigene RV Amplification Kit Catalog number 20 012 020 e 20 Verigene RV Amplification Trays MATERIALS NEEDED BUT NOT PROVIDED A Instruments and Equipment e Verigene Reader Catalog number 10 0000 02 e Verigene Processor SP with Amplification Catalog number 10 0000 07 e 70 C and 20 C freezer e 2 8 C refrigerator e Micro pipettors amp tips e Mini centrifuge e Cooling block B Consumables and Reagents e Nylon or Rayon tipped nasopharyngeal swabs Copan Innovation e Universal Transport Medium Catalog number 330C Copan Innovation Micro Test M5 Viral Transport Medium Catalog number R12515 Remel Inc Page 2 of 27 027 00024 02 Rev B A Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us STORAGE HANDLING STABILITY Component Storage Conditions Comments Extraction Tray 2 8 C Do not freeze Amplification Tray lt 20 C Keep frozen Test Cartridge 2 8 C Do not freeze Tip Holder Assembly 2 30 C Room Temperature PRECAUTIONS AND WARNINGS GENERAL e The RV is for in vitro diagnostic use only e The performance of the test with viruses infecting swine and other animal hosts has not been established e Federal law restricts this device to sale by or on the order of a physician or to a clinical laboratory its use is restricted to by or on the order of a physician e Performance characteristics of the RV
3. One 1 INFA H3 RSVA LP sample detected Influenza A and H3 but did not detect RSV A One 1 INFB RSVB LP sample detected Influenza B but did not detect RSV B One INFB RSVB LP sample detected Influenza B but did not detect RSV B One INFB RSVB HN sample detected RSV B but did not detect Influenza B Additional Positive Calls One 1 INFA H1 MT MP sample detected both Influenza A and H1 as expected but also detected H3 which was unexpected One 1 INFA H1 MT MP sample detected both Influenza A and H1 as expected but also detected H3 Flu B and RSV A which was unexpected One 1 INFA H1 MT HN sample detected Influenza A but did not detect H1 which was expected as this is a high negative sample However RSV A was also detected which was unexpected One INFA H3 RSVA LP sample detected both Page 15 of 27 027 00024 02 Rev B Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us Influenza A H3 and RSV A as expected for a low positive sample but also detected 2009 H1N1 which was not expected SOne INFA 2009H1N1 MT HN sample did not detect Influenza A and 2009 H1N1 which was expected as this is a high negative sample However Influenza B and RSV B were also detected which was not expected NOTE All the samples with additional positive calls gave the expected results for the intended virus there were no mis calls The source of the ad
4. especially at very low virus titers The RV is a qualitative test and does not provide the quantitative value of the detected organism The RV has not been evaluated for patients without signs and symptoms of upper respiratory infection The RV has not been evaluated for monitoring treatment of Influenza or RSV infection The RV has not been evaluated for screening of blood or blood product for the presence of influenza The affect of interfering substances has only been evaluated for those listed within Interference by substances other than those described can lead to erroneous results e The assay performance has not been established in individuals who received nasally administered or injectable influenza vaccine FluMist Influenza Vaccine Live Intranasal Medimmune contains live attenuated reassortants of each of the three strains A California 7 2009 H1N1 A Perth 16 2009 H3N2 and B Brisbane 60 2008 FluMist is administered intranasally FluLaval GlaxoSmithKline ID Biomedical is administered as an intramuscular shot and contains inactivated strains of each of the three strains A California 7 2009 H1N1 A Victoria 210 2009 H3N2 an A Perth 16 2009 like virus and B Brisbane 60 2008 In the dilution series for FluMist Influenza A and the H1 and H3 subtypes were not detected at or below an approximate 1 5x10 dilution while Influenza B was not detected at or below an approximate 1 5x10 dilution In the dilution series for FluLava
5. generally starting as early as October or November and ending as late as April or May Page 1 of 27 027 00024 02 Rev B a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us PRINCIPLES AND PROCEDURES OF THE RV AND THE VERIGENE SYSTEM The RV identifies virus specific nucleic acids for Influenza A virus Influenza B virus and Respiratory Syncytial Virus RSV The RV targets the following genes within the viruses matrix gene Influenza A hemagglutinin gene Influenza A subtypes H1 and H3 nucleoprotein gene Influenza A subtype 2009 H1N1 non structural gene Influenza B polymerase gene RSV A and RSV B For each target a set of primers amplifies a region of the gene and a set of probes located within the amplified region detects the generated amplicons by using gold nanoparticle based detection technology The entire RV is performed on the Verigene System which is a bench top sample to result molecular diagnostics workstation consisting of two instruments the Verigene Processor SP and the Verigene Reader The Verigene Processor SP automates the following steps of the RV i Sample Preparation Magnetic bead based viral RNA isolation from nasopharyngeal swab specimens obtained from symptomatic patients ii Target Amplification Multiplex RT PCR based amplification of the eluted viral RNA to generate virus specific amplicons iii
6. INFA UNSUBTYPEABLE Verified Influenza A event Repeat recommended see INTERPRETATION OF RESULTS section INFB Verified Influenza B RSVA Verified RSV A a RSVB Verified RSV B E Not Detected for all No Target Verified viruses analytes g Error Calls and Recourse Error Call Reason Recourse Notes Inhibition during Target No Call INT CTL 1 1C1 Not Detected o RV Amplification No Call INT CTL 2 1C 2 Not Detecta 2 Not Detected RepeatRve RV Processing and or Target Amplification Issues Processing and or Target No Call INT CTL IC1 and IC2 Not Detected Repeat RV Amplification Issues Ensure protective silver tape has been removed from the back of the Test Substrate Adjust Test Substrate repeat image analysis If the call persists repeat RV Reader unable to image No cal SNO GRID Test Substrate No Call VARIATION An inability to obtain the test result because of high No Call BKGD variability in the target Repeal RV ES l No Call NEG CTL specific signals Power cycle ProcessorSP Repeat Internal checks within the Processing Error Pre analytical error RV y crm Processor SP detected an unexpected event Page 8 of 27 027 00024 02 Rev B Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us EXPECTED VALUES While the timing and duration of influenza and RSV seasons can vary the
7. Isolate H1N1 5 Influenza A California 04 2009 H1N1 1 Influenza A Wisconsin 67 05 H3N2 0 1 H1 Influenza A Virginia 01 2006 H1N1 5 Influenza A Brisbane 59 2007 Clinical Isolate H1N1 5 2009H1N1 Influenza A 2009H1N1 Clinical Isolate H1N1 10 Influenza A California 04 2009 H1N1 1 H3 Influenza A Wisconsin 67 05 H3N2 0 1 INFB INFB Influenza B Wisconsin 2 2006 0 1 Influenza B Florida 02 06 1 RSVA RSVA RSV A Strain A2 10 RSVB RSVB RSV B Strain Wash 18537 62 1 Page 16 of 27 027 00024 02 Rev B A Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us D Analytical Reactivity The analytical reactivity of the RV was evaluated using multiple strains of Influenza A 7 seasonal H1 strains 7 seasonal H3 strains 4 2009 H1N1 strains 2 swine origin H1N1 strains 3 H5N1 strain and 2 H7N7 strain 8 Influenza B strains 2 RSV A strains and 3 RSV B strains Viral strains for Influenza A were selected to include strains representing temporal and geographical diversity In order to assess analytical reactivity cultured and titered strains of Influenza A H3 Influenza A H1 Influenza A 2009H1N1 Influenza B RSV A and RSV B were diluted in sample matrix Universal Transport Medium Copan at the concentrations listed in the adjoining Table and tested in triplicate in the RV All the Influenza A H3 Influenza A H1 and Influenza A 2009 H1N1 strains yielded e
