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Verigene EP Package Insert

1. 99 4 1265 1272 98 9 99 8 86 4 19 221 65 1 97 1 Do CHE So eo Prospectively Collected 84 2 99 9 Clinical Specimens 90 9 100 96 2 100 342 342 98 9 100 100 1293 1293 99 7 100 67 67 94 6 100 100 1 1 2 5 100 O D O D O 33 33 89 4 100 100 202 202 98 2 100 99 7 352 353P 98 4 100 D e D e 42 O O o D E D Q o C 2 S O 80 4 97 0 Page 22 of 40 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 MEME No Type EP Test Result Reference Method Results i PCR Amp BDS Results i PEE C 1 Fresh e Not Detected _ C jejuni subsp jejunia Positive for Campylobacter jejuni een i e ii Not Detected _ C jejuni subsp jejuni amp Proteus SPP 1 Negative for Campylobacterspp een Ded Simulated 2X i Not Detected Clar POSitive for Campylobacter lari een 1 o 1 Select NolDeteced Campylobacter oi a 1 i Fresh Campylobacter i Negative Positive for Campylobacter coli cene 2 Fresh Campylobacter M morganii subsp morganii amp N cinerea Positive for Campylobacter jejuni n 3 fresh Campylobacter i P aeruginosa UU Positive for Campylobacter jejuni nn
2. Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Verigene Enteric Pathogens Nucleic Acid Test EP Rx Only 20 005 023 Test Kit e 20 012 023 Amplification Kit LIU LKEY CODE NAN023 INTENDED USE The Verigene Enteric Pathogens Nucleic Acid Test EP is a multiplexed qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria viruses and genetic virulence markers from liquid or soft stool preserved in Cary Blair medium collected from individuals with signs and symptoms of gastrointestinal infection The test is performed on the automated Nanosphere Verigene System utilizing reverse transcription RT polymerase chain reaction PCR and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the following pathogenic bacteria and viruses Campylobacter Group composed of C coli C jejuni and C lari Salmonella species Shigella species including S dysenteriae S boydii S sonnei and S flexneri Vibrio Group composed of V cholerae and V parahaemolyticus Yersinia enterocolitica e Norovirus GI GIl e Rotavirus A In addition EP detects the Shiga toxin 1 gene and Shiga toxin 2 gene virulence markers Shiga toxin producing E
3. A i TOSO i Campylobacter E CO s s Positive for Campylobacter jejuni cei 9 i Fresh i Campylobacter Negative UU II L Positive for Campylobacter jejuni eni 6 Fresh i Campylobacter E coh Positive for Campylobacter jejuni n i Besh 00 Campylobacter Negative eee Positive for Campylobacter jejuni eni 8 i Fresh n Campylobacter M morganii subsp Morganii sss Positive for Campylobacter jejuni eni E NE 9 Fresh Campylobacter C braakii amp E cloacae subsp dissolvens amp N cinerea Positive for Campylobacter jejuni n 10 Fresh sss Campylobacter Negative s Positive for Campylobacter SPP cei Jt Fresh Campylobacter NegalvVe s Positive for Campylobacter spp 12 Fresh i Campylobacter Negative s Positive for Campylobacter jejuni n 19 i Presh _ 022p Campylobacter Negative s Positive for Campylobacterjejun eni 44 Fresh e Campylobacter NegaliVe s Positive for Campylobacter jejuni een 15 Fresh Campylobacter E coh Positive for Campylobacter jejuni n 16 Fresh sss Campylobacter Proteus SD s Positive for Campylobacter Coli eni ovs Afi Fresh Campylobacter M morganii subsp morganii Positive for Campylobacter jejuni re i Campylobacter and E PUN e 1 Select Salmonella Salmonella Positive for Campylobacter jejuni and Salmonella enterica i 1 1 2 Fresh _ NotDetected Salmonelaspp s Positive for Salmonella enterica rei L a 2 ji Fresh Not Detected Salmonelaspp Positive for Salmo
4. i Shigela i Negative e positive for ShigellaEIEC ecc 10 Fresh Shigella 1 NegaliVe e Positive for ShigellEIEC 7 i i Fresh Shgela ESCOl Q gg k 7 Positive for Shigella EIEC een J2 j Fresh i Shigella Negative t Not performed eccessi 13 i Fresh Shigella i NegalVe UU POSItVO for Shigella SPP rece 14 l Fresh Shigella Neate SG 7 I Sy O_DOU__ NK Positive for Shigella SPP eee 15 j Fresh i Shigella Negative ce Positive for Shigella Spp oui 19 i Fresh Shigela NegaliVe s Positive for ShigellEIEC iii m l 1 Frozen Shigela ete DPG amp 1g Positive for Shigella EIEC een RED 1 i Seld i Shigella sus ShigatoXin s Positive for Shiga toxin 1 A Simulated 2X Not Detected Vibrio parahaemolyticus s Positive for V parahaemolyticus sl 2 Simulated 2X Not Detected Vibrio cholerae U Positive for V cholera ceci B i 3 i Simulated 40X Not Detected Vibro cholerae Negative for V cholera resse 4 Simulated 40X Not Detected I Vibrio parahaemolyticus U II I Negative for V parahaemolyticus eni sui D Simulated 40X Not Detected Vibriocholerae Positive for V cholerae eccessi i pi 1 i Simulated 30X i a i Campylobacter lari i Not performed i q i One FP Campylobacter and one FN Campylobacter were processed together at the central reference testing site and may be a result of a sample mix up r i 1 On
5. 100 Norovirus Gl Norovirus Detected 93 8 100 100 15 16 16 16 16 16 69 8 99 8 79 4 100 79 4 100 100 100 100 Moderate 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 Norovirus GII Norovirus Detected 100 100 100 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 100 100 100 16 16 16 16 16 16 100 100 100 Negative Stool Matrix All Targets Not Detected 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 100 100 100 Clostridium difficile All Targets Not Detected 79 4 100 79 4 100 79 4 100 Page 30 of 40 100 100 100 Moderate 16 16 16 16 16 16 0 0 91000 1009 Rotavirus Rotavirus Detected aa HESS IUUD u QUOI 79 4 100 79 4 100 79 4 100 16 16 16 16 16 16 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free e a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 14 EP Reproducibility Performance Study Summary Call pate Total Agreement with Sample Expected Call Concentration 95 Cl Expected Result Initial 95 CI 89 90 90 90 90 90 Moderate 98 9 100 100 Escherichia coli Stx2 Shiga Toxin 2 Detected ow 25 010 ae 2621 0 97 8 100 98 9 92 2 99 7 96 0 100 94 0 100 89 90 90 90 88 90 Moderate 98 9 100 97 8 i 94
6. Centers for Disease Control and Prevention Web 14 July 2011 lt http www cdc gov ncidod eid vol5no1 altekruse htm 09 gt Campylobacter General Information Centers for Disease Control and Prevention 20 July 2010 Web 14 July 2011 http www cdc gov nczved divisions dfbmd diseases campylobacter Yersinia Enterocolitica Centers for Disease Control and Prevention 25 October 2005 Web Accessed 30 June 2014 Vibrio Illness Vibriosis Centers for Disease Control and Prevention 21 October 2013 Web Accessed 30 June 2014 http www cdc gov vibrio index html gt Shigellosis General Information Centers for Disease Control and Prevention http www cdc gov nczved divisions dfbmd diseases shigellosis Updated November 16 2009 Accessed December 20 2011 Burden of Norovirus Illness and Outbreaks Centers for Disease Control and Prevention 3 June 2014 Web Accessed 24 June 2014 lt http www cdc gov norovirus php illness outbreaks html gt Belliot G Lopman BA Ambert Balay K Pothier P 2014 The Burden of Norovirus gastroenteritis an important foodborne and healthcare related infection Clin Microbiol Infect doi 10 1111 1469 0691 12722 Guidance for Industry and Food and Drug Administration Staff Class Il Special Controls Guidance Document Norovirus Serological Reagents US Food and Drug Administration 9 March 2012 Web Accessed 24 June 2014 Rotavirus in the U
7. There were fourteen 14 Pre Analysis Errors these tests were repeated and valid test results were obtained for a PAE rate of 0 896 14 1863 There were 49 initial No Calls which were repeated once All except six 6 of these repeats generated a valid result yielding a final call rate for the study number of valid tests total tests conducted of 99 796 1794 1800 The percent detection rate of the Reproducibility Study across all sites was 98 1 There were 34 discordant calls observed versus expected Twenty six 26 of the 34 discordant results were observed with the low positive samples as expected For the moderate positive samples it is expected that the target s present in the sample will be detected approximately 9996 of the time in this study the targets were detected at an acceptable rate of 99 096 For the low positive samples it is expected that the target s present in the sample will be detected approximately 9596 of the time in this study the targets were detected at an acceptable rate of 96 896 The final study results for the negative panel members demonstrated 10096 agreement with the expected results The results of the Reproducibility Study are summarized in Table 14 Page 29 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service dl In the U S Phone 1 888 837 4436 toll free E a O S e re OR E Mail productsupport nanosphere us Out
8. set up one test at a time change gloves after handling a sample and decontaminate pipettes and sample tubes Note Store the original Cary Blair specimen at room temperature until completion of Verigene oystem testing 8 Upon Completion of a Test Run a he Verigene Reader will generate a ring to notify the user when the test is completed and the Processor SP will display a message indication Procedure Complete Ready to Open Drawer The Test Cartridge should be removed from the Processor SP upon completion of the test b Open the Drawer Assembly by pressing the OPEN CLOSE button c Capthe Amplification Tube for disposal d Remove the Test Cartridge and immediately orient to its side e While keeping the Test Cartridge on its side separate the Reagent Pack and keep the Test Substrate on its side for 30 60 seconds after removal as illustrated below to allow the final rinse to dry away from the analysis area Substrate Reagent Holder Pack EE Page 13 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Nanosphere Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 9 Analyze Results a b c Remove the protective tape from the back of the slide in the Substrate Holder Use the Reader s barcode s
9. 0 100 96 0 100 92 2 99 7 Salmonella enterica Salmonella Detected 89 90 90 90 86 90 98 9 100 95 6 94 0 100 96 0 100 89 0 98 8 89 90 90 90 88 90 Moderate 98 9 100 97 8 1 Shigella Detected 94 0 100 96 0 100 92 2 99 7 L d Shiga Toxin 1 Detected 88 90 90 90 86 90 97 8 100 95 6 92 2 99 7 96 0 100 89 0 98 8 90 90 90 90 89 90 ps s e basali Yersinia enterocolitica 96 0 100 96 0 100 94 0 100 Detected 89 90 90 90 80 90 EN i p pn 94 0 100 96 0 100 80 5 94 5 88 90 90 90 90 90 97 8 100 100 92 2 99 7 96 0 100 96 0 100 Campylobacter jejuni Campylobacter Detected 89 90 90 90 90 90 98 9 100 100 94 0 100 96 0 100 96 0 100 87 90 90 90 90 90 Moderate 96 7 100 100 su 90 6 99 3 96 0 100 96 0 100 nC DETER 88 90 90 90 90 90 97 8 100 100 92 2 99 7 96 0 100 96 0 100 87 90 88 90 86 88 Moderate 96 7 97 8 97 7 7 Norovirus GI Norovirus Detected as 3 E eee EN n p Een 90 6 99 3 96 0 100 89 0 98 8 81 90 87 90 86 87 Moderate 90 96 7 98 9 Norovirus GIl Norovirus Detected ee 32 3 oo a 0 pt 92 2 98 9 100 84 6 96 8 94 0 100 95 9 100 89 90 90 90 90 90 Moderate 98 9 100 100 94 0 100 96 0 100 96 0 100 Rotavirus Rotavirus Detected 88 90 90 90 87 90 97 8 100 96 7 92 2 99 7 96 0 100 90 6 99 3 1 87 90 90 90 90 90 egative Stool Matrix 96 7 100 100 Negative 90 6 99 3 96 0 100 96 0 100 86 90 90 90 90 90 Vibr
