Verigene CDF Package Insert
Contents
1. 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 Do not move the valve plate when removing the cartridge cover b The user must settle the reagents in the cartridge before loading into the Processor SP The optimal method for settling the reagents is to hold the Test Cartridge s reagent container on the side opposite the handle and tap the barcode end of the Cartridge with your index finger When tapping the cartridge allow the force of the tapping to move the cartridge and your right hand The tapping is more effective when the cartridge is held in the air so that it moves slightly Palm of hand on cover and fingers pulling on cartridge cover handle 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 Test Cartridge Page 9 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 w Customer Service or Technical Service y N a Nn O S p h re In the U S Phone 1 888 837 4436 toll free B OR E Mail productsupport nanosphere us2. Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 k Customer Service or Technical Service N a N O 5 p h A re In the U S Phone 1 888 837 4436 toll free 3 OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN022 instrument covers or panels are removed from the instrument If service is required contact Nanosphere Technical Support at 1 888 837 4436 or outside the U S contact your local Nanosphere distributor 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 WARNINGS AND PRECAUTIONS REAGENTS AND TEST CARTRIDGES A Toxicity of Reagents e Exposure to chemicals sealed inside the Test Cartridge is hazardous in case of skin contact and of ingestion Protective disposable gloves laboratory coats and eye protection should be worn when handling specimens Extraction Trays Amplification Trays and Verigene Test Cartridges e See Material Safety Data Sheets MSDS for toxicity information and are available upon request from Nanosphere Inc B Waste Disposal e The Amplification Tray contains amplification reagents and a microorganism Bacillus subtilis Dispose of the Amplification Tray in accordance with national state and local regulations e The Extraction Tray contains residual nucleic acids extraction r
3. CDF procedure immediately after preparation of the Stool Prep Buffer SPB specimen store the inoculated SPB specimen at 2 8 C and test within 24 hours including any necessary repeat testing If storing the SPB specimen prior to testing the specimen should be vortexed and microfuged prior to pipetting Should the time from preparation of the SPB specimen exceed 24 hours testing should be performed using a new preparation of SPB specimen with the original stool m i a B CDF Test Procedure Please refer to the Verigene System User s Manual for additional details on performing tests on the Verigene System as well as routine and daily maintenance 1 Test set up a Remove an Extraction Tray Tip Holder Assembly and Test Cartridge from the refrigerator Remove the Amplification Tray from the freezer and thaw at room temperature for 10 minutes Begin test run within 30 minutes or store thawed Amplification Tray at 2 8 C until ready to initiate testing Avoid subjecting Amplification Tray to multiple freeze thaw conditions 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 i i Press to open the gs Drawer Assembly Press to lift Drawer Clamp Page 5 of 29 Verigene Clostridium difficile Nucleic Acid
4. 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 CDF including an Extraction Tray Amplification Tray and Verigene Test Cartridge A separate Tip Holder Assembly contains two pipette tips that are used to transfer and mix reagents during the assay The user tests a specimen by loading the single use disposables 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 Test Cartridge into the Verigene Reader for optical analysis and generation of CDF test results Page 2 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service e N a nN O S p h re 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 NAN022 M MATERIALS PROVIDED Verigene CDF Nucleic Acid Test Kit Catalog number 20 005 022 e 20 Verigene CDF Test Cartridges Each Test Cartridge comes preloaded with all required reaction solutions including wash solutions oligonucleotide probe solution and signal amplification solution
5. h re 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 NAN022 Performance vs Direct Culture and Bi Directional Sequencing Relative to direct culture with Bi Directional Sequencing CDF demonstrated a sensitivity and specificity for toxigenic C difficile of 98 7 and 87 5 respectively CDF also demonstrated a 97 7 positive agreement and 97 8 negative agreement for 027 by sequencing The results are summarized in Table 7 Table 7 Verigene CDF test Performance vs Direct Culture amp Sequencing Direct Culture amp Sequencing Tox C difficile Tox C difficile fp Pee Tore atte es tot Tox C difficile Tox C difficile Verigene CDF Test Toxigenic C difficile Toxigenic C difficile PCR ribotype 027 Sensitivity 98 7 156 158 Pos Agreement 97 7 42 43 95 5 99 9 87 7 99 9 Specificity 87 5 1500 1715 Neg Agreement 97 8 1790 1830 85 8 89 0 97 0 98 4 Accuracy 88 4 1656 1873 Total Agreement 97 8 1832 1873 86 9 89 8 97 0 98 4 PPV 42 1 156 371 PPV 51 2 42 82 37 0 47 3 39 9 62 4 NPV 99 9 1500 1502 NPV 99 9 1790 1791 99 5 100 99 7 100 Of the 1 875 specimens evaluated two specimens were culture positive but were not sequenced because the isolate was either not sent or the result was inconclusive These two specimen