8. RV In all the samples tested a valid test result was provided indicating that the microorganisms did not negatively effect the RV processing Significantly all the microorganisms tested gave a Not Detected call for each analyte on the RV the microorganisms tested did not demonstrate cross reactivity with any of the analytes in the RV Viruses Strain pfumL INFA H1 H3 re INFB RSVA RSVB Human Adenovirus Type 1 Adenoid71 6210 Human Adenovirus Type2 Adenoid6 tox 2 oe Human Adenovirus Tye3 GB tox e Human Adenovirus Type4 Rk87 xa P Te Toe T Ye e Human Adenovirus Type5 Adenoid75 55410 Te Human Adenovirus Type7 Gomen 62x10 Human Adenovirus Type 11 Slobitski 1910 fo Human Adenovirus Type 14 dewt 554 Human Adenovirus Type 31 1315 354 fo Human Adenovirus Type 35 holden txt oe Human Coronavirus 0c43 ocas t o oe Human coronavirus 229E 220 tixto Human coronavirus NL63 ntes s940 o fo Page 19 of 27 027 00024 02 Rev B NS Cust Servi Technical Service Nanosphere o E Mail productsupport nanosphere us Ea EN EAS E ES T e
9. Test Cartridges e See Material Safety Data Sheets MSDS for toxicity information Material Safety Data Sheets MSDS are available upon request from Nanosphere Inc B Waste Disposal e Dispose unused reagents and waste in accordance with federal state and local regulations METHODS A Specimen Collection amp Storage e Use Nylon or Rayon tipped nasopharyngeal swabs for specimen collection Place swab into Viral Transport Medium Break swab shaft and cap the tube Inadequate or inappropriate specimen collection storage or transport may yield false negative results Training in specimen collection and handling is highly recommended because of the importance of specimen quality Store specimens refrigerated at 2 8 C for up to 72 hours before processing Store leftover specimens at lt 70 C Transport human respiratory specimens refrigerated at 2 8 C When transporting human respiratory specimens ensure that all applicable regulations for the transport of etiologic agents are met B RV Test Procedure This section has step wise instructions for testing samples using the RV assay on the Verigene System Please refer to the Verigene System User s Manual for additional details on performing tests on the Verigene System including daily maintenance 1 Create a Verigene Session a Login to the system as a user b From the Menu Bar Session tab select Start New Session The Session Setup window will appear c Touch t
10. Verigene Hybridization Test Gold nanoparticle probe based hybridization of the virus specific amplicons on a microarray Gold nanoparticle probes bound specifically to target containing spots on the microarray are silver enhanced and light scatter from the spots is measured on the Verigene Reader and further analyzed to make decisions regarding the presence Detected or absence Not Detected of a virus analyte The Verigene Reader also serves as the user interface and stores and tracks sample information throughout the assay process The Verigene Processor SP utilizes single use disposables to perform the RV including an Extraction Tray Amplification Tray and Verigene Test Cartridge A separate Tip Holder Assembly contains two pipette tips that are used to transfer and mix reagents during the assay The user tests a sample by loading the single use disposables into the Verigene Processor SP and pipetting the sample into the Extraction Tray The user initiates the test protocol on the Verigene Reader by scanning or entering the barcode ID located on the Test Cartridge along with sample information Following assay completion the user collects data on the Verigene Reader by scanning the barcode ID on the Test Cartridge and inserting it into the Verigene Reader for analysis MATERIALS PROVIDED A Verigene RV Test Kit Catalog number 20 005 020 e 20 Verigene RV Test Cartridges e 20 Verigene RV Extraction Trays with Tip Holder Assemblies
11. be Detected The recourse for the No Call INT CTL decision is to repeat the RV test In addition separate internal positive and negative controls are present within each Test Cartridge that inform regarding the proper functioning of the Test Cartridge External Controls Regardless of the choice of quality control materials all external quality control requirements and testing should be performed in conformance with local state and federal regulations or accreditation organizations as applicable and should Page 5 of 27 027 00024 02 Rev B A Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us follow the user s laboratory s standard quality control procedures External Controls can be prepared by using Influenza A Influenza B and RSV viral particles diluted in negative clinical matrix or Viral Transport Media It is recommended that the user refer to CLSI document C24 A2 Statistical Quality Control for Quantitative Measurements Principles and Definitions Approved Guideline Second Edition or other published guidelines for general quality control recommendations For further guidance on appropriate quality control practices refer to 42CFR 493 1202 c Control Description Function Inhibition Control Double stranded DNA target Controls for PCR inhibition due to sample IC1 present in the primer mix or process related inhibitors or due
12. case of INFA H3 and RSVA and Influenza B and RSV B For the Reproducibility Precision study the Test Panel comprised the 12 unique samples in duplicate for a total of 24 samples The Test Panel samples were then divided equally into Panel A 12 samples and Panel B 12 samples Viral Panel Virus Strain INFB RSVB INFA 2009H1N1 MT INFA H1 MT INFA H3 RSVA Level High Negative Low Positive Moderate Positive High Negative Low Positive Moderate Positive High Negative Low Positive Moderate Positive High Negative Low Positive Moderate Positive Samples o ON OD oO A O N allaxilx N O o OND A A O N ala o 12 At the two external study sites Test Panel A and Test Panel B were tested on separate days Testing each day involved 2 operators testing the same Test Panel in two replicate runs Both Test Panels A and B were tested for a total testing period of six non consecutive days The internal site ran a 12 day precision study and utilized the same four unique samples at the three levels The complete Test Panel Test Panel A and B was tested separately by two operators The precision study was run for a total testing period of 12 non consecutive days The cumulative results from the Reproducibility Precision Studies are summarized Data is presented for each individual site and then combined to provide collective results The Table contains the agreement between the expected resu
13. elements or spots are virus specific oligonucleotide sequences that bind to the amplified viral targets which in turn bind to gold nanoparticle probes via additional recognition elements A gold nanoparticle probe specific signal enhancement reaction deposits silver at the virus specific spots The scatter from the spots is detected by the Verigene Reader and registered as signal intensity In addition to the above recognition elements the Test Substrate has spots specific to positive control PC and negative control NC Three conditions were identified that together served as a single set of clinical cutoff criteria Condition 1 Noise Threshold Condition 2 Normalized Ratio to Negative Control Ratio to NC intensity at the virus specific recognition element normalized against the intensity values at the negative control elements Condition 3 Normalized Ratio to Positive Control Ratio to PC intensity at the virus specific recognition element normalized against the intensity values at the positive control elements The Noise Threshold was determined empirically The cut offs for the normalized ratios Ratio to NC and the Ratio to PC were determined by using ROC curves For a positive Detected decision the following criteria apply Condition 1 Signal intensity is above the noise threshold Condition 2 Ratio to NC gt 0 85 Condition 3 Ratio to PC 2 0 4 If any one of these criteria is not met a neg