10. 70x104 Campylobacter coli ATCC 43482 1 11x109 Campylobacter 3 70x10 1 11x10 Campylobacter lari ATCC 35222 3 70x104 Salmonella enterica subsp enterica serovar Typhi ATCC 9993 3 33x105 Salmonella 3 33x105 Salmonella enterica subsp arizonae ATCC 13314 3 33x105 Shigell Shigella dysenteriae Shiga Toxin 1 ATCC 29026 3 70x104 ii Shigella flexneri ATCC 25929 1 11x10 3 70x104 1 11x105 Shigella sonnei ATCC 29030 3 70x104 Shigella Shigella boydii ATCC 12035 1 11x10 Vibrio cholera ATCC 39315 1 11x105 Vibrio 3 70x104 1 11x105 Vibrio parahaemolyticus ATCC 49398 3 70x104 ATCC 700822 3 33x10 Versa Yersinia enterocolitica a 111x105 3 33x105 ATCC 23715 111x105 enterocolitica E coli Shiga Toxin 1 ATCC 43890 4 10x109 Shiga Toxin 1 4 10x105 3 70x10 E coli Shiga Toxin 2 ATCC BAA 176 1 11x105 Shiga Toxin 2 higa Toxin1 3 70x104 1 11x109 E coli Shiga Toxin 1 Shiga Toxin 2 ATCC 43895 3 70x104 Lu Shiga Toxin 2 Norovirus Norovirus GII ATCC D17219 1 67x108 1 67x108 ATCC VR 2550 1 11x10 1 11x10 Rotavirus Group A Rotavirus ATCC VR 2551 3 70x102 3 70x102 Page 32 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 D Analytical
11. S Centers for Disease Control and Prevention 12 May 2014 Web Accessed 24 June 2014 lt http www cdc gov rotavirus surveillance html gt Tate Jacqueline E et al Uptake Impact and Effectiveness of Rotavirus Vaccination in the United States Review of the First 3 Years of Postlicensure Data Pediatric Infectious Disease Journal 30 1 2011 Rotavirus Clinical Disease Information Centers for Disease Control and Prevention 22 Apr 2011 Web 14 July 2011 lt http www cdc gov rotavirus clinical html gt Chua K G rtler V Montgomery J Fraenkel M Mayall BC Grayson ML Campylobacter insulaenigrae causing septicaemia and enteritis J Med Microbiol 2007 56 Pt 11 1565 1567 Page 40 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014
12. Take the Extraction Tray out of the BSC and insert into the Extraction Tray Module on the Processor SP b The Extraction Tray can only be loaded in one location and orientation in the Drawer Assembly When the Extraction Tray is loaded correctly the Sample Loading Well is located at the right hand side of the Drawer Assembly Place the Extraction Tray in the Drawer Assembly and press down on the corners of the tray to ensure it is level The image below shows a properly loaded Extraction Tray Sample Loading Well fj Extraction Tray Y 3 Load the Tip Holder Assembly a The Tip Holder Assembly is a plastic holder that contains two Pipette Tips and a rubber Tip Seal Each Pipette Tip contains an O ring on top Page 8 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service A In the U S Phone 1 888 837 4436 toll free a O e TO OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 b Before using the Tip Holder Assembly check the top of each Pipette Tip for the O ring and confirm that the rubber Tip Seal is sitting straight and flush between the tips If either is missing replace with a new Tip Holder Assembly c Insert the Tip Holder Assembly into the Drawer Assembly The image below shows a properly loaded Tip Holder Assembly The Tip Holder Assem
13. all applicable regulations mandated by local state provincial and federal agencies for the handling transport of etiologic agents e National state and local public health authorities have published guidelines for notification of reportable diseases in their jurisdictions including Salmonella Shigella Vibrio and Shiga like Toxin producing E coli STEC stx1 stx2 to determine necessary measures for verification of results to identify and trace Page 18 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 outbreaks Refer to The CDC s Nationally Notifiable Disease Surveillance System http wwwn cdc gov nndss for additional information and resources Laboratories are responsible for following their state or local regulations for submission of clinical material or isolates on positive specimens to their state public health laboratories WARNINGS AND PRECAUTIONS INSTRUMENT A General Instrument Safety WARNING Use this product only as specified in this document Using this instrument in a manner not specified by Nanosphere may result in personal injury or damage to the instrument Anyone who operates the instrument must have e Received instruct
14. coli STEC typically harbor one or both genes that encode for Shiga toxins 1 and 2 EP is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness in conjunction with other clinical laboratory and epidemiological information however is not to be used to monitor these infections EP also aids in the detection and identification of acute gastroenteritis in the context of outbreaks Due to the limited number of positive specimens collected for certain organisms during the prospective clinical study performance characteristics for Yersinia enterocolitica Vibrio Group and Shigella species were primarily established with contrived specimens Concomitant culture is necessary for organism recovery and further typing of bacterial agents EP results should not be used as the sole basis for diagnosis treatment or other patient management decisions Confirmed positive results do not rule out co infection with other organisms that are not detected by this test and may not be the sole or definitive cause of patient illness Negative EP results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non infectious causes such as ulcerative colitis irritable bowel syndrome or Crohn s disease Page 1 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In t
15. rate was 98 196 314 320 Of the six 6 initial No Calls all 6 yielded a valid test result upon retesting for a final call rate of 100 320 320 The Pre Analysis Error rate was 0 9 3 333 The final study results for the negative panel members moderate positive samples and low positive samples agreed 99 796 with the expected results One inaccurate call was made involving a Salmonella enterica specimen whereby EP unexpectedly detected Stx2 in addition to Salmonella Additionally acceptable precision was observed for the panel members across multiple consumable lots days operators runs instruments and replicates The Reproducibility Study involved the testing of twenty 20 unique samples in triplicate by two 2 operators for five 5 non consecutive days at three sites for a total of ninety 90 tests per sample The twenty 20 member sample panel comprising representative strains of each of the eight 8 organisms targets detected by EP was prepared at two 2 different concentrations 18 positive samples two Norovirus strains tested and two 2 negative samples consisting of Negative Stool Matrix and Clostridium difficile This panel included for each strain a Low Positive sample which would be expected to produce a positive result approximately 95 of the time and a Moderate Positive sample which would be expected to yield a positive result approximately 9996 of the time A total of 1800 initial tests were conducted
16. s L Positive for Stx 1 GONO d di NM NN Fresh ss Shiga Toxin 1 and Norovirus Escherichia coli i positive for SIX 1 gene el meu d 3 i _ Fresh i Shiga Toxin 1 and Shiga Toxin 2 amp Citrobacter youngae s Positive for Stx 1 gene and Stx 2 gene i ue Oe asce Select i Shiga Toxin 1 and Campylobacter i Campylobacter sd Positive for Six 1 GENE sss 1 1 1 1 mMlated 19X i Shiga Toxin fand Salmonella Salmonella enterica subsp enterica _ Negative for Stx 1 gene and Six 2 gene ss c L2 1 2mulated 30X i Shiga Toxin 1 and Campylobacter i Campylobacter jejuni subsp jejuni Notperformed d 3 Simulated 2X Shiga Toxin pa and Yersinia x Yersinia enterocolitica x Positive for Stx 1 gene amp Stx 2 gene x q elsi Simulated 30X i NOt Detected Escherichia coli Shiga Toxin2 Positive for Stx 2 gene ei n T TEE Simulated 2X sss NOt Detected essi Escherichia coli Shiga Toxin 2 ris Positive for Stx 2 GENE s t NE ONE ij or l2 0 T Positive for Six 2 GENE ss ur AEN Fresh 1 Shiga Toxin 1 and Shiga Toxin 2 Citrobacter youngae 1 Positive for Stx 1 gene and Six 2 gene J U i A L mulated 31X Shiga Toxin 2 and Yersinia enterocolitica Yersinia enterocolitica Negalivefor Six 1 gene and Stx 2 gene i 2 Simulated 2X ATOAN Niga Ta 2 ana Yersinia Yersinia enterocolitica Positive for Stx 1 gene amp Stx 2 gene enterocol
17. study was performed to assess the potential inhibitory effect of endogenous and exogenous substances that can commonly be found in clinical stool specimens Four organisms representative of the target analytes detected by EP i e Campylobacter jejuni Escherichia coli Shiga toxin 1 Norovirus Gl and Rotavirus Group A were individually challenged at 3x LoD with 22 potentially interfering substances Table 18 at high medically relevant worst case concentrations None of the 22 substances tested showed any inhibitory effect on the detection of target enteric pathogens using EP Table 18 Exogenous Substances Evaluated for Interference Metronidazole Topical Cream 0 7596 Mucus Nasopharyngeal Swab Sample in UTM Nystatin Suspension Vaseline Original 100 Pure Petroleum Jelly Barium Sulfate Mucin from bovine submaxillary glands ion H Anti i 0 id wi i Preparation HY Anti Itch Hydrocortisone 1 Tums Antacid with Calcium Extra Strength 750 Type S Dehydrated Desitin Maximum Strength Original Paste Gaviscon Extra Strength Liquid Antacid Preparation H Hemorrhoidal Ointment H Carryover Cross Contamination Study The potential for carryover and cross contamination on the Verigene System was assessed by alternately testing nine representative high positive enteric pathogen samples Yersinia enterocolitica Shigella dysenteriae Stx1 Escherichia coli Stx2 Salmonella enterica Campylobacter jejuni and Vibrio cholera a
18. 0037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 STORAGE HANDLING AND STABILITY Table 1 Storage and Handling Storage Stool Prep Buffer SPB Tubes amp Swabs 20 30 C Do not freeze Sample Well Caps Test Test Cartridges A Shipped frozen Upon receipt store frozen Ap canon rays Do not re freeze after thawing Extraction Mm 2 8 C Do not freeze VERIGENE DAILY MAINTENANCE A Work Area Preparation Each day of testing and before and after sample preparation prepare the testing work area by sanitizing the biological safety cabinet BSC countertops vortex mixers Mini Centrifuges pipettes and any other equipment used for sample processing with a lint free decontaminating wipe B Verigene System Cleaning Prior to the start of testing each day perform the following steps for each instrument being used for testing IMPORTANT If there is liquid visible in the drawer assembly of the Verigene SP or anything out of the ordinary is observed do not proceed and immediately contact Nanosphere Technical Service 1 While wearing fresh gloves use a lint free decontaminating wipe to thoroughly wipe the drawer assembly of the Verigene SP For the Verigene Reader wipe down the user inter