6. identification of possible sources of a hypervirulent outbreak Traditionally cell cytotoxicity assays and enzyme immunoassays EIA have been used to detect CDI causing C difficile Cell cytotoxicity assays provide sensitive results but are labor intensive and time consuming EIAs on the other hand typically have low sensitivity 2 Comparatively molecular testing has demonstrated high accuracy and expedient assay turnaround times allowing for higher rates of C difficile detection in a clinically meaningful timeframe PRINCIPLES AND PROCEDURES OF CDF AND THE VERIGENE SYSTEM CDF 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 CDF sample analysis steps including i Specimen Preparation magnetic bead based bacterial DNA extraction from prepared stool specimens obtained from patients ii Target Amplification multiplex PCR based amplification to generate specific amplicons ili 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 assessed optically with high efficiency by the Verigene Reader The Verigene Reader also serves
7. the OPEN CLOSE button on the Processor SP The Processor SP will automatically verify that each consumable is properly loaded and begin 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 set up one test at a time change gloves after handling a sample and decontaminate pipettes and sample tubes between tests 8 Upon Completion of a Test Run a The Verigene Reader will generate a ring to notify the user when the test is completed and the Processor SP will display a message indicating 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 Cap the 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 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 Holder Substrate Holder 9 Analyzing Results a Remove the protective tape from the back of the slide in the Substrate Holder b Use the Readers barcode scanner to read the barcode on the Substrate When the barcod
8. 502 NPV 99 8 1787 1791 97 8 99 1 99 4 99 9 Of the 1 875 specimens evaluated two specimens were culture positive but were not sequenced because the isolate was either not sent or the result was inconclusive These two specimens were not included in the performance characteristics above Of the 120 specimens that were toxigenic C difficile positive by Verigene CDF testing but toxigenic C difficile negative by direct enriched toxigenic culture 90 were positive by bi directional sequencing for tcdC 6 were positive by bi directional sequencing for tcdB and 24 were negative by bi directional sequencing for both tcdC and tcaB Page 21 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re 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 NAN022 M B Precision and Reproducibility Precision and reproducibility of CDF was evaluated using a 7 member panel consisting of two different C difficile strains ATCC 43255 Toxigenic Wild Type and ATCC 1805 Toxigenic Mutant at 3 different concentrations The panel included 1 negative control along with 2 high negative low positive samples expected to produce a negative result approximately 20 to 80 of the time 2 low positive sample
9. 7 strains of C difficile by the CDF test is solely for epidemiological purposes and is not intended to guide or monitor treatment for C difficile infections Concomitant culture is necessary only if further typing or organism recovery is required BACKGROUND INFORMATION AND CLINICAL UTILITY Clostridium difficile is a gram positive spore forming bacillus that can cause mild to severe diarrhea pseudomembranous colitis sepsis and even death Recently C difficile infection CDI has become more prevalent and more severe worldwide The colonic flora in a healthy adult generally resists colonization by C difficile Yet CDI among relatively young and healthy individuals are increasing Antibiotic exposure is an important risk factor for CDI The most common way to acquire this organism is through a hospital acquired infection When exposed to most types of antibiotics the intestinal flora in humans become unbalanced allowing toxigenic forms of C difficile that are acquired to proliferate The toxigenic forms of C difficile those that produce toxins A and B encoded by the tcdA and tcdB genes within the pathogenicity locus PaLoc appear to play a large role in causing disease Although toxin A is well correlated with pathogenicity some strains of toxigenic C difficile do not produce toxin A but still carry it The majority of CDIs are caused by toxigenic C difficile strains that produce both toxins but there are still cases that involve varia
10. 8 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 Page 26 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a nN O S p h re Inthe 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 NAN022 TEST KIT LABELING The contents of a Test Kit may use EN 980 graphical symbols The symbols are defined below EF Catalog number In vitro diagnostic medical device Upper Limit Temperature limitation ft Upper and Lower Limit Temperature limitation w Consult instructions for use Key code Use this key code to obtain instructions for i KEY CODE use at www e labeling eu xn x Harmful F rel Flammable Page 27 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re 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 NAN022 i PATENTS AND TRADEMARKS The Verigene Reader may be protected by US patent 7 110 585 and other pending US and foreign patent applications T