14. fall and winter months are the peak times in the US RSV infections in the US typically occur during annual community outbreaks in the late fall winter and early spring and there may be variation in the timing of outbreaks between regions and between communities in the same region During the 2008 2009 and 2009 2010 flu seasons 21 of 519 543 samples and 20 of 456 302 samples tested for influenza were positive for either Influenza A or Influenza B based on data from laboratories in the US which collaborated with World Health Organization WHO and National Respiratory and Enteric Virus Surveillance System NREVSS During the 2006 2007 and 2007 2008 flu seasons the prevalence was 13 of 179 268 samples tested and 18 of 225 329 samples tested respectively According to data reported to the NREVSS the prevalence of RSV was 15 in the 404 798 samples tested for RSV during the 2008 2009 season and 16 in the 369 944 samples tested during the 2007 2008 season In the RV multi site methods comparison study which analyzed 1022 samples collected during the 2008 2009 and 2009 2010 flu season the prevalence of Influenza A was 30 0 for Influenza B was 4 3 and of RSV was 10 2 No dual infections were detected by culture and DFA but 0 2 or 2 specimens of the total infections were found to contain dual infections by the RV and confirmed subsequently by bi directional sequencing one specimen was positive for RSV A and RSV B and a sec
15. hospitalized and between 3 000 and 49 000 people die of complications each year depending on the severity of the season Symptoms include fever cough headache body aches congestion and fatigue Flu can lead to serious complications such as pneumonia bronchitis sinus infections and a general worsening of chronic conditions In the spring of 2009 a novel quadruple reassortant virus now known as 2009 H1N1 Influenza emerged in North America and quickly spread becoming a global pandemic by the summer of 2009 According to CDC estimates the virus infected between 43 million and 89 million people between April 2009 and April 2010 Importantly this Influenza A subtype was found to be susceptible to the antiviral drug oseltamivir brand name Tamiflu 2 while antiviral resistance varied among other Influenza A subtypes Thus treatment decisions may be impacted by the timely availability of Influenza A subtyping information Respiratory Syncytial Virus RSV infection is the most common cause of bronchiolitis and pneumonia in children under 1 year of age in the United States Each year 75 000 to 125 000 children in this age group are hospitalized due to RSV infection Symptoms of RSV infection include coughing sneezing runny nose fever and decrease in appetite RSV is also recognized as a serious contributor to respiratory ailments in the aged and immunocompromised demographic Flu and RSV occur as seasonal outbreaks in the United States
16. this product in relation to the manufacture or use of nucleic acid arrays may be covered by one or more of the following patents owned by Oxford Gene Technology Limited or Oxford Genet Technology IP Limited together OGT US patents 6 054 270 5 700 637 European patent 0 373 203 Japan patents 3 393 528 and 3 386 391 and pending patents The purchase of this product does not confer the purchaser any rights or licenses under any of OGT s patents This product is sold under licensing arrangements between Nanosphere Inc and Life Technologies Corporation IP Holdings Inc The purchase of this product conveys to the buyer limited non transferable rights to use the Platinum Tfi One Step q RT PCR SuperMix which includes SuperScript III Reverse Transcriptase and UDG decontamination technology owned by Life Technologies wherein both the Platinum Tfi One Step q RT PCR SuperMix and the UDG decontamination technology are solely for activities by the purchaser in detection of respiratory infectious agents within the field of human diagnostics No other rights are conveyed Further information on purchasing licenses may be obtained by contacting the Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 Email outlicensing invitrogen com Verigene and the Nanosphere logo are registered trademarks of Nanosphere Inc Platinum and SuperScript are registered trademarks of Life Technologies Corporation Copyrig
17. to Amplified with every RT PCR reagent failures reaction Process Control MS2 bacteriophage with an Controls for sample isolation step or IC2 intact viral RNA genome nucleic acid extraction step and the Added automatically to each RT PCR step test sample including external positive and negative controls External Positive Any of the three viral particles Serve as external controls for the extraction Control Influenza A Influenza B or target amplification and detection steps RSV Used to verify reagent performance a during installation system validation and when troubleshooting dictates see Verigene System User s Manual b to verify the performance of a new lot batch of reagents c when the integrity of storage conditions is in question External Negative Viral Transport Media Controls for reagent and or environmental Control contamination TROUBLESHOOTING Refer to the Troubleshooting section of the Verigene System User s Manual LIMITATIONS e Performance characteristics of this product were determined with nasopharyngeal swab specimens only e A trained health care professional should interpret assay results together with the patient s medical history clinical signs and symptoms and the results of other diagnostic tests e Viral nucleic acid may persist in vivo independent of virus viability Detection of analyte target s does not imply that the corresponding virus es are infectious or are the cau
18. 009H1N1 Influenza A California 04 2009 RSV A RSV A Strain A2 RSVB RSV B Wash 18537 62 G Competitive Inhibition RV Page 22 of 27 027 00024 02 Rev B Customer Service or Technical Service Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us Nanosphere H Fresh vs Frozen Freezing virus containing samples can potentially compromise viral integrity which may translate to a lower effective concentration in frozen samples In order to determine the impact of freezing samples on RV performance unfrozen negative nasopharyngeal swab samples were obtained and spiked with Influenza A Influenza B and RSV viruses to represent all the analytes in the RV Each virus was tested at multiple concentrations Paired comparisons were conducted for each sample by testing it before and after freezing The impact of 2 additional freeze thaw cycles on the RV for a total of 3 freeze thaw cycles was also assessed The Fresh vs Frozen comparison study involved testing a total of 60 negative nasopharyngeal NP swab samples across 6 unique virus types representing all the targets and analytes in the RV The negative NP swab samples were not pooled each virus was represented by 10 unique NP swab samples 10 x 6 60 unique samples Each virus type was tested at 2 or 3 different concentrations to represent levels 1 log above the limits of detection 2 logs above the limit of detection and greater than 2 logs abo
19. ANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGMENT ARE PROVIDED BY NANOSPHERE INC Nanosphere Inc shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product and its components Page 26 of 27 027 00024 02 Rev B a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us BIBLIOGRAPHY Thompson W W Shay D K Weintrau E ef al Mortality associated with influenza and respiratory syncytial virus in the United States JAMA 2003 289 179 186 2 Jansen A G S C Sanders E A M Hoes A W et al Influenza and respiratory syncytial virus associated mortality and hospitalizations Eur Respir J 2007 30 1158 1166 a Falsey A R Hennessey P A Formica M A ef al Respiratory syncytial virus infection in elderly and high risk adults New Engl J Med 2005 352 1749 1759 E Mahoney J B Detection of Respiratory Viruses by Molecular Methods Clin Microbiol Rev 2008 21 716 747 5 Q amp A Seasonal Influenza Flu The Disease Centers for Disease Control and Prevention Updated September 10 2010 Retrieved October 22 2010 from http www cdc gov flu about ga disease htm Seasonal Influenza Flu Flu Symptoms amp Severity Centers for Disease Control and Prevention Updated September 8 2010 Retri
20. ELEEE EE EEE EEE EET Page 18 of 27 027 00024 02 Rev B a Customer Service or Technical Service Nanosphere o E Mail productsupport nanosphere us rra LL RSV A Strain A2 1x10 z e a RSV A Strain Long 1X10 z EN sa l C C E E RSV B Strain Wash 18537 62 1X10 i gt g E 3 RSV B Strain B 1 Wild Type 1X10 5 E z y i RSV B Strain 9320 1X10 a 3 i i ALL In addition to the above testing both swine origin H1N1 strains were tested at 1 and 2 logs higher concentrations 1x10 and 1x10 TCID50 mL No cross reactivity to 2009 H1N1 was observed In addition to the above testing the H5 and H7 strains were tested at 1 and 2 logs higher concentration No cross reactivity to any sub types was observed E Analytical Specificity Cross Reactivity Analytical specificity studies were performed to assess potential cross reactivity of the RV with respiratory pathogens and other microorganisms commonly present in the respiratory tract A total of 53 organisms of interest were identified as respiratory pathogens with which the majority of the population may be infected These organisms included 24 bacterial strains that were tested between 10 to10 cfu mL and 29 virus strains that were tested between 10 to10 TCID50 mL The microorganisms were diluted into a sample matrix Universal Transport Medium Copan at the concentration levels described in the Table below and tested subsequently in the