19. 014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e GO OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 F Microbial Interference Two representative bacterial organisms detected by EP Campylobacter jejuni and Escherichia coli Shiga toxin 1 and two representative viral organisms Norovirus GI and Rotavirus Group A were evaluated for potential interference in the presence of fourteen 14 potentially interferent microorganisms not detected by EP Bacteroides fragilis Prevotella oralis Prevotella melaninogenica Bifidobacterium bifidum Clostridium perfringens Enterobacter aerogenes Enterococcus faecalis Escherichia coli Klebsiella pneumonia Lactobacillus acidophilus Staphylococcus aureus Blastocystis hominis Entamoeba histolytica and Candida albicans These 14 microorganisms represent the most prevalent bacteria known to be present in the human colon and therefore the most likely to be encountered in stool specimens tested with EP These normal flora organisms were tested at a concentration of 10 CFU mL with the exception of the parasites Blastocystis hominis and Entamoeba histolytica which were tested at 9x1 0 cells mL and 7x10 cells mL respectively No interference was observed with EP for any of the samples tested G Interference Exogenous Substances A comprehensive interfering substances
20. 100 Salmonella 100 100 enterica subsp 2 2 1 1 salamae 15 8 100 2 5 100 Salmonella spp or O aos 20 23 N A N A N A not identified 66 4 97 2 Page 27 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service dl In the U S Phone 1 888 837 4436 toll free a a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 11 Clinical Mixed Specimen Combinations Detected by EP Multiple Target Combinations Detected by EP Reference Test Target 1 Target 2 Target 3 Fast RSI Discrepant Identification AM Specimens Y enterocolitica Shiga Toxin 1 m 2 LX J a Stx 1 gene Stx 2 gene Campylobacter Shiga Toxin 1 Stx 1 gene Salmonella nme MEM 1 Salmonella ee m oo ES Campylobacter Campylobacter Vbio NA 1 1 Vr _ Salmonella Shiga Toxin 1 i a a a Stx 1 gene Shigella Shiga Toxin 1 Shigella Y enterocolitica Shiga Toxin 2 TE Stx 2 gene Stx 1 gene Salmonella Norovius OO NA ia Shiga Toxin 1 Norovirus orovirus 1 Sixt gene 2 1 Norovius TOTAL ae oe a a me Table 12 Clinical Mixed Specimen Combinations Detected by Reference Methods Multiple Target Combinations by Reference Test Detected by EP Discrepant Target 1 Target 2 Target 3 Total Specimens Discrepant Targets M 9 OO NA
21. 3 tests run for a total final valid test rate of 98 7 The twenty six 26 specimens which yielded a final No Call result were not included in the valid dataset utilized in the comparative test result data analysis Therefore 98 7 1940 1966 of the valid specimens were analyzed in this clinical evaluation to establish clinical performance of the test 1294 of which were prospectively collected fresh specimens 34 of which were prospectively collected frozen specimens 203 of which were selected frozen specimens and 409 of which were simulated frozen specimens The clinical performance of EP is summarized below in Table 6 for the five bacterial targets n21940 in Table 7 for the Stx Combined targets Table 8 for the Stx1 and Stx2 targets n21940 and Table 9 for the two viral targets n21942 Table 10 contains additional genus group level specific EP performance data stratified by individual species within each genus i e Campylobacter Group Salmonella spp Shigella spp and Vibrio Group In total there were 25 mixed specimens that were detected by EP and 11 mixed specimens detected by the reference comparator methods Table 11 lists the distinct mixed specimen combinations detected by EP in the clinical study and Table 12 lists the distinct mixed specimen combinations detected by the reference comparator methods which were all detected by EP Page 21 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B Oc
22. 95 2 ro 6 6 26 27 96 1 Campylobacter coli Salmonella enterica dysenteriae Shiga Vibrio Cholerae Yersinia enterocolitica Escherichia coli Shiga Toxin 2 Norovirus Gl Rotavirus Shigella dysenteriae Shiga 3 3 3 3 3 3 3 21 21 100 Toxin 1 Escherichia coli Bonum 2c 20 21 95 2 6 6 26 27 96 1 For the Low Titer Campylobacter coli and High Titer E coli Stx2 sample EP did not detect Campylobacter in one of the three replicates although Shiga Toxin 2 was correctly identified in all cases An additional 6 replicates were tested and the expected result was obtained for both analytes in all replicates b In one of three replicates Low titer organism was not detected High titer organism was correctly identified For the Low Titer Rotavirus and High Titer Y enterocolitica combination EP correctly identified both organisms in 2 of the 3 replicates and missed the detection of Rotavirus in one replicate An additional 6 replicates were tested and the expected result was obtained for both analytes in all replicates Page 37 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e e OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 J Cutoff Verification Target mean intensity values
23. ARKS The Verigene Reader may be protected by US patent 7 110 585 and other pending US and foreign patent applications The Verigene Processor SP may be protected by US patents 7 396 677 and 7 625 746 and other pending US and foreign patent applications The Verigene Test Cartridge and or its method of 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 7 321 829 7 695 952 7 773 790 8 323 888 and other pending US and foreign patent applications Methods for analysis of results by the Verigene Reader are made possible under license of US Patent Nos 5 599 668 and 5 843 651 owned by Abbott Laboratories Verigene and the Nanosphere Logo are registered trademarks of Nanosphere Inc Copyright 2014 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 a
24. CHARACTERISTICS The results of the analytical and clinical studies conducted to establish the performance characteristics of EP are provided below A Clinical Performance A method comparison study was conducted at multiple external clinical study sites to evaluate the performance of EP Bacterial results were compared to reference culture followed by bacterial biochemical identification Stx1 and Stx 2 specimens were identified using enriched culture and EIA with positive specimens typed for bacterial virulence markers by PCR amplification followed by confirmatory bi directional sequencing EP viral test results were evaluated by comparison to a composite of three RT PCR methods and bi directional sequencing BDS Subjects included individuals whose routine care called for enteric pathogen testing There were 1940 evaluable specimens enrolled in the clinical trial 78 specimens resulted in an initial EP No Call for a No Call rate of 4 096 78 1940 specimens Sixteen 16 specimens incurred an initial Pre Analysis Error PAE and three 3 specimens incurred a PAE upon repeat yielding a Pre Analysis Error rate of 0 996 19 2063 tests run for a total initial valid test rate of 95 196 Of the 78 initial No Calls 52 yielded a valid test result upon retesting and of the 16 initial PAEs thirteen 13 specimens yielded a valid call upon repeat The final No Call rate was 1 396 26 1940 specimens and the final Pre Analysis Error rate was 0 196 3 206
25. ELE CEPT TEEPE EEE E ECCT EE Identified by Prospectively Collected o c D E D Q o C 2 O STX Combined x No add i Identified by EP test as x Was u ed Method s x PCR Amp BD Sequencing Results if applicable A Fe 5 UU Shiga Toxin 1 L Negative eee Posilive for Stx GONO eee de os ANNAM MM Shiga Toxin 1 and Norovirus Escherichia COM sd Positive for Stx 1 gene esl m T 3 luu ED nismo ga Toxin 1 and Shiga Toxin2 Citrobacter youngae mi OVE for Six 1 gene and Stx 2 gene sss De e in niga Toxin 1 and Campylobacter Campylobacter te Positive for Stx 1 gene sss o i MUTATE 13X 1 388 Toxin land Salmonella i Salmonella enterica subsp enterica Negative for Six 1 gene and Stx 2 gene i e 14 E a Simulated SOX i Shiga Toxin fand Campylobacter i Campylobacter jejuni subsp jejuni o ri x 3 x Simulated 2X x j pice s x Yersinia enterocolitica Positive for Stx 1 gene amp Stx 2 gene di b bmulted 30X i Not Detected Escherichia coli Shiga Toxin 2 Positive for Stx 2 GONO e 1 Simulated 2X Not Detected 1 Escherichia coli Shiga Toxin 2 Positivefor Stx 2 gene cei 6 x MN EE CREE Shiga Toxin 2 sss Negative ds Positive for Six 2 gene ess T o mam le scie higa Toxin 1 and Shiga Toxin 2 i Citrobacter youngae Positive for Stx 1 gene and Stx 2 gene re 2 io Mulated 31X Shiga Toxin 2 and Yersinia enterocolitica Yersinia enterocolitica Di Negative for Stx 1 gene and
26. Reactivity Inclusivit Analytical reactivity of EP was demonstrated with a comprehensive panel of one hundred and eleven 111 clinically relevant bacterial strains and forty one 41 clinically relevant viral strains representing temporal geographical and phylogenic diversity for each claimed target Table 16 For the Stx1 and Stx2 targets Shiga toxin producing organisms tested included the vast majority of serotypes isolated in the U S and those that are outbreak related All 111 bacterial strains generated the expected result when tested in triplicate at a concentration of three times LoD The majority of viral strains were also detected at three times LoD The only exceptions were a few Norovirus GII strains that required concentrations between 10x and 50x LoD for 100 detection Considering the in silico analysis EP is expected to detect most of the Norovirus GII strains Nevertheless noroviruses are extremely diverse genetically and detection of some strains may occur with reduced sensitivity Norovirus strains GII 9 GIl 14 and GIV 1 and Rotavirus A strains G4 G5 G10 G11 and G15 are predicted to be detected based on in silico analysis Norovirus GIl 11 is not expected to be detected by EP Additionally based on in silico analysis rare Norovirus genotypes GlI 6 and GIl 13 are predicted to either be not detected by EP or to be detected with reduced sensitivity Inclusivity to Norovirus strains GIl 8 and Rotavirus G7 G21 and G24 could no
27. Shiga Toxin 1 Shiga TOAN 2 N Salmonella 9 anna TOTAL po f 9 Page 28 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 B Precision and Reproducibilit The Precision Study involved the daily testing of twenty 20 unique samples in duplicate by two 2 operators for four 4 non consecutive days for a total of sixteen 16 tests per sample In total the study yielded 320 test results The twenty 20 member sample panel comprising representative strains of each of the nine 9 organisms targets detected by EP was prepared at two 2 different concentrations 18 positive samples two Norovirus strains tested and two 2 negative samples consisting of Negative Stool Matrix and Clostridium difficile This panel included for each strain a Low Positive sample which would be expected to produce a positive result approximately 9596 of the time and a Moderate Positive sample which would be expected to produce a positive result approximately 9996 of the time The results of the precision study are summarized in Table 13 which provides the percent agreement between the expected results and the obtained results for each sample tested The initial call