11. A construct serves as a hybridization control and is referred to as the Internal Processing Control 1 INT CTL 1 This control material and detection oligonucleotides are included within the Extraction Tray and the Test Cartridge If the signals from INT CTL 1 are not valid a no call result No Call INT CTL 1 will be obtained and the test should be repeated Bacillus subtilis serves as an extraction amp amplification control and is referred to as the Internal Processing Control 2 INT CTL 2 This control is automatically added by the Processor SP to each specimen prior to the extraction step The primers and detection oligonucleotides are included within the Extraction Tray Amplification Tray and the Test Cartridge If the signals from INT CTL 2 are not valid then a no call result No Call INT CTL 2 will be obtained and the test should be repeated If the result is No Call INT CTL 2 then the likely cause of the failure is in either the extraction or the amplification part of the procedure The CDF algorithm utilizes the results from both positive controls and an additional negative control while determining the presence or absence of any other target on the panel If either of the positive controls or the negative control are not valid then a no call result will be obtained and the test should be repeated Table 3 Processing Controls Controls for proper hybridization steps due to specimen or process related inhibitors or due to r
12. Customer Service or Technical Service N a N O 5 p h A re 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 NAN022 ls Verigene Clostridium difficile Nucleic Acid Test CDF 20 005 022 Test Kit e 20 012 022 Amplification Kit li KEY CODE NANO22 INTENDED USE The Verigene Clostridium difficile Nucleic Acid Test CDF is a qualitative multiplexed in vitro diagnostic test for the rapid detection of toxin A tcdA toxin B tcdB and tcdC gene sequences of toxigenic Clostridium difficile and for presumptive identification of PCR ribotype 027 strains from unformed liquid or soft stool specimens collected from patients suspected of having C difficile infection CDI Presumptive identification of the PCR ribotype 027 strain of C difficile is by detection of the binary toxin cdt gene sequence and the single base pair deletion at nucleotide 117 in the tcdC gene The tcdC gene encodes for a negative regulator in C difficile toxin production The test is performed on the Verigene System and utilizes automated specimen preparation and polymerase chain reaction PCR amplification combined with a nanoparticle based array hybridization assay to detect the toxin gene sequences associated with toxin producing C difficile The CDF test is indicated for use as an aid in the diagnosis of CDI Detection of PCR ribotype 02
13. Detected Wild type Toxigenic Clostridium difficile Detected Not Detected Wild type Toxigenic Clostridium difficile Detected Detected Not Detected Detected era amp PCR ribotype 027 Clostridium difficile Wild type Toxigenic Clostridium difficile Detected Wild type Toxigenic Clostridium difficile Detected Not Detected ae amp PCR ribotype 027 Clostridium difficile Wild type Toxigenic Clostridium dificile Detected Page 12 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 nN Customer Service or Technical Service y N a N O 5 p h re 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 NAN022 Error calls related to an invalid test are listed in the Table 2 below together with the appropriate recourse which should be taken by the user Table 2 Invalid Calls and Recourse Ensure protective silver tape has been removed from back of Test Substrate Ensure Test Substrate is seated properly l in the Substrate Holder Repeat image No Call NO GRID Reader unable to image Test Substrate analysis by selecting Menu and Enter Barcode and then scanning the Substrate barcode If the No Call persists repeat CDF from original stool specimen tcdC is not detected but tcdA and or No Call IND tcdB is Detected or tcdC is mutant and Bina
14. Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN022 7 Loading the Sample a At the Reader 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 Hybridization Amplification and Extraction options are selected see image below b Inthe subsequent dialogue box select or de select Toxigenic C diff and or Ribotype 027 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 Pipette 100 uL from the SPB tube avoiding any solids at the bottom of the tube into the bottom of the Sample Loading Well in the Extraction Tray refer to image for Sample Loading Well location If pipette tip clogs when loading re centrifuge the SPB tube for an additional 30 seconds Sample Loading Well Page 10 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 w Customer Service or Technical Service eS N a nN O S p h re 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 NAN022 d Close the Drawer Assembly by pressing
15. Shipped Frozen Upon receipt store Amplification Trays frozen Do not re freeze after thawing Page 3 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service w y N a nN O S h re 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 NAN022 METHODS A Specimen Collection Processing and Storage Inadequate or inappropriate specimen collection storage or transport may yield false negative results Because of the importance of specimen quality training in specimen collection and handling is highly recommended 1 2 3 Collect an unformed liquid or soft stool specimen in a sterile container from the patient suspected of having C difficile infection Store the stool specimen at 2 8 C Specimens must be tested within 48 hours of collection Sanitize vortex mixers centrifuges pipettes countertops and any other equipment used for sample processing with a lint free decontaminating cloth or comparable sanitizer Put on fresh gloves Remove one Stool Prep Buffer Tube green top with clear preloaded liquid buffer from the CDF Stool Preparation Sample Kit immediately apply the Sample ID and place into the hood with a sterile flocked swab and sterile paddle Observe the consistency of the specimen If specimen is l
16. Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service w eS N a Nn O S p h re 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 NAN022 2 Loading the Extraction Tray a Prior to loading the Extraction Tray thoroughly shake the Tray to resuspend the magnetic beads which have settled during storage Check for complete resuspension by visually inspecting the well containing the beads The well containing the magnetic beads is easily distinguished as the beads are black in color Following adequate resuspension gently tap the tray on the bench to ensure that the reagents settle to the bottom of each well b The Extraction Tray can only be loaded in one location and orientation in the Drawer Assembly When loaded correctly the Sample Loading Well is located in the front right hand corner 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 3 Loading 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 b Before using the Tip Holder Assembly check the top of each Pipette Tip for the O ring and confir
17. U S Contact your local Nanosphere distributor i www e labeling eu NAN022 i D Analytical Reactivity Inclusivity Analytical reactivity of CDF was demonstrated with a comprehensive panel of sixty three 63 independently confirmed C difficile strains at three times the LoD i e 3 750 CFU mL of stool These 63 strains comprise a wide range of different toxinotypes including the hypervirulent 027 strain toxinotype Ill These include Toxinotypes 0 I HL IV V VIII IX X XI XII XXI XXII IX XXIII and XIV XV All tests correctly reported the expected results for the detection of the gene sequences for toxigenic C difficile and the Mutant 027 strain with one exception CDC 2009048 toxinotype XIV XV as classified by the CDC is associated with a non 027 strain However CDF reported detection of the tcdA tcdB binary and tcdC MUT targets as would be expected for a PCR ribotype 027 strain Subsequent sequencing of the tcdC gene verified the presence of the A117 deletion E Analytical Specificity Cross reactivity Ninety four 94 microorganisms including two 2 non toxigenic C difficile strains and fourteen 14 non C difficile Clostridium species were tested with CDF to determine analytical specificity In addition the cross reactivity of Clostridium botulinum was evaluated by in silico analysis Each bacterial strain was prepared in a Negative Stool Matrix and tested in triplicate in concentrations of 5x10 CFU mL st
18. acked punctured previously used or anyway 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 all applicable regulations mandated by local state provincial and federal agencies for the handling transport of etiologic agents WARNINGS AND PRECAUTIONS INSTRUMENTS 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 Ensure that anyone who operates the instrument e Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Reads and understands all applicable Material Safety Data Sheets MSDS 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 Page 15 of 29 Verigene
19. c Acid Test CDF 027 00035 01 Rev A December 2012 k Customer Service or Technical Service N a N O 5 p h re 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 NAN022 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 The 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 CDF provides a qualitative result for the presence Detected or absence Not Detected of the CDF target genes as listed in Table 1 The image analysis of the Test Substrate provides light signal intensities from the target specific capture spots Their presence is verified before a valid result is provided as described below Table 1 Calls for Valid Tests ia v omr e Not Detected Not Detected Toxigenic Clostridium difficile Not Detected Detected Wild type Toxigenic Clostridium difficile Detected Not
20. difficile PCR ribotype 027 Sensitivity 98 7 154 156 Pos Agreement 97 5 39 40 95 5 99 8 86 8 99 9 Specificity 87 6 1500 1713 Neg Agreement 97 8 1787 1828 85 9 89 1 97 0 98 4 Accuracy 88 5 1654 1869 Total Agreement 97 7 1826 1869 87 0 89 9 96 9 98 3 PPV 42 1 154 367 PPV 48 2 39 81 36 9 47 2 36 9 59 5 NPV 99 9 1500 1502 NPV 99 9 1787 1788 99 5 99 9 99 7 100 Verigene CDF Test Of the 1 875 specimens evaluated six specimens were culture positive but were not PCR ribotyped because the isolate was either not sent or the result was inconclusive These six specimens were not included in the performance characteristics above Page 18 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re 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 NAN022 Performance vs Enriched Culture and PCR Ribotyping Relative to enriched culture with PCR ribotyping CDF demonstrated a sensitivity and specificity for toxigenic C difficile of 91 8 and 92 5 respectively CDF also demonstrated a 91 4 positive agreement and 98 5 negative agreement for 027 by PCR ribotyping The results are summarized in Table 6 Table 6 Verigene CDF test Performa