21. Pe ire Epstein Barr Virus B95 8 a z S E a z NN Human Parainfluenza Type3 C243 ima fo 2 fo Human ParainfluenzaType4 M25 zoa fo fo Bacteria Strain cfulmL INFA H1 H3 200 NFB RSVA RSVB Acinetobacter Baumanni laa p CeCe Ce 2 Bordetella bronchiseptica 800 p o o Po o 1 Bordetella pertussis amar METI O ET O E E O E Haemophilus influenzae aa foe foe Klebsielapneumoniae Br T T Te fe Te Lactobacillus acidophius Laa T o oe e Ce Te fe Listeria inocua axe e TT Moraxella catarhais RAR ee fe e Neisseria gonorrhoeae s p CUT Ul fe fe Pseudomonas aeruginosa Boston41501 351 To fo Page 20 of 27 027 00024 02 Rev B a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us Staphylococcus epidermidis 3 7x10 5 z E Streptococcus pneumoniae 9 0x10 z z z B Streptococcus agalactiae O90R 2 0x10 7 E 5 Streptococcus pyogenes 1 3x10 a S Streptococcus salivarius 5 5x10 a z g F Determination of Clinical Cut Off and Call Algorithm The RV uses a microarray based platform in which the viruses the inhibition control IC1 and process control IC2 are represented by recognition elements on the Test Substrate The recognition
22. Remove the Reagent Pack from the Substrate Holder NOTE Handle only the Substrate Holder Do not touch the substrate s surface q Re scan the RV Test Cartridge identification number into the Verigene Reader using the attached barcode scanner r Immediately before analysis remove the protective tape from the back of the Substrate Holder and insert the Substrate Holder into the Verigene Reader for analysis Refer to the Verigene System User s Manual for specific instructions on starting the analysis and obtaining results s Open the Drawer Clamp and remove the used trays and Tip Holder Assembly from the Verigene Processor SP t Dispose of each consumable in the appropriate waste receptacle QUALITY CONTROL Quality control as a component of an overall quality assurance program consists of tests and procedures for monitoring and evaluating the analytical performance of a measurement system to ensure the reliability of patient test results A Quality Control Verigene System The Verigene System uses a series of automated on line quality measurements to monitor instrument functionality software performance fluidics test conditions reagent integrity and procedural steps each time a test is performed A series of automated on line procedural checks guide the user through the testing process each time a test is performed RV test barcode and sample information are linked upon entry into the Verigene Reader to help prevent misreportin
23. T and upon repeat culture DFA A specimen was negative for RSV by RV and by sequencing but positive for RSV by culture and NAAT assay 2 specimens were negative for RSV by RV and positive for RSV by culture The specimens were negative for RSV by NAAT assay and failed sequencing 34 specimens were positive for RSV A or RSV B by RV but negative by culture 1 was positive for RSV A and 3 were positive for RSV B by RV and by sequencing 3 specimens failed subtype sequencing and are not included in the Subtyping Tables f1 specimen was positive for both RSV A and RSV B dual infection by RV and was culture positive for RSV By sequencing specimen was positive for both RSV A and RSV B Page 13 of 27 027 00024 02 Rev B Nanosphere Customer Service or Technical Service Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us B Reproducibility Precision Studies The Reproducibility Precision study was conducted at three sites two external and one internal to investigate the inter laboratory reproducibility of the RV The reproducibility panels see Table below were developed by using 6 unique virus strains that together represented all of the analytes in the RV The 6 virus strains were combined to generate combinations such that each virus strain was represented at 3 levels HN High Negative LP Low Positive and MP Moderate Positive For two of the unique samples virus strains were combined as in the
24. a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us Verigene Respiratory Virus Plus Nucleic Acid Test on the Verigene System REF REF 20 005 020 Test Kit e 20 012 020 Amplification Kit INTENDED USE The Verigene Respiratory Virus Plus Nucleic Acid Test RV on the Verigene System is a qualitative nucleic acid multiplex test intended to simultaneously detect and identify multiple respiratory virus nucleic acids in nasopharyngeal NP swab specimens from individuals with signs and symptoms of respiratory tract infection The following virus types and subtypes are identified using the RV Influenza A Influenza A subtype H1 Influenza A subtype H3 2009 H1N1 Influenza B Respiratory Syncytial Virus RSV subtype A and RSV subtype B The test is not intended to detect Influenza C virus Detecting and identifying specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and laboratory findings Negative results for Influenza A Influenza B or RSV do not preclude influenza virus or RSV infection and should not be used as the sole basis for diagnosis treatment or patient management decisions Conversely positive results do not rule out bacterial infection or co infection with other viruses The agent detected may n
25. ative Not Detected decision is provided Criteria set for each of the three conditions are required to be met for a Detected call For a result the decision tree verifies the presence of IC1 and 1C2 in conjunction with each of the viruses see Schematic Both IC1 and IC2 signal intensities have to meet the detection criteria for a valid call A valid Call is made only after both inhibition control IC1 and process control IC2 are verified during analysis of each test signifying that the extraction and target amplification processes performed correctly If 1C1 or IC2 are Not Detected a No Call INT CTL 1 or a No Call INT CTL 2 is provided respectively If both IC1 and IC2 are Not Detected a NO CALL INT CTL result is provided The following exception exists IC2 detection alone is sufficient for a valid call if any of the viral targets are also detected there is no requirement for IC1 to also be Detected The recourse for the No Call INT CTL decision is to repeat the RV test In addition separate internal positive and negative controls are present within each Test Cartridge that inform regarding the proper functioning of the Test Cartridge Page 21 of 27 027 00024 02 Rev B Customer Service or Technical Service Nanosphere o E Mail productsupport nanosphere us RV Decision Process RV Decision Process Internal Controls fail Verify Internal Contro
26. ditional positives is likely cross contamination artifacts during the Test Panel preparation There were 14 No Calls and 2 pre analytical errors in the study These 16 samples were repeat tested successfully In this study the No Call rate was 1 6 14 864 and the pre analysis error failure rate was 0 2 2 864 Out of the 864 samples tested the percent agreement for all panel members for the combined sites ranged from 97 2 100 95 Cl range from 90 3 99 7 to 95 0 100 0 respectively C Analytical Sensitivity RV The analytical sensitivity of RV was assessed and confirmed by using virus strains with established titers to represent all analytes in the test In order to determine the Limits of Detections LODs the strains were serially diluted into negative pools of nasopharyngeal NP swab samples around the estimated LOD and replicates were tested in the RV The lowest concentration level where all the replicates were detected was chosen as the LOD For confirmation 20 replicate samples were prepared at the LOD level chosen Confirmation of LOD required that at least 19 of the 20 samples tested gave a Detected call for the analyte The confirmed LOD levels for the different analytes and the associated virus strains are presented Virus Analyte Strain LOD TCID50 ML INFA INFA Influenza A Virginia 01 2006 H1N1 1 Influenza A 2009H1N1 Clinical Isolate H1N1 10 Influenza A Brisbane 59 2007 Clinical