28. Stx 2 gene rri i i 2 Simulated 2X 5092 Toxin 4 Sniga TOR n ean ers Yersinia enterocolitica Positive for Stx 1 gene amp Stx 2 gene enterocolitica Page 24 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 Table 8 Summary of Clinical Test Performance n 1940 Compared to Broth Enrichment EHEC EIA and Stx 1 and STX 2 typing Agreement 95 CI Agreement 95 CI Specimen Type Specimen Type 100 99 8 100 99 8 Fresh 4 4 1287 1290 Fresh 1294 6 6 1286 1288 39 8 100 99 3 100 54 1 100 99 4 100 100 100 Frozen 34 34 34 Frozen 89 7 100 Prospectively Collected Prospectively Collected 34 34 89 7 100 Clinical Specimens Clinical Specimens 96 6 99 4 57 593 348 350 88 3 99 6 98 0 99 9 100 99 5 100 100 Selected 203 9 9 193 194 Selected 203 10 10 193 193 66 4 100 97 2 100 69 2 100 98 1 100 100 99 2 Simulated 50 50 356 359 Simulated 92 9 100 97 6 99 8 BEER E EEE EEE IIC OOO UO OOO VAIO CLE COLE CEC ELE EE CEE OTO SOSIO SOSIO No Simultes on denied by EP estas __ _Refrenc Comparaor Methods as PCR AMIBD Sequencing Results applica l 21 TE ie MIA TOXIN Ii NEGARE
29. TEC 0157 H7 accounts for about 75 of these illnesses Patients with STEC are at risk of developing a condition known as hemolytic uremic syndrome HUS a severe complication that can be fatal and is characterized by renal failure hemolytic anemia and thrombocytopenia Approximately 8 of the persons diagnosed with an 0157 H7 STEC infection develops HUS Non O157 STEC has also been responsible for illness in the US and throughout the world The most commonly identified non O157 serogroups responsible for illness in the U S include O26 O45 O103 0111 0118 0121 and O145 If undiagnosed or under reported incidences of STEC infections in the US are accounted for an estimated 96 534 STEC 0157 and 168 698 non O157 infections occur annually The CDC recommends testing all stool cultures for shiga toxins and as of July 1 2013 the Joint Commission mandates all member labs must test all stool cultures for 0157 STEC using at least selective or differential media QSA 04 06 01 EP 6 Salmonella spp Infection with nontyphoidal Salmonella spp causes diarrhea and fever Approximately one million cases are known to occur annually in the U S The Salmonella serotypes that cause a majority of human illness in the U S includes Typhimurium Enteritidis and Newport Most cases of Salmonella infection or salmonellosis are typically self resolving however patients at risk Including children elderly and immunocompromised may require antimicrobial
30. bly can only be loaded in one location and orientation in the Drawer Assembly For orientation there are two holes on the deck of the Drawer Assembly that fit each Pipette Tip and the opening to the Tip Seal should face away from the Processor SP p d f Tip Holder Assembly 4 Loading the Amplification Tray a Remove the cap from the Amplification Tube from the thawed Amplification Tray and save the cap to re cap the tube when processing is complete b Insert the Amplification Tray into the Drawer Assembly The image below shows a properly loaded Amplification Tray The Amplification Tray can only be loaded in one location and orientation in the Drawer Assembly When loaded properly the tray sits flat Amplification Tray Page 9 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 N a O h e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free distributor i www e labeling eu NAN023 C Lower and latch the Drawer Clamp over the Trays while supporting the Drawer with the opposite hand The image below shows a closed Drawer Clamp over properly loaded trays and Tip Holder Assembly The Drawer Clamp will latch onto the Drawer Assembly when closed properly and the user will be unable to lift the Drawer Clamp without pressing the sliver latc
31. ca cC 9 9 5 5 Je 2 5 100 85 8 99 9 66 4 100 40 0 100 66 4 100 47 8 100 Campylobacter 100 100 100 100 jejuni subsp 5 5 1 1 Shigella flexneri N A 16 16 5 5 doylei 47 8 100 2 5 100 79 4 100 47 8 100 Campylobacter 90 0 100 100 100 100 jejuni subsp 18 20 21 21 Shigella sonnei 2 2 11 11 5 5 jejuni 68 3 99 8 83 9 100 15 8 100 71 5 100 47 8 100 93 3 100 0 100 68 1 99 8 47 8 100 0 97 5 54 1 100 Salmonella Genus Prospective Prospective Fresh Frozen Selected Simulated Analytical Fresh Frozen Selected Simulated Analytical 87 0 98 3 100 100 i 100 100 91 1 100 iii 20 23 58 59 67 67 31 31 gie 2 2 1 1 51 56 10 10 66 4 97 2 90 9 100 94 6 100 88 8 100 15 8 100 2 5 100 80 4 97 0 69 2 100 Gina n 100 84 2 100 tohi 2 2 N A Vibrio cholerae N A N A 16 19 5 5 yp 15 8 100 60 4 96 6 47 8 100 Simonei 100 100 Vibrio 100 100 94 6 100 bongori N A N A 2 2 1 1 parahaemolyticus 2 2 1 1 35 37 5 5 15 8 100 2 5 100 15 8 100 2 5 100 81 8 99 3 47 8 100 Salmonella oo 100 ina N A 58 59 1 1 N A 90 9 100 2 5 100 Salmonella 100 enterica subsp 1 1 arizonae 2 5 100 Salmonella 100 100 rds subsp ex se T B 1 co larizonae 9 Salmonella cp 100 100 enterica subsp 52 52 25 25 enterica 93 2 100 86 3 100 Salmonella 100 100 s subsp a oe T n 2n outenae 5 9 Salmonella 100 100 enterica subsp 3 3 1 1 indica 29 2 100 2 5
32. canner to read the barcode on the Substrate Holder When the barcode is accepted a prompt to load the Substrate Holder into the Reader will be displayed Immediately insert the Substrate Holder into the Reader When the load substrate prompt occurs it will only display for 20 seconds The analysis will only start if the Substrate is loaded during the animated prompt To properly insert the Substrate Holder into the Reader hold the Substrate Holder by the handle with the barcode facing away from you Next insert the Substrate Holder into the Substrate Compartment The Substrate Compartment is designed to place the Substrate Holder in the correct position Do not force the Substrate Holder in but do insert it into the Substrate Compartment as far as it will go comfortably Close the door of the Substrate Compartment The analysis will automatically begin A small camera icon will appear on the Reader to indicate that analysis has begun Once the analysis is completed by the Reader the camera icon will be replaced with an upward facing arrow and the Reader rings Confirm that a result other than No Call NO GRID has been generated by touching the Substrate icon for the test A Substrate producing a No Call NO GRID result should be reanalyzed Once the scan is complete dispose of the used Test Substrate and the used Reagent Pack To access the remaining used consumables raise the Drawer Clamp remove and dispose the used Extract
33. checks guide the user through the testing process each time a test is performed The EP barcode and specimen information are linked upon entry into the Verigene Reader to help prevent misreporting of results Page 16 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e GO OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 Assay Controls EP is a specimen to result detection system wherein nucleic acids are isolated from unformed stool specimen and specific detection is performed on an oligonucleotide array housed within the Test Cartridge To prevent reagent dispensing errors all reagents are prepackaged in single use disposables including Stool Prep Buffer Tubes reagent trays and cartridges Several layers of controls built into EP ensure that failures at any step within the test are identified during the procedure or in the end point image analysis of the Test Cartridge Internal Processing Controls An artificial DNA construct serves as a target hybridization control and is referred to as the Internal Processing Control 1 INT CTL 1 If the INT CTL1 is not valid a No Call INT CTL 1 result will be obtained and the test should be repeated MS2 Phage serves as a specimen isolation and amplifica
34. cluding an Extraction Tray Amplification Tray and 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 specimen by loading the single use consumables into the Verigene Processor SP pipetting the prepared specimen into the Extraction Tray and initiating the protocol on the Verigene Reader by scanning or entering Test Cartridge ID and specimen information Following assay completion the user inserts the substrate slide portion of the Test Cartridge into the Verigene Reader for optical analysis and generation of test results Page 3 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e GO OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 MATERIALS PROVIDED Verigene EP Test Kit Catalog number 20 005 023 e 20 Verigene EP Test Cartridges Each Test Cartridge comes preloaded with all required reaction reagents including wash solutions oligonucleotide probe solution and signal amplification solutions required to generate a test result The Test Cartridges are labeled as EP 20 006 023 e 20 Verigene EP Extraction Trays with Tip Holder Assemblies Each Extraction Tray comes preloaded with all required reagents including lysis b
35. cted at least one target in 11 296 149 1328 of prospectively collected specimens In routine practice prevalence rates may vary depending on the institution geographical location and patient population Table 5 Prevalence of Organisms Detected by EP Clinical Study Observations US Geographic Region Division Sw w w x Ww amp Gmwmw R 9 7 5 5 2 9 x o 3s 34 2 2 39 monete P 3 3 1 6 m 1 s 2 2 3 wa m 9 6 9 dre w 28 9 u We m s 9 fo 2 2 x 1 6 0 YwexwWm Posi o 9 9 0 o ww LC 3 m o 3 1 p 7 x w 3 0 9 6 9 s ma o I s s w 3 0 9 6 9 Wem P o 2 2 s w ww Wm me s 9 3 3 wl 1 I9 Geographic Areas Reference Manual US Census Bureau Chapter 6 https www census gov geo reference pdfs GARM Ch6GARM pdf Webpage last revised 9 16 2013 Page 20 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e e OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 PERFORMANCE
36. e TP Salmonella and one FN Salmonella were processed together at the study testing site and may be a result of a sample mix up Page 23 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 Table 7 Summary of Clinical Test Performance n21940 Compared to Broth Enrichment EHEC EIA STX Combined A t 95 CI Specimen Type da SEO Eee J Positive 100 99 7 Fresh 1294 TIT 1283 1287 4 59 0 100 99 2 99 9 100 Frozen 34 34 34 89 7 100 100 99 5 Selected 203 13 13 189 190 75 3 100 97 1 100 98 2 98 7 Simulated 409 107 109 296 300 93 5 99 8 96 6 99 6 SEER TEPER TEPER EEE PETER TEE CRETE ETT PETE TETCUTETEEE UTE TEEPUEE TET TTETETCUTE TEC ELTETETETTETETELTEEETEEEEEETET TE EEE EE REECE EE ECE CEE ECCT LEE ECEEEEEECEE CEE TCE TCE TET ELEC TE CE RR ASA NR RR NGA CECT EEE CERT EEE CERT EEE RR ARA RR ARA RRPR ER RR ERPA RR SG RR RR RR ER ERR RR RR ERR ER SR ERR RR RR ERR SERA SER GR S REN ERR RR RARE ERR EEE ECE INR EEE CEE CEE EE EEE ECE EEE EEE EE EE EET ECE REED Cre EE EE EE EE EE EE ERE EE EEE EE EE EIRE ERRAR EERE EE CEE PEEL EEE EEE TEE EE EE TEE PEER EET CEE EE EE EE EP EE CER CPE PERE EEE PECL CEE PEELE EERE PEER EE CEE EEE CETL EL CEE EET EE P
37. ed No Call INT CTL 2 indicating lysis extraction or amplification issue INT CTL 1 and INT CTL 2 not detected No Call INT CTL indicating lysis extraction Repeat EP amplification or target hybridization issue Ensure Test Substrate is seated properly in the substrate holder Repeat image l analysis by selecting Menu and Enter No Call NO GRID Reader unable to image Test Substrate Barcode and then scanning the Substrate barcode If the No Call persists repeat EP from original stool specimen No Call VARIATION Reader unable to obtain test result No Call BKGD because of high variability in the target Repeat EP No Call NEG CTL specific signals ay Or Dd seco Power cycle the Processor SP and repeat Processing Error within the Processor SP detected an EP y P unexpected event Repeat tests should be from the Cary Blair preserved stool specimen into a new Stool Prep Buffer tube 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 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