21. dy results are summarized in Table 10 Page 22 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h A re 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 NAN022 Table 10 Reproducibility Study Results i 9 Total Agreement with Expected Result Se emm rovs Espoos Tetai Agreement with Expected eeu CDF 100 100 100 100 100 Negative Negative juan 30 30 30 30 30 30 90 90 Stool Matrix 9 88 4 100 88 4 100 88 4 100 96 0 100 Moderate 100 100 100 100 100 Positive Positive 30 30 30 30 30 30 90 90 MP 88 4 100 88 4 100 88 4 100 96 0 100 Toxigenic Low Q5 100 100 100 100 Wild Type Positive i 30 30 30 30 30 30 90 90 C difficile LP 88 4 100 88 4 100 88 4 100 96 0 100 High 20 80 30 33 30 16 70 26 70 4 Negative Posi fae 9 30 10 30 5 30 24 90 HN 14 7 49 4 17 3 52 8 5 6 34 7 17 9 37 0 Positive Moderate 100 100 100 100 100 Positi 30 30 30 30 30 30 90 90 ositive 88 4 100 88 4 100 88 4 100 96 0 100 Positive MP Toxigenic Low 959 96 70 96 70 100 97 80 Positi 29 30 29 30 30 30 88 90 osi
22. e 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 v Touch the Processing option 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 Cover s 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 8 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 nN N h Customer Service or Technical Service A In the U S Phone 1 888 837 4436 toll free a n O S p re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN022 6 Loading a 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 cartridge cover by bending the cover away and over the Reagent Pack edge Ensure that the valve plate is not moved during cover removal see illustration below
23. e is accepted a prompt to load the Substrate Holder into the Reader will be displayed c Immediately insert the Substrate Holder into the Reader d Scanning the barcode ensures that the test result is associated with the correct sample 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 e To properly insert the Substrate into the Reader hold the Substrate by the handle with the barcode facing away from you Next insert the Substrate Holder into the Reader substrate compartment The compartment is designed to place the Holder in the correct position Do not force the holder in but do insert it into the compartment as far as it will go comfortably Close the door of the substrate compartment f The analysis will automatically begin A small camera icon will appear on the Reader to indicate that analysis has begun g Once the analysis is completed by the Reader the camera icon is replaced with an upward facing arrow and the Reader rings h 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 rescanned and reanalyzed Use the Interpretation of Results section to analyze results i Once the scan is complete dispose of used Test Substrate Page 11 of 29 Verigene Clostridium difficile Nuclei
24. e the reliability of patient test results Page 13 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 nN Customer Service or Technical Service y N a N O 5 p h A re 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 NAN022 Verigene System The Verigene System uses a series of automated on line quality measurements to monitor instrument functionality software performance fluidics test conditions reagent integrity and procedural steps each time a test is performed A series of automated on line procedural checks guide the user through the testing process each time a test is performed CDF test barcode and specimen information are linked upon entry into the Verigene Reader to help prevent misreporting of results Assay Controls CDF is a specimen to result detection system wherein DNA is 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 levels of controls built into CDF ensure that failures at any step within CDF are identified during the procedure or in the end point image analysis of the Test Cartridge An artificial DN
25. eagent failures Artificial DNA construct along with the detection oligonucleotides are included within the Extraction Tray and the Test Cartridge Internal Processing Control INT CTL 1 Intact Bacillus subtilis along with primers included within the Amplification Tray and Controls for specimen isolation or Internal Processing detection oligonucleotides included within the nucleic acid extraction step and Control INT CTL 2 Extraction Tray and Test Cartridge The INT amplification steps including CTL 2 is added to each test specimen at the possible PCR inhibition beginning of the procedure Page 14 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re 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 NAN022 External Controls Regardless of the choice of quality control materials all external quality control requirements and testing should be performed in conformance with local state and federal regulations or accreditation organizations as applicable and should follow the user s laboratory s standard quality control procedures TROUBLESHOOTING Refer to the Troubleshooting section of the Verigene System User s Manual LIMITATIONS e A trained health care professional should interpret assay res
26. eagents and residual sample The lysing reagents lysis enzymes and chaotropic salts in the Extraction Tray and Stool Prep Buffer tube are expected to render the residual sample non infectious limited studies to confirm non infectivity have been performed It also contains a residual volume of the sample buffer which contains formamide a teratogen It is recommended to dispose the Extraction Tray and the Stool Prep Buffer tube in biohazardous waste e All of the Test Cartridge waste reagents including the purified DNA 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 Individual MSDS with more information is available for the Stool Prep Buffer Tube Test Cartridge Amplification Tray and Extraction Tray at www e labeling eu and at www nanosphere us EXPECTED VALUES A total of 1875 prospectively collected unformed stool specimens were obtained from five large hospitals geographically distributed across the United States The number and percentage of toxigenic C difficile positive cases by culture calculated by age are presented in Table 4 In routine practice prevalence rates may vary depending on the institution geographical location and patient population Table 4 Observed Prevalence of Toxigenic C difficile by Age Group Toxigenic C difficile Age Group years Prevalence ia ich ae incl