27. ductsupport nanosphere us FDA cleared Nucleic Acid Amplification Test NAAT Subtyping results for Influenza A and RSV specimens and discordant results between the RV and the reference method were confirmed and or analyzed respectively by using bi directional sequencing at an independent reference laboratory described in Table footnotes Samples with an initial No Call result were re tested successfully by following the recommendations in the Interpretation of Results section A total of 34 samples 3 3 generated a No Call result all but two of the samples 0 2 resolved upon retest A total of 25 samples 2 4 resulted in a pre analysis error pre ae A pre ae may occur due to a number of causes including a user initiated procedure termination for example procedure termination upon realizing that an incorrect sample was loaded or an instrument initiated procedure termination for example because internal checks detect an unexpected event or non availability of the Test Substrate for analysis due to test consumable breakage etc All the specimens with an initial pre ae result resolved upon retest Influenza A A Influenza A Results RV vs Culture DFA Culture DFA INFA Positive Negative Total Total Positive ami 48 359 Sensitivity 98 7 96 8 99 5 95 CI RV Negative 7 659 663 Specificity 93 2 91 1 94 8 95 CI Total 315 707 10229 B Influenza A Subtype H3 Results RV vs C
28. e Test Cartridge by holding the Substrate Holder handle with the left hand pressing the palm of the right hand along one edge and using fingers to lift up the opposite edge Page 4 of 27 027 00024 02 Rev B a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us i Before loading the Test Cartridge into the Verigene Processor SP scan the RV Test Cartridge identification number into the Verigene Reader using the attached barcode scanner Upon being prompted enter sample information into the Verigene Reader j The Verigene Reader will display the viral targets for which results will be reported upon successful completion of the assay De select any undesired targets from the list NOTE Once the test process is initiated the results for any de selected targets cannot be retrieved k Select viruses and or virus subtypes to be tested Tap the Test Cartridge and insert into Verigene Processor SP NOTE If the Test Cartridge is not seated properly the drawer will not close m Pipette 200 uL of sample into the Sample Well of the Extraction Tray n Close the drawer of the Verigene Processor SP by pressing the open close button on the front of the instrument The Verigene System will perform a series of checks and automatically initiate the test process o Once the test process is complete remove the RV Test Cartridge from the Verigene Processor SP p
29. eved October 22 2010 from http www cdc gov flu about disease symptoms htm 7 The 2009 H1N1 Pandemic Summary Highlights April 2009 April 2010 Centers for Disease Control and Prevention Updated August 3 2010 Retrieved October 29 2010 from http www cdc gov h1n1flu cdcresponse htm CDC Estimates of 2009 H1N1 Influenza Cases Hospitalizations and Deaths Centers for Disease Control and Prevention Updated May 14 2010 Retrieved October 29 2010 from http www cdc gov h1n1flu estimates 2009 h1n1 htm E Key Facts About Antiviral Drugs and Influenza Flu Centers for Disease Control and Prevention Updated September 9 2009 Retrieved October 29 2010 from http www cdc gov flu protect antiviral keyfacts htm 1 Centers for Disease Control and Prevention 2008 Update Influenza Activity United States September 28 November 29 2008 Respiratory Syncytial Virus Activity United States July 2008 December 2009 MMWR 57 49 1329 1332 11 RSV Frequently Asked Questions Centers for Disease Control and Prevention Updated October 17 2008 Retrieved October 22 2010 from http www cdc gov rsv about fag html 12 Seasonal Influenza Flu Past Weekly Surveillance Reports Centers for Disease Control and Prevention website Updated October 15 2010 Retrieved October 26 2010 from http www cdc gov flu weekly pastreports htm 13 2006 07 U S Influenza Season Summary Centers for Disease Control and Prevention websi
30. g of results Quality Control RV The RV is a sample to result detection system wherein viral RNA is isolated from nasopharyngeal swabs and amplified prior to specific detection on a microarray housed within the Test Cartridge All reagents are prepackaged in single use disposable reagent trays and cartridges to prevent reagent dispensing errors Several layers of controls built into the RV ensure that failures at any step within the RV are identified during the procedure or in the end point image analysis of the Test Cartridge An MS2 bacteriophage sample processing control IC2 is added to each sample automatically prior to extraction to monitor failure in the Sample Extraction and Target Amplification steps see Table below An Inhibition Control IC1 is included in the Primer Mix reagent and added automatically to monitor PCR inhibition A valid Call is made only after both inhibition control IC1 and process control IC2 are verified during analysis of each test signifying that the extraction and target amplification processes performed correctly If IC1 or IC2 are Not Detected a No Call INT CTL 1 or a No Call INT CTL 2 is provided respectively If both IC1 and IC2 are Not Detected a NO CALL INT CTL result is provided The following exception exists C2 detection alone is sufficient for a valid call if any of the viral targets are also detected there is no requirement for IC1 to also
31. have been determined only with nasopharyngeal swab specimens e f infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to 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 e Handle supplies reagents and kits with powder free gloves at all times to avoid contamination and change gloves between removal of used disposables and loading of new disposables e Handle samples carefully Open one tube or sample at a time to prevent sample contamination e Biological samples such as tissues body fluids and blood of humans and other animals are potentially infectious When handling and or transporting human respiratory specimens follow all applicable regulations mandated by local state provincial and federal agencies for the transport of etiologic agents e The detection of viral nucleic acid is dependent upon proper specimen collection handling transportation storage and preparation including extraction Failure to observe proper procedures in any one of these steps can lead to incorrect results PRECAUTIONS AND WARNINGS INSTRUMENTS A General Instrument Safety WARNING Use this product only as spec
32. he Session ID button and enter information by using the data entry keyboard The Session ID can be any unique identifier in a format defined by the lab The operator ID is automatically entered as the currently logged in operator d Touch the Processing option on the Navigation Bar at the bottom of the screen 2 Performing a RV test a For each sample to be tested retrieve an RV Test Kit and an RV Amplification Kit from storage and set aside at room temperature Each RV Test requires an RV Test Cartridge RV Extraction Tray and Tip Holder Assembly and an RV Amplification Tray b Open the Drawer Assembly of the Verigene Processor SP by pressing the open close button on the front of the instrument c Lift the Drawer Clamp so that the trays and Tip Holder Assembly can be inserted into the Verigene Processor SP d Shake the Extraction Tray to mix the reagents and tap to settle the reagents Place the Extraction Tray onto the Extraction Module e Remove the Tip Holder Assembly from the plastic pouch and place the assembly onto the Tip Module f Thaw the Amplification Tray gently vortex and tap to settle the reagents Place the Amplification Tray onto the Amplification Module g Lower the Drawer Clamp over the trays and latch it to secure the trays in their positions NOTE If the Drawer Clamp is not latched appropriately the drawer will not close h Set the RV Test Cartridge on a level surface and remove the cover that seals th