38. es mimicus Cedecea davisae Porphyromonas asaccharolytica tubiashii amalonaticus Prevotella melaninogenica vulnificus 3 strains Citrobacter freundii mirabilis aldovae sedlakii Proteus vulgaris aleksiciae bifermentans penneri bercovieri bolteae stuartii frederiksenii butyricum Providencia alcalifaciens TA intermedia difficile 2 strains rettgeri kristensenii difficile non tox aeruginosa 2 strains mollaretii haemolyticum Pseudomonas fluorescens pseudotuberculosis methylpentosum putida ruckeri Clostridium nexile Ruminococcus bromii rohdei noyvi liquefaciens Serratia orbiscindens marcescens perfringens aureus scindens e epidermidis Type 4 Group E septicum agalactiae 090R sordellii Streptococcus dysgalactiae spiroforme mutans Type 5 Group C sporogenes Parasites Adenovirus Type 14 Group B2 aerofaciens hominis Type 26 Group D piger parvum Type 31 Group A farda histolytica Type 37 Group D aerogenes lamblia Type 40 Group F cloacae Human Cell Line Human 4 faecalis Colon epithelial cells C Serovar Group Type 1 Group C Type 2 Group C Type 3 Group B1 Collinsella Desulfovibrio Edwardsiella Enterobacter Enterococcus faecium Fungal Strain Coxsackievirus B4 __ Candidaalbicans ytomegalovirus Sub species masoucida and sub species salmonicida 2 strains Echovirus 11 Enterovirus 68 Sapovirus 2 strains Page 35 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2
39. face screen and the door of the analysis compartment lt is not necessary to change gloves between instruments however do not use the same lint free decontaminating wipe to clean different instruments 2 If needed dry the Verigene SP drawer assembly with a lint free cloth prior to loading EP consumables Page 5 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e GO OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 METHODS A Specimen Collection amp Storage Inadequate or inappropriate specimen collection storage or transport may yield false negative results Due to the importance of specimen quality training of personnel in the correct manner to perform specimen collection and handling is highly recommended 1 Collect stool preserved in Cary Blair medium by using the medium manufacturers recommended collection procedure or collect unpreserved and unformed liquid or soft stool specimens and place as soon as possible into the Cary Blair medium by using the medium manufacturers recommended collection procedure 2 It is recommended that Cary Blair preserved specimens be stored refrigerated at 2 8 C until EP testing is completed for up to 48 hours after collection For repeat testing prepare the s
40. free distributor i www e labeling eu NANO023 REFERENCES 1 2 Bryce J Boschi Pinto C Shibuya K et al WHO estimates of the causes of death in children Lancet 2005 365 1147 1152 Herikstad H Yang S Van Gilder TJ et al A population based estimate of the burden of diarrhoeal illness in the United States FoodNet 1996 7 Epidemiol Infect 2002 129 9 17 Centers for Disease Control and Prevention CDC National Shiga toxin producing Escherichia coli STEC Surveillance Overview Atlanta Georgia US Department of Health and Human Services CDC 2012 Mead PS Slutsker L Dietz V et al Food related illness and death in the United States Emerg Infect Dis 1999 5 607 25 Slutsker L Ries AA Greene KD Wells JG Hutwagner L Griffin PM Escherichia coli O157 H7 diarrhea in the United States clinical and epidemiologic features Ann Intern Med 1997 126 505 13 Bad Bug Book Foodborne Pathogenic Microorganisms and Natural Toxins US Food and Drug Administration 2012 Web Accessed 30 June 2014 lt http www fda gov downloads Food FoodbornelllnessContaminants UCM297627 pdf gt Diagnosis and Treatment Salmonella Centers for Disease Control and Prevention 27 Sept 2010 Web 14 July 2011 lt http www cdc gov salmonella general diagnosis html gt Altekruse Sean F Norman J Stern Patricia Fields and David L Swerdlow Campylobacter jejuni An Emerging Foodborne Pathogen Emerging Infectious Diseases 5 1 1999
41. h Note If the Drawer Clamp is not latched properly the Processor SP will display an error message on the Status Display when the user attempts to close the Drawer Assembly Lower the Drawer Clamp 5 Ordering a Test a All tests must be ordered through the Verigene Reader No test can be processed on the Verigene Processor SP without the user entering the Test Cartridge ID and Sample ID into the Verigene Reader V Log into the Verigene Reader To start a new Session proceed to the next step iii To order a test in a previously created session select the desired Session from the drop down SESSION menu then proceed to step v Note Up to 60 Test Cartridges can be entered into a single session From the Menu Bar SESSION tab select Start New Session where the Session Setup window will appear Touch 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 laboratory The operator ID is automatically entered as the currently logged in user Touch the Processing tab on the Navigation Bar at the bottom of the screen b Enter the Test Cartridge ID by scanning the barcode using the barcode scanner attached to the Reader The user may manually enter in the Test Cartridge ID by selecting MENU and Enter Barcode and then keying in the Test Cartridge ID number with the Reader s keyboard C optional Scan the Test Cartridge C
42. he U S Phone 1 888 837 4436 toll free N a O S h e e OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 BACKGROUND INFORMATION AND CLINICAL UTILITY Acute diarrhea caused by bacterial and viral infection represents a significant worldwide healthcare burden The World Health Organization estimates that diarrhea causes or is a major contributor to approximately one quarter of all post neonatal childhood deaths The Centers for Disease Control and Prevention CDC estimate that 1 4 episodes of diarrhea occur per person per year in the USA Though most cases of diarrhea caused by enteric bacteria and viruses are self resolving and not life threatening some can have serious implications Because clinical treatment decisions are often made based on the identity of the infecting pathogen and in some cases the presence of an accompanying virulence gene it is important to identify these targets quickly Using multiplex molecular methods it is possible to determine this information from a single nucleic acid test targeting the following analytes Shiga toxins 1 and 2 Shiga toxin producing Escherichia coli STEC also referred to as enterohemorrhagic E coli EHEC and verocytotoxic E coli VTEC cause approximately 265 000 illnesses annually in the U S with more than 3 600 associated hospitalizations and 30 deaths A particularly virulent strain of S
43. idization options are selected see image below x Assign Sample Page 12 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Nanosphere Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 b In the subsequent dialogue box select or de select the bacterial viral or toxin gene targets from the list to activate or de activate results reporting for those targets Press Yes to confirm The Verigene Reader will automatically default to the selected targets for the next test run Note Once a test run is started results for de selected targets cannot be retrieved c fusing optional Cap Protocol sample has already been loaded Skip to step 7d Pipette 200 uL of the prepared specimen in the Stool Prep Buffer into the Sample Loading Well of the Extraction Tray Sample Loading Well d Close the Drawer Assembly by pressing the OPEN CLOSE button on the Processor SP The Processor SP will automatically verify that each consumable is properly loaded and being sample processing e Confirm countdown has started on the Processor SP display screen before leaving the area f In order to set up additional tests on other Processor SP instruments follow the same procedure To avoid contamination and sample mix ups
44. ils on performing tests on the Verigene oystem as well as routine and daily maintenance 1 Test set up after Specimen Processing a Remove an Extraction Tray Tip Holder Assembly and Test Cartridge from the refrigerator Hemove the Amplification Tray from the freezer and thaw at room temperature for a minimum of 10 minutes and begin the test run within 30 minutes after removal from the freezer Do not refreeze the Amplification Tray once it has been thawed Page 6 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S A e OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO23 b The image below shows an empty Verigene Processor SP Open the Drawer Assembly by pressing the black OPEN CLOSE button located on the front of the Verigene Processor SP Open the Drawer Clamp by pressing in the silver latch and lifting the Clamp prior to loading the consumables f Press to open the Drawer Assembly Press to lift Drawer Clamp 2 Loading the Extraction Tray a Prior to loading the Extraction Tray thoroughly shake the Extraction Tray to resuspend the magnetic beads which will have settled during storage Check for complete resuspension by visually inspecting the well containing the beads The
45. inding buffer wash solutions and buffer solutions necessary to extract nucleic acids and generate a test result The Extraction Trays are contained within a carrier labeled as EP 20 009 023 e Verigene EP Stool Preparation Sample Kit Each Kit contains 20 tubes containing Verigene EP Stool Prep Buffer SPB and 20 swabs packaged in a resealable bag The Kit is labeled as EP 30 001 023 e 20 Verigene Sample Well Caps The Caps come packaged in strips of 5 Caps and are contained within a plastic bag The bag is labeled as 40 001 001 Verigene EP Test Amplification Kit Catalog number 20 012 023 e 20 Verigene EP Amplification Trays Each Amplification Tray comes preloaded with all required reagents including enzymes and buffers necessary to amplify nucleic acids and generate a test result as well as an amplification tube The Amplification Trays are contained within a carrier labeled as EP 20 011 023 MATERIALS NEEDED BUT NOT PROVIDED Instruments and Equipment Verigene Reader Catalog number 10 0000 02 Verigene Processor SP Catalog number 10 0000 07 2 8 C Refrigerator lt 20 C Freezer lt 70 C Freezer Optional Micro pipettors amp filtered tips Vortex Table top quick spin Mini Centrifuge Decontamination wipes spray or comparable sanitizer Biological Safety Cabinet BSC Verigene Extraction Tray Holder Catalog number 421 00019 01 Optional Page 4 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 0