27. ested had any inhibitory effect on the detection of C difficile using CDF Page 24 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service e N a nN O S p h re 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 NAN022 lhe Table 13 Potential Interfering Substances Tested l PreparationH Hemorrhoidal Aluminum Hydroxide Reagent Palmitic Acid Free Acid Sigma Walgreens Enema Mineral Oil Grade Laxative F Whole Blood ee OPEEP ON y AJINA Immodium AD Anti Diarrheal Contraceptive Gel Nasopharyngeal Swab Sample in Universal Transport Media Dulcolax Laxative Suppositories Pepto Bismol Max Strength UTM Ex lax Maximum Strength Nystatin Suspension Dimenhydrinate Sumulani Laxative O i FET Monitstat 3 en I Aid Antibiotic intment PreparationH Medicated Wipes Wet Ones Antibacterial Hand Wipes Metronidazole Topical Cream Vagisil Anti ltch Creme Maximum Strength K Y Personal Lubricant Jelly Naproxen Sodium PreparationH Anti Itch Vaseline Original 100 Pure Mucin from bovine submaxillary Hydrocortisone 1 Petroleum Jelly glands Type I S Dehydrated Desitin Maximum Strength Sarna Anti ltch Lotion Sensitive Barium Sulfate Original Paste Eevee pele Equi Bile bovine dried unfractioned Cary Blai
28. he 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 2012 Nanosphere Inc All rights reserved NOTICE TO RECIPIENTS ABOUT LIMITED LICENSE OR RELATED The receipt of this product from Nanosphere Inc or its authorized distributor includes limited non exclusive license under patent rights held by Nanosphere Inc Such license is solely for the purposes of using this product to perform the proprietary nucleic acid analysis method for which it was intended from Nanosphere Inc or its authorized distributor For avoidance of doubt the foregoing license does not include rights to use this product for agriculture or veterinary medicine applications Except as expressly provided in this paragraph no other license is granted expressly impliedly or by esto
29. iquid proceed to subsection A If specimen is a soft solid proceed to subsection B A Liquid Stool a Thoroughly mix the stool in the original container with a sterile paddle for 5 seconds b Transfer 150 uL of specimen into the Stool Prep Buffer tube c Screw the cap finger tight on to the Stool Prep Buffer tube and set aside B Soft Stool a Thoroughly mix the stool in the original container with a sterile paddle for 5 seconds b Dip the flocked swab into the specimen until flocked tip is fully immersed in specimen See swabs illustrated below c Once evenly coated transfer swab to the Stool Prep Buffer tube and break swab at the pre formed scored breakpoint d Leave scored swab in the Stool Prep Buffer tube and screw the cap finger tight on to Stool Prep Buffer tube Incorrect Correct Incorrect Inadequate Adequate Excessive Specimen Specimen Specimen Page 4 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 i D Customer Service or Technical Service eS N a nN O S D h re 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 NAN022 Vortex the Stool Prep Buffer tube for a minimum of 15 seconds Microfuge the specimens for a minimum of 30 seconds Put on fresh gloves before starting the CDF Test Procedure 0 If not running the Verigene
30. l errors four motor stalls two tip failures one cracked slide and one cartridge not detected The eight specimens that experienced pre analytical errors and the 46 No Call specimens all reported results upon repeat testing however two of the No Call specimens required a second repeat test Repeat testing of the 17 No Call IND specimens called all but two specimens Therefore two specimens had a final Indeterminate call and were not included in the clinical data analysis of evaluable results Thus 1 875 specimens were analyzed in this clinical evaluation Page 17 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re 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 NAN022 Performance vs Direct Culture and PCR Ribotyping Relative to direct culture with PCR ribotyping CDF demonstrated a sensitivity and specificity for toxigenic C difficile of 98 7 and 87 6 respectively CDF also demonstrated a 97 5 positive agreement and 97 8 negative agreement for 027 by PCR ribotyping The results are summarized in Table 5 Table 5 Verigene CDF test Performance vs Direct Culture amp PCR Ribotyping Direct Culture amp PCR Ribotyping TGR nee Tort am o re e e Toxigenic C difficile Toxigenic C
31. m that the rubber Tip Seal sitting straight and flush between the tips If either is missing replace with a new Tip Holder Assembly Page 6 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 nN N h Customer Service or Technical Service A In the U S Phone 1 888 837 4436 toll free a n O S p re OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN022 c Insert the Tip Holder Assembly into the Drawer Assembly The image below shows a properly loaded Tip Holder Assembly The Tip Assembly 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 Processor SP Tip Holder Assembly 4 Loading the Amplification Tray a After thawing gently vortex lt 5 seconds the Amplification Tray and gently tap the tray on the bench to settle the reagents Remove the cap from the Amplification Tube 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 7 of 29 Ve