33. ht 2010 Nanosphere Inc All rights reserved NOTICE TO RECIPIENTS ABOUT LIMITED LICENSE OR RELATED The receipt of this product from Nanosphere Inc or its authorized distributor includes limited non exclusive license under patent rights held by Nanosphere Inc Such license is solely for the purposes of using this product to perform the proprietary nucleic acid analysis method for which it was intended from Nanosphere Inc or its authorized distributor For avoidance of doubt the foregoing license does not include rights to use this product for agriculture or veterinary medicine applications Except as expressly provided in this paragraph no other license is granted expressly impliedly or by estoppels LIMITED PRODUCT WARRANTY Nanosphere Inc warrants that this product will meet the specifications stated on the product information sheet If any component of this product does not conform to these specifications Nanosphere Inc will at its sole discretion as its sole and exclusive liability and as the users sole and exclusive remedy replace the product at no charge or refund the cost of the product provided that notice of nonconformance is given to Nanosphere Inc within sixty 60 days of receipt of the product This warranty limits Nanosphere Inc liability to the replacement of this product or refund of the cost of the product NO OTHER WARRANTIES OF ANY KIND EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION IMPLIED WARRANTY OF MERCH
34. ified in this document Using this instrument in a manner not specified by Nanosphere may result in personal injury or damage to the instrument Ensure that anyone who operates the instrument e Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Reads and understands all applicable Material Safety Data Sheets MSDS Electrical Shock Hazard WARNING Severe electrical shock can result from operating the instrument without its instrument covers or back panels in place Do not remove instrument covers or panels High voltage contacts are exposed when instrument covers or panels are removed from the instrument If service is required contact Nanosphere Technical Support at 1 888 VERIGENE 837 4436 Maintenance of the Verigene Reader and Verigene Processor SP For general maintenance and cleaning instructions please refer to the Verigene System User s Manual Page 3 of 27 027 00024 02 Rev B Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us PRECAUTIONS AND WARNINGS REAGENTS AND TEST CARTRIDGES A Toxicity of Reagents e Exposure to chemicals sealed inside the Test Cartridge is hazardous in case of skin contact and of ingestion Protective disposable gloves laboratory coats and eye protection should be worn when handling specimens Extraction Trays Amplification Trays and Verigene
35. l the H1 subtype was not detected at or below the 1 1x10 dilution Influenza A and the H3 subtype were not detected at or below an approximate 1 1x10 dilution and Influenza B was not detected at or below an approximate 1 1x10 dilution e The assay performance has not been established in immunocompromised individuals INTERPRETATION OF RESULTS RV provides a qualitative result for the presence Detected or absence Not Detected of the following viruses analytes Influenza A Influenza B RSV A RSV B For Influenza A positive samples RV provides subtyping information as H1 H3 or 2009 H1N1 In order to obtain results the intensities from target specific oligonucleotide spots on the microarray are imaged and analyzed on the Verigene Reader Additionally two Internal Controls IC1 inhibition control and IC2 process controls guide decisions regarding the validity of the test process and their presence is verified before a valid result is provided Together they ensure that the isolation and amplification steps performed satisfactorily If the internal controls fail verification a No Call INT CTL 1 for IC 1 failure or a No Call INT CTL 2 for IC2 failure or a No Call INT CTL for failure of both IC1 and IC2 are provided If the internal controls are verified the presence or absence of individual viruses is reported based on the cut off criteria For each valid test a Detected or Not Detected result is pro
36. l Spray 10 viv Luffa opperculata Similasan Sinus Relief 1 0 viv Dexamethasone Dexamethasone BO mL sis Budesonide Budesonide 2 Mometasone Furoate Mometasone Furoate 2 5 pg mL Fluticasone propionate Fluticasone propionate 5 pg mL Sulphur Boiron 4 5 mg mL Menthol Menthol 0 5 mg mL Oseltamamivir Phosphate Tamiflu 33 ug mL Galphimia Glauca Homeopathic Remedy Boiron 115 g mL Histaminum Hydrochloricum Boiron 115 pg mL Influenza A and subtypes H1 and H3 No detection at or below 5x10 dilution Influenza B No detection at or below 5x10 dilution FluMist Influenza Vaccine Live Intranasal Medimmune Influenza A and subtypes H1 and H3 No detection at or below 1x10 dilution Influenza B No detection at or below 1x10 dilution See below for additional information In addition to the interferents the impact of two sets of Influenza vaccines in the RV was also assessed FluMist Influenza Vaccine Live Intranasal Medlmmune contains live attenuated reassortants of each of the three strains A California 07 2009 H1N1 A Perth 16 2009 H3N2 and B Brisbane 60 2008 FluMist is administered intranasally FluLaval GlaxoSmithKline ID Biomedical is administered as an intramuscular shot and contains inactivated strains of each FluLaval GlaxoSmithKline ID Biomedical Page 24 of 27 027 00024 02 Rev B Customer Service or Technical Service e N a n O S p h e
37. ls verification No Call INT CTL CS Repeat RV A Internal Controls verified proceed to viral identification IF IF Target Signal gt Noise Threshold AND Target Signal lt Noise Threshold OR Ratio to NC gt 0 85 AND Ratio to NC lt 0 85 OR Ratio to PC gt 0 4 Ratio to PC lt 0 4 Competitive inhibition or interference in the RV was assessed in clinically relevant co infections A set of 12 unique high titer low titer combination samples each containing a virus at a high titer and another virus at a low titer were generated The samples represented combinations of Influenza A subtypes H1 H3 and 2009 H1N1 and RSV A B viruses see the Table below The levels for individual strains were chosen based on the limits of detection determined for the individual strains The high titers for the strains were at least 3 log orders higher than their limits of detection and the low titers were held close to the limits of detection S i High Titer Low Titer ample Virus TCID50 mL Virus TCID50 mL 1 RSVA 2x10 INFA H3 5 2 RSVA 2x10 INFA H1 25 3 RSVA 2x10 INFA 2009H1N1 10 4 RSVB 5X10 INFA H3 5 5 RSVB 5X10 INFA H1 25 6 RSVB 5X10 INFA 2009H1N1 10 7 INFA H3 2 5X10 RSVA 100 8 INFA H3 2 5X10 RSVB 5 9 INFA H1 2x10 RSVA 100 10 INFA H1 2x10 RSVB 5 11 INFA 2009H1N1 1x10 RSVA 100 12 INFA 2009H1N1 1x10 RSVB 5 Viral Strains INFA H3 Influenza A Wisconsin 67 05 INFA H1 Influenza A Virgina 01 2006 INFA 2
38. lts and the obtained results for each virus strain in the Test Panel These are grouped further based on their concentration levels MP LP and HN NOTE Though some samples in the Test Panels contained combinations of viruses results are stratified by individual virus strains Page 14 of 27 027 00024 02 Rev B a Customer Service or Technical Service Nanosphere o E Mail productsupport nanosphere us Site Site 1 Site 2 Site 3 All 3 Sites lo Level Agreement Agreement Agreement PIE et Hi 95 Cl MP 12 12 12 12 46 48 70 72 97 2 90 4 99 2 INFA H1 MT LP 12 12 12 12 48 48 72 72 100 94 9 100 HN 11 12 12 12 48 48 71 72 98 6 92 5 99 8 MP 12 12 12 12 48 48 72 72 100 94 9 100 INFA H3 LP 12 12 12 12 47 48 71 72 98 6 92 5 99 8 HN 12 12 12 12 48 48 72 72 100 94 9 100 MP 12 12 12 12 48 48 72 72 100 94 9 100 oe E 12 12 12 12 48 48 72 72 100 94 9 100 HN 12 12 12 12 47 48 71 72 98 6 92 5 99 8 MP 12 12 12 12 48 48 72 72 100 94 9 100 INFB LP 12 12 12 12 48 48 72 72 100 94 9 100 HN 12 12 11 12 48 48 71 72 98 6 92 5 99 8 MP 12 12 12 12 48 48 72 72 100 94 9 100 RSVA LP 12 12 12 12 46 48 70 72 97 2 90 4 99 2 HN 12 12 12 12 48 48 72 72 100 94 9 100 MP 12 12 12 12 48 48 72 72 100 94 9 100 RSVB LP 11 12 12 12 47 48 70 72 97 2 90 4 99 4 HN 11 12 12 12 48 48 71 72 98 6 92 5 99 8 Expected Calls One 1 INFB RSVB HN detected Influenza B