46. io parahemolyticus 89 0 98 8 96 0 100 96 0 100 Page 31 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 C Analytical Sensitivity Limit of Detection Analytical sensitivity LoD of the EP was determined for 20 strains of Enteric Pathogens representing all nine EP reportable target analytes The LoD was defined as the concentration at which the test produces a positive result greater than 95 of the time Serial dilutions of the strains were tested and the putative LoD confirmed with 20 replicates To ensure the accuracy of the LoD determination if the initial detection rate was 100 a further 20 replicates were performed at the next lower concentration until lt 95 was achieved The LoDs for the 18 strains tested and the corresponding LoD ranges for the EP reportable target are shown in Table 15 below Table 15 EP Limit of Detection LoD units Campylobacter spp Salmonella spp Shigella spp Vibrio Spp Y enterocolitica and E coli in CFU mL LoD units for Norovirus is copies mL LoD units for Rotavirus IS TCIDso mL Representative Organism Tested Strain Number Organism LoD ng Target LoD Campylobacter jejuni subsp jejuni ATCC 43429 3
47. ion and Amplification Trays and the Tip Holder Assembly Page 14 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free lt a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 10 Printing Results a Touch the Substrate icon in the Session s Processing screen A window displaying the results will open Touch the Print option on this screen to print a Detail Report b A Summary Report is available by moving to the Results screen of the Session on the bottom Navigation Bar go to MENU then select Print Summary The Summary Report will provide the results for all tests processed within the current Session C Detail Reports can also be viewed and printed from the Results window First select the desired test from the list go to MENU and then touch Print Detail INTERPRETATION OF RESULTS EP provides a qualitative result for the presence Detected or absence Not Detected of the EP target genes The image analysis of the Test Substrate provides light signal intensities from the target specific capture spots as well as the negative control background and imaging control spots The mean signal intensity of a target is compared to the assay s signal detection threshold to make a call Table 2 lists the possible te
48. ions in both general safety practices for laboratories and specific safety practices for the instrument e Read and understood all applicable Material Safety Data Sheets MSDS B 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 1 888 837 4436 C Maintenance of the Verigene Reader and Verigene Processor SP For routine and daily maintenance instructions please refer to the Verigene System User s Manual WARNGINGS AND PRECAUTIONS REAGENTS AND TEST CARTRIDGES A Material Safety Data Sheets e Material Safety Data Sheets MSDS for the Test Cartridge Amplification Tray and Extraction Tray are available at www e labeling eu www nanosphere us or upon request from Nanosphere Inc B 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 Test Cartridges e See Material Safety Data Sheets MSDS for toxicity information C Waste Disposal e The Amplification Tray contains amplification reagents a
49. itica Page 25 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service A In the U S Phone 1 888 837 4436 toll free e a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 9 Summary of Viral Target Clinical Test Performance n21940 Compared to Reference Methods real time RT PCR and two conventional PCRs with bi directional sequencing RE Agreement 95 CI e Agreement 95 CI dali GAMA 94 9 99 6 l 99 9 Fresh 1294 37 392 1250 12554 Fresh 1294 1290 12919 82 7 99 4 99 0 99 9 4 99 99 6 100 100 100 Frozen 34 b 33 33 Frozen 34 34 34 0 97 89 7 100 89 7 100 100 99 5 98 0 100 Selected 203 18 18 184 185 Selected 50 51 f 152 152 81 5 100 97 0 100 89 6 100 97 6 100 100 100 Simulated 409 409 409 Simulated 409 409 Prospectively Collected Prospectively Collected cO c D E D Q o Ev 2 E O Clinical Specimens Norovirus Rotavirus 99 1 100 99 1 100 Composite Comparator No SID Specimen Type EP test result Method Result m I D WW wa NiDwede i EMEN 205611381 Fresh Not Detected 1 _____Norovirus GI b o dd 065602 Frozen x Not Detected x Norovirus Gl C 1 973241 Selected Rotavirus Norovirus Rotavirus D 77 TN w MEM Tr RN a ed 2541010491 s EIER Norovirus b Negati
50. labeling eu NAN023 Analytical Specificity Exclusivit One hundred and fifty eight 158 organisms consisting of 134 bacterial organisms 18 viruses four 4 parasites one 1 fungal organism and one 1 human cell line were tested with EP to determine analytical specificity see Table 17 Eight 8 organisms including Astrovirus and Sapovirus 2 strains Campylobacter hominis and all four parasites were tested as genomic DNA RNA To rule out cross reactivity between the analytes detected by EP nine 9 organisms representing all of the EP detected targets were tested at elevated concentrations of 5 x 10 CFU mL for bacteria and at least 100x LoD for viruses The exclusivity of the following was evaluated by in silico analysis only 15 species of Vibrio not associated with human infection four 4 non pathogenic strains of Escherichia coli Yersinia pestis Clostridium botulinum Norovirus genotypes genogroups GIV 2 GII and GV and Rotavirus genogroups B C D and NADRV with the exception of porcine strains within genogroup C All of the organisms tested yielded the expected Not Detected results indicating that there was no cross reactivity with EP with the exception of Campylobacter insulaenigrae that yielded a single positive result 1 9 for Campylobacter In silico analysis also indicates a potential for low level cross reactivity While Campylobacter insulaenigrae has been isolated primarily from marine mammals in rare ca
51. n organizations as applicable and should follow the user s laboratory s standard quality control procedures TROUBLESHOOTING Refer to the Troubleshooting section of the Verigene System User s Manual Page 17 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e GO OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 LIMITATIONS e Atrained 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 Inrare instances Campylobacter insulaenigrae may yield a false positive Campylobacter detected result e The following 15 species of Vibrio each of which are NOT associated with infections in humans and therefore unlikely to be encountered in human stool were shown NOT to be detected by EP based upon in silico analysis only V anguillarum V brasiliensis V coralliilyticus V crassostreae V cyclitrophicus V ichthyoenteri V kanaloae V nigripulchritudo V ordalii V orientalis V rotiferianus V rumoiensis V scophthalmi V splendidus and V tasmaniensis e The following 27 species of Vibrio each of which are NOT associated with infections in humans and therefore unlikely to be e
52. ncountered in human stool were not evaluated for exclusivity by empirical testing or in silico analysis due to a lack of genome sequence information V aerogenes V aestuarianus V chagasii V diabolicus V diazotrophicus V ezurae V fortis V gallicus V gazogenes V gigantis V halioticoli V hepatarius V hispanicus V litoralis V mediterranei V mytili V natriegens V navarrensis V neonatus V nereis V pacini V pectenicida V pomeroyi V proteolyticus V ruber V superstes and V xuii e EP is expected to be inclusive to most strains of the Norovirus Gl GII and GIV genotypes known to cause disease in humans based on empirical testing and supplemented by in silico analysis However due to the high genetic diversity within Noroviruses some strains may not be detected or may be detected with reduced sensitivity by EP Refer to the Analytical Sensitivity Inclusivity section and Table 16 for details e EP inclusivity to Norovirus strains GII 9 GIl 14 and GIV 1 was evaluated by in silico analysis only Rare Norovirus genotypes Gll 6 and Gll 13 were determined to either be detected at reduced sensitivity or predicted to not be detected by EP based on in silico analysis For GIl 8 EP inclusivity is unknown as in the absence of sequence information in silico analysis could not be performed e Norovirus Gll 11 is not expected to be detected by EP based on in silico analysis e Norovirus Gill GIV 2 and GV are not expec
53. nd the internal controls Dispose the Amplification Tray in accordance with national state and local regulations e The Extraction Tray contains residual nucleic acids extraction reagents and residual sample It also contains a residual volume of the sample buffer which contains formamide a teratogen Dispose the Extraction Tray and Stool Prep Buffer tube in accordance with national state and local regulations Page 19 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 e All of the waste reagents including the purified nucleic acids are contained within the Test Cartridge There is a very small amount of residual formamide lt 1 v v Dispose the Test Cartridge in accordance with national state and local regulations EXPECTED VALUES Prevalence In the EP Methods Comparison study 1328 prospectively collected fresh and frozen specimens were obtained from seven medium to large sized healthcare institutions geographically distributed across the United States The number and percentage of positive cases positivity rate determined by EP stratified by geographic region for each of the organisms detected by the test are presented in Table 5 Overall EP dete
54. nella enterica cei WM Q 3 Fresh 0 Not Detected Salmonella spp e Positive for Salmonella enterica sl go 1 0m NotDetected Salmonela spp e ne 1 1 Fresh Salmonella Negative s Negative for Salmonella spp 2 fresh Salmonella NegalVe Positive for Salmonella enterica een 3 i Fresh l Salmonella E COl s Positive for Salmonella enterica cei h 4 Fresh Salmonella C freundii amp Proteus SD Negative for Salmonella pp sl 9 o Fresh Salmonella Negative Positive for Salmonella enterica en A 6 Fresh Salmonella P alcalifaciens s Negative for Salmonella pp iL fresh Salmonella Negative Positive for Salmonella enterica eni de 1 1 l Frozen Salmonella Profeus spp Positive for Salmonella enterica een j i 1 Select _ Salmonella Campylobacter O a Positive for Campylobacter jejuni a k i Fresh Not Detected 9higela DD Positive for Shigela EIEC j i Fresh Shigella Negative s Positive for ShigellEIEC O O 2 Fresh 1 Shigella Negative s positive for ShigellaEIEC a 3 Fresh Shigella NegatiVe s Positive for ShigellaEIEC iii 9 l Fresh Shigella NEGATIVE Ojy Oaaaaaa aaa aaa Positive for Shigella EIEC een 5 i Fresh il Shigella i Negative s Positive for ShigellaEIEC a I i Fresh Shigella NegatiVe e Positive for ShigellaEIEC 9 Fresh 9higela A hydrophila cavieae amp P putida k Positive for Shigella EIEC e 9 i Fresh
55. observed with EP were examined for the testing of the sixteen 16 bacterial samples and three 3 viral samples used to establish the Limit of Detection of the assay In addition the cut off data set included the test results of two 2 negative samples With replicates of 20 for each sample and ten target spot groups evaluated per test a total of 6160 data points 1320 expected positive were assessed to verify the assay cut off CONTACT INFORMATION In the United States Nanosphere Inc 4088 Commercial Avenue Northbrook IL 60062 Customer and Technical Service 1 888 VERIGENE 837 4436 Outside of the United States Please contact your local Nanosphere distributor TEST KIT LABELING The contents of a Test Kit may use EN 980 graphical symbols The symbols are defined below Catalog number E CE eme 00000 m fim Upper Limit Temperature limitation do Upper and Lower Limit Temperature limitation Meo Consult instructions for use i KEY CODE Key code Use this key code to obtain instructions for use at www e labeling eu Xn x Harmful Flammable Page 38 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e e OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 PATENTS AND TRADEM