32. nce vs Enriched Culture amp PCR Ribotyping Enriched Culture amp PCR Ribotyping Tox C difficile Tox C difficile Gael Toes Neg Total Tox C difficile Tox C difficile Verigene CDF Test Toxigenic C difficile Toxigenic C difficile PCR ribotype 027 Sensitivity 91 8 247 269 Pos Agreement 91 4 53 58 87 9 94 8 81 0 97 1 Specificity 92 5 1480 1600 Neg Agreement 98 5 1783 1811 91 1 93 7 97 8 99 0 Accuracy 92 4 1727 1869 Total Agreement 98 2 1836 1869 91 1 93 6 97 5 98 8 PPV 67 3 247 367 PPV 65 4 53 81 62 2 72 1 54 0 75 7 NPV 98 5 1480 1502 NPV 99 7 1783 1788 97 8 99 1 99 4 99 9 Of the 1 875 specimens evaluated six specimens were culture positive but were not PCR ribotyped because the isolate was either not sent or the result was inconclusive These six specimens were not included in the performance characteristics above Of the 120 specimens that were toxigenic C difficile positive by Verigene CDF testing but toxigenic C difficile negative by direct enriched toxigenic culture 90 were positive by bi directional sequencing for tcdC 6 were positive by bi directional sequencing for tcdB and 24 were negative by bi directional sequencing for both tcdC and tcaB Page 19 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p
33. nts missing toxin A A B The toxins are pro inflammatory enterotoxins causing the majority of symptoms associated with CDI The PCR ribotype 027 strain exhibits increased toxin production This is thought to be a result of a mutation in the tcdC gene which is also located within the PaLoc that causes a negative regulation in toxin synthesis This growth in toxin production is believed to cause an increase in spore production which leads to improved persistence in the intestines Along with toxins A and B binary toxin cat is also produced Although the role of binary toxin in the pathogenesis is unclear it is recognized as a virulence factor in the hypervirulent strain The PCR ribotype 027 strain shows hypervirulence especially in an outbreak setting Accurate strain identification is Critical for understanding the ever changing epidemiology of C difficile and for the prevention of future outbreaks in hospitals The presumptive identification of PCR ribotype 027 strains could potentially assist in early detection Page 1 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 w Customer Service or Technical Service y N a nN O S p h re 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 NAN022 and prevention of a PCR ribotype 027 outbreak and may aid in the
34. ool Two 2 organisms Cryptosporidium parvum and Giardia lamblia were tested using genomic DNA at a concentration of 1x10 copies of gDNA For the viruses Echovirus 11 and Coxsackievirus were tested at 5x10 PFU mL stool Adenovirus Enterovirus Cytomegalovirus and Rotavirus were also tested using genomic DNA at a concentration of 1x10 copies of gDNA Noroviruses were tested as clinical samples Analytical specificity was observed to be 100 including that determined by in silico analysis F Microbial Interference Microorganisms that may be encountered in clinical stool samples but not detected by CDF were tested to evaluate the potential for microbial interference CDF was tested against the same ninety four 94 organisms that were used for analytical specificity at the same medically relevant concentrations using two strains of toxigenic C difficile ATCC BAA 1805 toxinotype III and ATCC 43325 toxinotype 0 at 1 5x LoD and 3x LoD respectively No interference was observed with CDF for any of the samples tested G Interference The potential inhibitory effects of thirty four 34 products exogenous substances shown in Table 13 that are possibly encountered in stool samples was evaluated Each interfering substance was evaluated at its worst case concentration against two C difficile strains ATCC 1805 ATCC 43225 Additionally Cary Blair media was tested None of the thirty four 84 substances or the Cary Blair media t
35. orical Perspectives on Studies of Clostridium difficile and C difficile Infection Clinical Infectious Diseases 46 1 S4 S11 doi 10 1086 521865 2 Curry S R Marsh J W Muto C A O Leary M M Pasculle A W amp Harrison L H 2006 tcdC Genotypes Associated with Severe TcdC Truncation in an Epidemic Clone and Other Strains of Clostridium difficile Journal of Clinical Microbiology 45 1 215 221 doi 10 1128 JCM 01599 06 Hensgens M Keessen E Squire M Riley T Koene M de Boer E et al 2012 Clostridium difficile Infection in the Community A Zoonotic Disease Clinical Microbiology and Infection 18 7 635 645 doi 10 1111 1469 0691 2012 03853 x Kelly C P amp LaMont J T 2008 Clostridium difficile More Difficult Than Ever New England Journal of Medicine 359 1932 1940 doi 10 1056 NEJMra0707500 LaSala P Svensson A Mohammad A amp Perrotta P 2012 Comparison of Analytical and Clinical Performance of Three Methods for Detection of Clostridium difficile Archives of Pathology amp Laboratory Medicine 136 5 527 531 PMID 22540301 Matamouros S England P amp Dupuy B 2007 Clostridium difficile Toxin Expression is Inhibited by the Novel Regulator TcdC Molecular Microbiology 64 5 1274 1288 PMID 17542920 Salcedo J Keates S Pothoulakis C Warny M Castagliuolo l LaMont J et al 1997 Intravenous Immunoglobulin Therapy for Severe Clostridi
36. ppel 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 28 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service e N a nN O S p h re 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 NAN022 i REFERENCES Bartlett J G 2008 Hist