39. ond specimen was positive for Influenza A and RSV A It is recommended that the samples undergo repeat testing if nucleic acids from all three analytes Influenza A Influenza B and RSV are detected in a single sample The age distribution of the patient population in the RV multi site methods comparison study is presented in the Table below Age Categorization yrs Number of Subjects Proportion 0 2 269 26 3 3 5 118 11 5 6 11 147 14 4 12 18 124 12 1 19 64 297 29 1 265 67 6 6 All Ages 1022 100 PERFORMANCE CHARACTERISTICS OF RV A Method Comparison Studies for RV A total of 1022 nasopharyngeal swab specimens were collected prospectively during the 2008 2009 and 2009 2010 respiratory seasons for routine influenza or RSV testing by DFA culture methods The residual specimens were frozen and later tested at three clinical sites between Site 1 314 Site 2 385 Site 3 323 using the RV The results for each target including the Sensitivity and Specificity for the comparison study are presented in the following Tables The RV performance was compared to a culture based reference method followed by direct fluorescent antibody DFA identification of all culture positive specimens A small number of specimens see footnotes in the 2x2 tables were tested by using an Page 9 of 27 027 00024 02 Rev B A Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail pro
40. or Influenza A 2009H1N1 1 specimen was negative by RV for Flu A and positive for Flu B Initial culture DFA of the specimen was Flu A positive By sequencing the specimen was positive for Flu B and negative for Flu A By NAAT and upon repeat culture DFA the specimen was positive for Flu B and negative for Flu A and RSV 1 specimen was negative for Flu A and positive for RSV B by RV By initial culture DFA the specimen was Flu A positive By sequencing the specimen was positive for RSV B and negative for Flu A By NAAT and upon repeat culture DFA the specimen was positive for RSV and negative for Flu A and Flu B f2 specimens were negative by RV but Flu A positive by culture By sequencing both specimens were negative for Flu A Both specimens were negative for Flu A and negative for Flu B and RSV B by NAAT 25 specimens failed sequencing or were not subtyped by the RV assay and are not included in the Subtyping Tables Page 11 of 27 027 00024 02 Rev B a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us Influenza B A Influenza B Results RV vs Culture DFA results Culture DFA INFB Positive Negative Total Total Positive 43 ge 46 Sensitivity 100 91 8 100 95 Cl RV Negative 0 976 976 Specificity 99 7 99 1 99 9 95 Cl Total 43 979 1022 B Influenza B Discordant Summary 1 specimen was positive for Influenza B by RV and negati
41. ot be the definite cause of disease The use of additional laboratory testing and clinical presentation must be considered in order to obtain the final diagnosis of respiratory viral infection Performance characteristics for Influenza A Virus were established when Influenza A H3 A H1 and 2009 H1N1 were the predominant Influenza A viruses circulating These characteristics may vary when other Influenza A viruses are emerging If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities specimens should be collected with appropriate infection control precautions used specifically for novel virulent influenza viruses and sent to 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 BACKGROUND INFORMATION AND CLINICAL UTILITY The respiratory tract is one of the most common sites for infections as it comes into contact with pathogens frequently Influenza A and B viruses and RSV are collectively responsible for a majority of respiratory illnesses and cause significant morbidity and mortality Infections with Influenza A and B viruses often result in the respiratory illness commonly referred to as the flu Flu is highly contagious and according to the CDC 5 20 of the population contract the flu each year Over 200 000 people are
42. re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us of the three strains A California 7 2009 H1N1 A Victoria 210 2009 H3N2 an A Perth 16 2009 like virus and B Brisbane 60 2008 Both vaccine samples were diluted into a sample matrix Universal Transport Medium Copan and log dilution series were generated for each vaccine Both Influenza vaccines gave Detected signals in the RV for Influenza A H3 H1 and Influenza B As the viruses in these vaccines were reassortants neither vaccines gave a Detected call for 2009 H1N1 In the dilution series for FluMist Influenza A and the H1 and H3 subtypes were not detected at or below an approximate 1 5x10 dilution while Influenza B was not detected at or below an approximate 1 5x10 dilution see Table In the dilution series for FluLaval the H1 subtype was not detected at or below the 1 1x10 dilution Influenza A and the H3 subtype were not detected at or below an approximate 1 1x10 dilution and Influenza B was not detected at or below an approximate 1 1x10 dilution In all the other samples tested a valid test result was provided indicating that the interferents did not present a problem during the different steps of the RV Moreover in each of these samples the expected calls were obtained for each of the analytes tested none of the interferents at the tested concentration prevented the RV from making the correct Detected calls for the vi
43. rus strains and analytes present in samples CONTACT INFORMATION Nanosphere Inc 4088 Commercial Avenue Northbrook IL 60062 Customer Service and Technical Service Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us TEST KIT LABELING The contents of a Test Kit may use symbols defined below on labels Catalog number Use by YYYY MM DD Batch code Serial number e El El In vitro diagnostic medical device Page 25 of 27 027 00024 02 Rev B a Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us PATENTS AND TRADEMARKS The Verigene Reader may be protected by US patent 7 110 585 and other pending US and foreign patent applications The Verigene Processor and SP Processor may be protected by US patent 7 396 677 and 7 625 746 and other pending US and foreign patent applications The Verigene Test Cartridge and or its method use may be protected by one or more of the following US patents 6 506 564 6 602 669 6 645 721 6 673 548 6 677 122 6 720 147 6 730 269 6 750 016 6 767 702 6 759 199 6 812 334 6 818 753 6 903 207 6 962 786 6 986 989 7 695 952 and other pending US and foreign patent applications Methods for analysis of results by the Verigene Reader are made possible under license of US patents 5 599 668 and 5 843 651 owned by Abbott Laboratories The use of
44. sative agents for clinical symptoms e The detection of viral nucleic acid is dependent on proper specimen collection handling transport storage and preparation including extraction Failure to observe proper procedures in any of these steps could lead to incorrect results e There is a risk of false negative results due to sequence variants in the viral targets of the assay procedural errors amplification inhibitors in the specimen or inadequate viral concentration for amplification e A specimen yielding a negative result may contain respiratory viruses other than Influenza A Influenza B or RSV e There is a risk of false positive results due to cross contamination by target viruses their nucleic acids or amplified product or from non specific signals in the assay Page 6 of 27 027 00024 02 Rev B Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us e Performance characteristics of the RV in a paired comparison study of fresh and 3x freeze thaw cycles demonstrated positive percent agreement of 100 for Influenza A Influenza A subtype H1 Influenza A subtype H3 Influenza A subtype 2009 H1N1 Influenza B RSV A and RSV B The negative percent agreement was 100 Influenza A Influenza A subtype H1 Influenza A subtype H3 Influenza A subtype 2009 H1N1 Influenza B RSV A and RSV B However freeze thaw cycles may compromise specimen stability
45. te Report prepared August 10 2007 Retrieved December 5 2008 from http www cdc gov flu weekly weeklyarchives2006 2007 06 07summary htm 14 2007 08 U S Influenza Season Summary Centers for Disease Control and Prevention website Report updated October 6 2008 Retrieved December 5 2008 from http www cdc gov flu weekly weeklyarchives2007 2008 07 08summary htm 15 Centers for Disease Control and Prevention 2010 Respiratory Syncytial Virus Activity United States July 2008 December 2009 MMWR 59 8 230 233 18 Centers for Disease Control and Prevention 2008 Brief Report Respiratory Syncytial Virus Activity United States July 2007 December 2008 MMWR 57 50 1355 1358 Page 27 of 27 027 00024 02 Rev B