56. overs 2D barcode using a barcode gun style scanner to display the Test Cartridge s Reference Number Expiration Date and Lot Number on reports Note The wand style barcode scanner will not read 2D barcodes Page 10 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S e e OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO23 6 Load the Test Cartridge a Hold the Test Cartridge by the handle with one hand using the other hand apply pressure with the palm of the hand and remove the Test Cartridge cover by bending the cover away and over the Reagent Pack edge see illustration below Alternatively an opener may be used to remove the Test Cartridge cover Ensure that the valve plate is not moved during cover removal Do not remove the Test Cartridge cover until immediately prior to inserting the Test Cartridge into the Processor SP Pull here to remove cartridge cover Palm of hand on cover and fingers pulling on cartridge cover Do not move the valve plate when removing the cartridge cover Pull opener up to remove cartridge If using opener insert to edge of cartridge cover b Settle the reagents in the Test Cartridge before loading into the Processo
57. parahaemolyticus is responsible for approximately 4 500 cases of illness in the United States annually Vibrio parahaemolyticus typically causes watery diarrhea along with nausea vomiting and abdominal cramps Treatment is not usually required in a majority of cases of infection with V parahaemolyticus V cholerae causes cholera an infection in the small intestines resulting in profuse watery diarrhea and vomiting V cholerae is relatively uncommon in developed countries causing less than 100 cases annually in the U S An Page 2 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 estimated 3 5 million cases of cholera occur each year worldwide resulting in over 100 000 deaths Antibiotics can be used to shorten the duration of symptoms associated with cholera Shigella spp Infections with Shigella are known as shigellosis with 14 000 cases reported in the United States each year Patients infected with Shigella a genus of gram negative bacteria often develop fever stomach cramps and bloody diarrhea There are four serogroups of Shigella S dysenteriae S flexneri S boydii and S sonnel S sonnei and S flexneri are the most common causes of
58. pecimen in a new Stool Prep Buffer as described in the Specimen Processing section see Section B B Fresh Cary Blair Preserved Specimen Processing Put on fresh gloves 2 For each Cary Blair preserved specimen to be tested place one sterile flocked swab and one uncapped Stool Prep Buffer tube place the cap to the side for recapping later into a biological safety cabinet BSC 3 Wipe down the outside of the specimen vial with a lint free decontaminating wipe 4 Invert the vial containing the Cary Blair preserved specimen twice and vortex the specimen for 5 10 seconds to ensure homogeneity 5 To prepare the Stool Prep Buffer tube dip the provided flocked swab into the Cary Blair preserved specimen vial until the flocked tip is fully immersed in specimen Once evenly coated transfer the swab to the Stool Prep Buffer tube and break swab at the pre formed scored breakpoint Leave the swab in the otool Prep Buffer tube and screw the cap finger tight on to Stool Prep Buffer tube 6 Recap the original Cary Blair preserved specimen container and set aside 7 Repeat steps 1 6 for each specimen changing gloves between each specimen 8 Vortex each Stool Prep Buffer tube for 15 20 seconds 9 Spin all prepared Stool Prep Buffer tubes in the Mini Centrifuge for 30 35 seconds 10 Put on fresh gloves before continuing the procedure C Verigene EP Test Procedure Please refer to the Verigene System User s Manual for additional deta
59. pplications Except as expressly provided in this paragraph no other license is granted expressly impliedly or by estoppel 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 MERCHANTABILITY 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 39 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Nanos here OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll
60. r SP The optimal method for setting the reagents is to hold the Test Cartridge s reagent container on the side opposite the handle and tap the barcode end of the Test Cartridge with your index finger When tapping the Test Cartridge allow the force of the tapping to move the Test Cartridge and your right hand The tapping is more effective when the Test Cartridge is held in the air so that it moves slightly Page 11 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e eC OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO23 c Insert the Test Cartridge into the Hybridization Module of the Processor SP until it reaches a stopping point The image below shows the user loading a Test Cartridge into the Verigene Processor SP Note If the Test Cartridge is not inserted properly the Processor SP will display a message on the information screen when the user attempts to close the Drawer Assembly If this occurs remove the Test Cartridge from the Hybridization Module and re insert the Test Cartridge 7 Loading the Sample a Enter the Sample ID by scanning or manually enter the Sample ID using the Reader s touch screen keyboard Press Yes to confirm the Sample ID Ensure the Extraction Amplification and Hybr
61. s infection and the number of hospitalizations in the United States since 2006 when an estimated 60 000 children were hospitalized each year Antiviral drugs are ineffective against rotavirus and the best treatment is management of dehydration PRINCIPLES AND PROCEDURES OF VERIGENE EP AND THE VERIGENE SYSTEM EP is performed using the Verigene System which is a bench top sample to result molecular diagnostics workstation consisting of two modules the Verigene Processor SP and the Verigene Reader The Verigene Processor SP automates the EP sample analysis steps including i Specimen Preparation Cell lysis and magnetic microparticle based nucleic acid extraction from prepared stool specimens obtained from patients ii Target Amplification Multiplex PCR and RT PCR based amplification of the extracted nucleic acids to generate target specific amplicons iii Hybridization amplicon hybridization to target specific capture DNA in a microarray format and mediator and gold nanoparticle probe hybridization to captured amplicons Silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold silver aggregates that are imaged optically with high efficiency by the Verigene Reader The Verigene Reader also serves as the user interface and central control unit for the Verigene System storing and tracking information throughout the assay process The Verigene Processor SP utilizes single use consumables to perform EP in
62. ses it may cause septicemia and gastroenteritis in humans Page 34 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 17 Organisms Tested for Exclusivity Campylobacter Vibrio and Yersinia Bacteria Species Not Detected by EP Species Gems Species Genus Species Abiotrophia defectiva coli 3 strains concisus nai baumannii coli EAEC CUIVUS Iwoffli T coli EPEC 2 strains fetus Escherichia 7 butzleri coli ETEC 2 strains gracilis Arcobacter cryaerophilus fergusonii hominis allosaccharophila hermannii hyointestinalis bestiarum varium Campylobacter insulaenigrae caviae hepaticus lanienae Helicobacter U encheleia pylori 4 strains mucosalis Jenae enteropelogenes Klebsiella oxytoca rectus eucrenophila pneumoniae showae hydrophilia acidophilus sputorum jandaei Lactobacillus reuteri upsaliensis salmonicida rhamnosus alginolyticus veronii lactis campbellii Alcaligenes faecalis grimontii cincinnatiensis Bacillus cereus us grayi fluvialis Listeria m caccae monocytogenes Vibrio furnissii fragilis Morganella morganii harveyi Bacteroides TERET merdae Peptostreptococcus anaerobius metschnikovii stercoris shigelloid
63. shigellosis in the United States Although patients with mild Shigella infections usually recover without antibiotic treatment antibiotics may be used to treat severe cases of shigellosis Antidiarrheal agents can worsen illness and should therefore be avoided Norovirus _Noroviruses are highly contagious and cause on average 19 21 million cases of acute gastroenteritis each year 3 Norovirus illnesses cost two billion dollars annually in the United States ranking norovirus in the top five pathogens for enteric illnesses Infection with Norovirus a single stranded non enveloped RNA virus causes nausea vomiting diarrhea and abdominal pain Norovirus infections constitute a major disease burden which leads to high rates of hospitalization and mortality in children and the elderly Five distinct norovirus genogroups have been described Gl GV but human pathogens have been described only from genogroup I genogroup ll Il and genogroup IV however genogroup IV norovirus is a rare cause of disease in the United States Rotavirus Globally Rotavirus is the leading cause of severe diarrhea in infants and young children Rotavirus a double stranded non enveloped RNA virus is a cause of viral gastroenteritis and is most commonly seen in infants and young children Symptoms include fever vomiting and watery diarrhea and may last upwards of 8 days after initial infection Vaccination efforts have greatly reduced the incidence of rotaviru
64. side the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 13 EP Precision Performance Study Summary Call Rate Total Agreement with Sample Expected Call Concentration 95 eri CI Expected Result i 95 CI Initial 5 100 100 100 Moderate 16 16 16 16 16 16 0 0 91000 1009 Escherichia coli Stx2 Shiga Toxin 2 Detected US s es E a A 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 93 8 100 100 0 0 9 4010 95 1009 Salmonella enterica Salmonella Detected DS Ah 2 ee sa o oe n o 15 16 16 16 15 16 69 8 99 8 79 4 100 69 8 99 8 16 16 100 100 Shigella Detected 79 4 100 79 4 100 79 4 100 Shigella dysenteriae Sk1 Shiga Toxin 1 Detected 100 100 100 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 100 100 100 16 16 16 16 16 16 BO Yersina enterocolitica 79 4 100 79 4 100 79 4 100 Yersinia enterocolitica Detected 100 100 100 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 100 100 100 16 16 16 16 16 16 0 0 91000 1009 Campylobacter jejuni Campylobacter Detected vs a a i I E ee x 16 16 16 16 16 16 79 4 100 79 4 100 79 4 100 100 100 100 16 16 16 16 16 16 0 0 91000 1009 Vibrio parahemolyticus Vibrio spp Detected 2 DS A vM E ee DE 14 16 16 16 16 16 61 7 98 5 79 4 100 79 4 100 N 93 8 100 100 Moderate 15 16 16 16 16 16 69 8 99 8 79 4 100 79 4
65. st results generated by EP representing identification of bacterial viral and or genetic virulence marker nucleic acid sequences targets Their presence is verified before a valid result is provided as described below Table 2 Calls for Valid Tests Organism Gene Taraet Genes Test Result Reported as Detected Genus Group Species Toxin Gene _ Salmonella species mob Samna Shigella species pah Shgela Vibrio Group rfbL tht amaA Vibro Yersinia Yersinia enterocolitica recN Shiga Toxin 1 Shiga Toxin 1 Shiga Toxin 1 1 Shiga Toxin 2 REM NEN RN Shiga Toxin 2 Levi region Ta Fi TEA NotDeieeeg E i QC coli C jejuni and C lari S dysenteriae S boydii S sonnei and S flexneri V cholerae and V parahaemolyticus Page 15 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free N a O S h e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Error calls related to an invalid test are listed in the Table 3 below together with the appropriate recourse that should be taken by the user Table 3 Invalid Calls and Recourse Seer rei MM Eon E Rara p Repeat EP indicating target hybridization issue Internal Control 2 not detect
66. t 5x10 CFU mL and Norovirus GI and GII and Rotavirus Group A at 100x LoD followed by testing a negative stool sample The high titer sample was alternated with the negative sample three times on nine unique Verigene SP Processors No carryover or cross contamination was observed Page 36 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service A In the U S Phone 1 888 837 4436 toll free e a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO023 l Competitive Inhibition In order to assess competitive inhibition for EP binary combinations of all test panel organisms representing all possible dual infections were evaluated Contrived samples were prepared in Negative Stool Matrix NSM with one panel organism present at a Low Positive titer 3x LoD and a second organism present at a High Positive titer gt 10 CFU mL stool The performance of EP was evaluated with each of the 56 unique sample combinations tested in replicates of three 3 The results of the Competitive Inhibition testing are summarized in Table 19 No evidence of competitive inhibition was observed at the titers tested Table 19 Results from the Competitive Inhibition Study Organisms at High Titer 100x LoD Total Detection Rate Organism at low titer D i x U Campylobacter coli ae 20 21
67. t be evaluated due to a lack of available sequences Table 16 Organisms Tested for Inclusivity Reportable Total Number of Species Genogroups Tested Target Type Target Organisms Strains Name Total LoD Tested Number of Strains Number Concentration IX C coli 5 C jejuni subsp jejuni 4 C jejuni subsp dii edad Doylei 1 C lari 5 S bongori 1 S enterica subsp various 5 Salmonella S enterica subsp enterica serovar various 25 Bacterial Shigella S boydii 5 S dysenteriae 5 4 S flexneri 5 S sonnei 5 V cholerae 5 V parahaemolyticus 5 Yersinia Y enterocolitica 7 enterocolitica Shiga toxin 2 E coli 16 GI 13 including Gl 1 GI 2 2 strains Gl 3 2 strains GI 4 2 strains GI 5 GI 6 and GII 3 1 GIl 4 4 GII 5 Norovirus E Viral GII 1 GII 2 GII 3 2 GII 3 3 GlI 4 2 GII 4 3 Gll 4 5 GII 10 GII 12 2 Group A 12 including G1 4 strains G2 3 strains G3 66 G8 G9 G12 Two 2 strains contain Shiga Toxin 1 Five e strains contain both Shiga Toxin 1 and Shiga Toxin 2 a Shiga toxin 1 S dysenteriae 2 a E coli 17 b Toxin er m a an Page 33 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 E Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free Nanos here OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e
68. ted to be detected based on in silico analysis e EP inclusivity to Rotavirus A genotypes G4 G5 G10 G11 and G15 was evaluated based on in silico analysis only Inclusivity of EP to Rotavirus A genotypes G7 G21 and G24 is unknown representative strains were not available for empirical testing and in the absence of sequence information in silico analysis could not be performed e Rotavirus genogroups B D and NADRV are not expected to be detected by EP based on in silico analysis In addition Rotavirus genogroup C strains are not expected to be detected by EP with the exception of porcine strains within this genogroup WARNING AND PRECAUTIONS GENERAL e EP is for in vitro diagnostic use e Caution Federal law restricts this device to sale by or on the order of a physician or to a clinical laboratory e Never use any Tips Trays Tubes or Test Cartridges that have been broken cracked punctured previously used or visibly damaged using damaged material may lead to No Call or false results 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 specimens carefully Open one tube or specimen at a time to prevent specimen contamination e Biological specimens such as stool tissues body fluids and blood of humans are potentially infectious When handling and or transporting human specimens follow
69. therapy to resolve symptoms Antimicrobial therapy can prolong the duration of non typhoidal Salmonella and is only recommended for patients with the severe symptoms Campylobacter spp Campylobacter infection is the most common cause of bacterial gastroenteritis Antibiotics are generally not prescribed unless symptoms are severe Delaying treatment for several days while waiting for lab tests to confirm the presence of C jejuni can reduce the effectiveness of the therapy The CDC reports that 14 cases of Campylobacter infection are diagnosed per 100 000 people in the United States annually with many more cases going undiagnosed Campylobacter infections occur more frequently in summer months and the organism is more frequently recovered in young children over any other age group Infections with Campylobacter are more often than not self resolving and do not require antimicrobials for treatment Yersinia enterocolitica The disease yersiniosis is caused by Y enterocolitica which is an infection that can resemble Crohn s disease or appendicitis with symptoms including diarrhea and fever There is an estimated one culture confirmed case of Y enterocolitica infections per 100 000 persons in the United States each year Yersiniosis is self limiting and does not generally require antibiotics The CDC monitors the frequency of Y enterocolitica infections through the foodborne disease active surveillance network FoodNet Vibrio Spp Vibrio
70. tion control and is referred to as the Internal Processing Control 2 INT CTL 2 This control is automatically added by the Verigene SP to each specimen prior to the extraction step If the process control is not valid a No Call INT CTL 2 result will be obtained and the test should be repeated If both INT CTL 1 and INT CTL 2 are not detected a No Call INT CTL result is generated These internal controls are not utilized for the detection of positive samples Additional positive controls are immobilized on the Test Slide These are used to determine that hybridization was performed correctly The EP algorithm requires that these controls be valid before decisions regarding the absence of any other target on the panel can be determined If these controls are not detected a No Call result will be obtained and the test should be repeated Table 4 Internal Processing Controls Internal Process Control Artificial DNA construct with detection Controls for target hybridization INT CTL 1 oligonucleotides related issues Intact MS2 Phage along with primers and detection oligonucleotides Added to each test specimen Internal Process Control INT CTL 2 Controls for lysis extraction and target amplification 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 accreditatio
71. tober 2014 Nanosphere Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 6 Summary of Bacterial Target Clinical Test Performance n 1940 Compared to Reference Methods Culture and Conventional Biochemical and Automated Phenotypic Identification Agreement 95 CI Specimen Type 90 9 98 7 Fresh 1294 20 22 1255 127249 79 8 98 9 97 9 99 2 Fon oe Selected m Simulated Fen Selected 5B Simulated n Fn Selected 5 Simulated Prospectively Collected 15 8 100 891 100 Campylobacter spp Clinical Specimens Salmonella spp 86 8 99 9 98 5 67 68 92 1 100 66 7 2 3k 9 4 99 2 96 6 100 100 341 341 98 9 100 98 8 1275 1291 98 0 99 3 97 1 33 34m 84 7 99 9 Prospectively Collected o lt D E D SI o Ev 2 O Shigella spp Vibrio Spp 63 1 100 97 2 100 92 9 100 99 0 100 100 1294 1294 99 7 100 100 34 34 89 7 100 100 202 202 98 2 100 100 350 350 99 0 100 gt o gt O O D oo oo O Clinical Specimens Y enterocolitica 100 09 59 93 9 100 Verigene Enteric Pathogens Nucleic Acid Test EP Specimen Type NEHME 7 Agreement 95 CI
72. ve sse dL 3 DBIA E Fresh i s Norovirus e Negative NE E NE 066181 i Fresh ie NOFOVIFUS i a Negative Norovirus RM oos Shiga Toxins oo i e u 067641 u Fresh Not Detected Rotavirus u f 1 u 970381 Selected Not Detected Rotavirus u g 1 067631 Fresh Rotavirus Negative Reference method positive for Norovirus and Rotavirus RT PCR endpoint PCR with subsequent bi directional sequencing must be positive Page 26 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service A In the U S Phone 1 888 837 4436 toll free e a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN023 Table 10 Summary of Genus Group Level Test Performance Versus Reference Method s Stratified by Species Campylobacter Genus Shigella Genus Eo Prospective Organism Fresh Eo Selected Simulated Analytical Fec ozb Selected Simulated Analytical 91 7 97 5 98 5 100 66 7 100 100 100 m 22 24 39 40 67 68 15 15 pa 2 3 8 8 50 50 20 20 Py 72 0 99 0 86 8 99 9 86 3 100 78 2 100 g 9 4 99 2 54 1 100 92 9 100 83 2 100 m 100 100 100 100 100 100 100 p x oli 3 3 3 3 18 18 5 5 Shigella boydii 2 2 14 14 5 5 29 2 100 29 2 100 81 5 100 47 8 100 15 8 100 76 8 100 47 8 100 100 97 3 100 100 100 100 se nd 1 36 37 9 9 4 4 r
73. well containing the magnetic beads is easily distinguished as the beads are black in color Following adequate resuspension gently tap the tray on the counter to ensure that the reagents settle to the bottom of each well Optional Cap Protocol If not using the Optional Cap Protocol proceed to step 2b Remove one cap from the strip of caps and place inside the BSC li After shaking and tapping the Extraction Tray place the Extraction Tray in the Extraction Tray Holder inside the BSC Refer to image below for visual of the Extraction Tray Holder lli Pipette 200uL of the prepared sample into the bottom of the Sample Loading Well in the Extraction Tray Refer to the image below for Sample Loading Well location Extraction Tray Holder Extraction Tray Sample Loading Well Page 7 of 40 Verigene Enteric Pathogens Nucleic Acid Test EP 027 00037 01 Rev B October 2014 Customer Service or Technical Service In the U S Phone 1 888 837 4436 toll free a O S e re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NANO23 iv After sample loading place the Sample Well Cap over the Sample Loading Well Take precaution to handle only the edges of the Cap and firmly press down until the Cap is fully inserted into the Sample Loading Well Sample Well Cap in Packaging Pressing down on edges of cap Extraction Tray with cap inserted V

Verigene EP Package Insert

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