46. ulture DFA Sequencing Culture DFA Sequencing INFA H3 Positive Negative Total Total Positive 108 0 108 Sensitivity 100 96 6 100 95 Cl RV Negative 0 909 909 Specificity 100 99 6 100 95 Cl Total 108 909 1017 Page 10 of 27 027 00024 02 Rev B A Customer Service or Technical Service e N a n O S p h e re Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us C Influenza A Subtype H1 Results RV vs Culture DFA Sequencing Culture DFA Sequencing INFA H1 Positive Negative Total Total Positive 39 1 40 Sensitivity 100 91 0 100 95 CI RV Negative 0 977 977 Specificity 99 9 99 4 100 95 Cl Total 39 978 1017 D Influenza A Subtype 2009 H1N1 Results RV vs Culture DFA Sequencing Culture DFA Sequencing INFA 2009 H1N1 Positive Negative Total Total Positive 206 0 206 Sensitivity 99 5 97 3 99 9 95 Cl RV Negative 1 810 811 Specificity 100 99 5 100 95 Cl Total 207 810 1017 E Influenza A and Subtype Discordant Summary a1 specimen was positive for Influenza A by both culture and RV No sub typing was observed on the RV result Also the specimen failed subtype sequencing for H1 H3 and 2009 H1N1 P4 specimens were Influenza A positive by RV No sub typing was observed on the RV All 4 specimens were culture negative and failed sequencing 1 specimen was Influenza A H1 by RV and positive for Influenza A by culture Sequencing resulted in a positive result f
47. ve for Influenza B by culture DFA This specimen was tested by NAAT assay and found to be positive for Influenza B Specimen was positive for Influenza B by sequencing and upon repeat culture DFA P2 specimens were positive for Influenza B by RV and negative for Influenza B by culture Both specimens were positive for Influenza B by sequencing RSV A RSV Results RV vs Culture DFA results Culture DFA RSV Positive Negative Total Total Positive 104 ges 109 Sensitivity 97 2 92 1 99 0 95 Cl RV Negative que 910 913 Specificity 99 5 98 7 99 8 95 Cl Total 107 915 1022 Page 12 of 27 027 00024 02 Rev B Nanosphere Customer Service or Technical Service Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us B RSV A Subtype Results RV vs Culture DFA Sequencing Culture DFA Sequencing RSVA Positive Negative Total Positive 57 0 57 RV Negative 0 962 962 Total 57 962 1019 C RSV B Subtype Results RV vs Culture DFA Sequencing Culture DFA Sequencing RSV B Positive Negative Total Positive 53 0 53 RV Negative 0 966 966 Total 53 966 1019 D RSV Discordant Summary Total Sensitivity 100 93 7 100 95 Cl Specificity 100 99 6 100 95 Cl Total Sensitivity 100 93 2 100 95 Cl Specificity 99 9 99 6 100 95 Cl 1 specimen was positive for RSV B by RV and by sequencing but negative for RSV by culture DFA Specimen was positive for RSV by NAA
48. ve the limit of detection for all but 2 viruses Each unique sample was tested 4 times once while fresh or unfrozen and once after each of the three 3 freeze thaw cycles Logistically after a sample was spiked with the appropriate virus an aliquot of this sample was tested in the RV while the sample was fresh The residual sample was frozen by placing the sample at or below 70 C For the first freeze thaw the residual sample was thawed and an aliquot was tested in the RV This constituted the first freeze thaw cycle This process was repeated for the second and third freeze thaw cycles Thus the study involved testing 60 samples x 4 separate tests 1 fresh 3 frozen 240 samples The performance of each virus analyte after each freeze thaw cycle was compared to the corresponding result obtained when the sample was fresh or unfrozen The results from this paired comparison showed 100 agreement between the results obtained while the sample was fresh and those obtained after each freeze thaw cycle at all the concentration levels tested Overall the results showed that the RV detected the viruses analytes correctly even after the sample was subjected to 3 sets of freeze thaw cycles The viral analytes yielded a positive percentage agreement of 100 after each freeze thaw cycle when compared to the results from the corresponding fresh samples Carry Over Cross Contamination Study The carry over study
49. vided for each of the viruses analytes in the RV If the specimen contains a novel Influenza A virus that is unsubtypeable for H1 H3 or 2009 H1N1 RV provides a Detected result for Influenza A and a Not Detected result for H1 H3 and 2009 H1N1 the unsubtypeable result for Influenza A is an inferred result or inferred unsubtypeable A fresh specimen should be tested for confirmation of the inferred unsubtypeable result For assays that differentiate between Influenza A subtypes a positive test result for Influenza A in the presence of a negative test result for an Influenza subtype i e H1 or H3 or 2009 H1N1 necessitates immediate notification of appropriate local state or federal public health authorities to determine necessary measures for verification of results in accordance with the MMWR notice http www cdc gov mmwr preview mmwrhtml mm5613a4 htm and http www cste org ps 2007pdfs novelfluanndssjan10final23 pdf to determine whether the questionable Flu A specimen represents a novel strain of Influenza A Page 7 of 27 027 00024 02 Rev B Customer Service or Technical Service N anos p h ere Phone 1 888 837 4436 toll free E Mail productsupport nanosphere us Dy Calls for Valid Tests Virus Internal Controls Calls Notes Status INFA H1 Verified Influenza A H1 INFA H3 Verified Influenza A H3 INFA 2009H1N1 Verified Ima 2099 H1N1 Inferred unsubtypeable Influenza A strain rare
50. was performed to assess the carry over cross contamination of the RV by alternately running High Positive samples followed by High Negative samples Multiple Verigene Processors SP s used in the study and each Processor SP was subjected to a comprehensive set of tests Based on the collective data there was no evidence of cross contamination Moreover there was no evidence of any cross over within the Verigene Processor SP modules when high titer samples were alternated with low titer samples Interferences The potential inhibitory effect of interfering substances or interferents that may be encountered in nasopharyngeal specimens on the RV was assessed The viral strains and the titers used in the studies are listed along with the interferents and the amount employed in the tests in the following two Tables Concentration Unique Sample Viral Strain Expected Detected Calls TCID50 mL Influenza A Wisconsin 67 05 Influenza A H3 2 INFA H3 RSV A RSV A Strain A2 RSVA 100 Influenza B Wisconsin 2 06 Influenza B 5 INFB RSV B RSV B Wash 18537 62 RSVB 10 INFA H1 Influenza A Virginia 01 2006 Influenza A H1 20 INFA 2009 H1N1 A California 04 2009 Influenza A 2009 H1N1 20 Page 23 of 27 027 00024 02 Rev B A Customer Service or Technical Service Nanosphere o E Mail productsupport nanosphere us Active tere Se Amt Human Blood Human Blood 5 viv Oxymetazoline Anefrin Nasal Spray 10 viv NaCl Saline Nasa
51. xpected subtyping results Two swine origin H1N1 strains of non human origin Influenza A Swine lowa 15 30 and Influenza A Swine 1976 31 gave a Detected result for Influenza A and for H1 but gave a Not Detected result for 2009 H1N1 Higher concentrations 1 and 2 logs did not cross react with 2009 H1N1 demonstrating good assay specificity The H5 and H7 Influenza strains in this study were grown in culture titered and inactivated before they were tested in the RV The H5 and H7 strains gave a Detected result for Influenza A and a Not Detected result for H1 H3 and 2009 H1N1 As expected these strains were unsubtypeable in the RV Due to the inactivation process used for the H5 and H7 strains a loss in the original titer was noted While lower dilutions than those reported in the Table below were not tested higher dilutions 1 and 2 logs higher concentrations were examined No cross reactivity with H1 H3 or 2009 H1N1 was observed at any of these higher dilutions again demonstrating good assay specificity Virus Strains eet INFA H1 H3 g INFB RSVA RSVB Influenza A I A New Caledonia 20 99 1X10 j 7 7 i i 3 Influenza A PR 8 34 H1N1 1X10 7 Influenza A1 FM 1 47 H1N1 5X10 E z Influenza A NWS 33 H1N1 1X10 E 7 Influenza A1 Denver 1 57 H1N1 1X10 z Influenza A Hawaii 15 01 H1N1 1X10 gt z O MENE AN E Influenza A Virginia 01 2006 H1N1 1X10
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