37. r Medium Phillips Genuine Milk of Tums Antacid with Calcium Extra aa Magnesia Saline Laxative Strength 750 H Cutoff Verification Analytical testing of 59 strains of C difficile comprising of a range of toxinotypes and non toxinogenic strains was performed in duplicate with CDF to verify the cut off values of the two tiered filter algorithm Using the established cut off levels for the assay CDF correctly detected the expected analytes for all of the samples tested I Carryover Cross Contamination The potential for carry over and cross contamination of CDF on the Verigene system was assessed by alternately testing a high positive C difficile sample toxigenic amp Mutant Clostridium difficile strain BAA 1805 at 5x10 CFU mL followed by testing a negative sample comprising only of CDF negative stool matrix The high titer sample was alternated with the negative sample three times on three unique Verigene SP Processors for a total of eighteen individual tests No carry over or cross contamination was observed Page 25 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a nN O S D h re 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 NAN022 CONTACT INFORMATION In the United States Nanosphere Inc 408
38. rigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 w Customer Service or Technical Service eS N a nN O S D h re 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 NAN022 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 in the silver latch Lower the Drawer Clamp 5 Ordering a Test a All tests must be ordered through the Verigene Reader No tests can be processed on the Verigene Processor SP without the user entering the Test Cartridge ID and Sample ID to the Verigene Reader i Log in to the Verigene Reader li If the user would like to start a new Session proceed to the next step ili If the user would like to order a test in a previously created session they can select the desired Session from the drop down SESSION menu then proceed to step v Up to 60 cartridges can be entered into a single session iii From the Menu Bar SESSION tab select Start New Session where the Session Setup window will appear iv Touch Session ID button and enter information by using th
39. ry is not detected INT CTL 1 Not Detected Probable failure during the target No Call INT CTL 1 hybridization part of the procedure only This control does not require extraction or amplification to work properly INT CTL 2 Not Detected Probable failure during extraction or No Call INT CTL 2 amplification part of the procedures Repeat CDF This control requires proper extraction amplification and hybridization INT CTL 1 and INT CTL 2 Not Detected Probable failure during the target No Call INT CTL hybridization part of the procedure Other failures during extraction or amplification may also have occurred No Call VARIATION Reader unable to obtain test result No Call BKGD because of high variability in the target No Call NEG CTL specific signals Pre analytical error Internal checks Processing Error within the Processor SP detected an Power cycle Processor SP repeat CDF unexpected event F irst repeat test should use the SPB sample if within 24 hours of preparation or the original stool specimen if gt 24 hours from SPB preparation Second repeat test if needed should use the original stool specimen the original specimen should be stored at 2 8 C and tested within 48 hours of collection 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 ensur
40. s expected to yield a positive result 95 of the time and 2 moderate positive samples expected to yield a positive result 100 of the time Precision Precision of CDF was determined internally at Nanosphere by testing each of the 7 specimens daily in duplicate by 2 operators for 12 non consecutive days for a total of 48 replicates per specimen 1 site x 2 operators site x 2 replicates operator x 12 days 48 replicates per specimen The Precision Study results are summarized in Table 9 Table 9 Precision Study Results Panel Expected Total Agreement with 3 100 i CDF Negative Stool Negative y 1 nie 48 48 J 92 6 100 Moderate Positive 100 bes 2 a 48 48 A PORE 92 6 100 97 9 Low Positive LP 95 Positive 47 48 88 9 100 Toxigenic Wild Type C difficile 12 5 High Negative HN ade ae a 4 7 25 3 ai T 92 6 100 igeni 95 8 M C Low Positive LP 95 Positive 46 48 85 6 99 5 20 8 High Negative HN 8 jac 10 5 35 0 95 Two sided Exact Binomial Confidence Interval calculation Reproducibility Reproducibility of CDF was determined at three external sites by testing the same 7 member sample panel in triplicate daily by two operators per site for five non consecutive days for a total of sixty replicates per specimen 3 sites x 2 operators site x 2 replicates operator x 5 days 90 replicates per specimen The Reproducibility Stu
41. s required to generate a test result The Test Cartridges are labeled as CDF 20 006 022 e 20 Verigene CDF Extraction Trays with Tip Holder Assemblies Each Extraction Tray comes preloaded with all required solutions including lysis binding 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 CDF 20 009 022 e Verigene CDF Stool Preparation Sample Kit Each Kit contains 20 tubes containing CDF Stool Prep Buffer SPB and 20 swabs packaged in a resealable bag The Kit is labeled as CDF 30 001 022 Verigene CDF Amplification Reagent Kit Catalog number 20 012 022 e 20 Verigene CDF Amplification Trays Each Amplification Tray comes preloaded with all required solutions including enzymes and buffers necessary to amplify nucleic acids and generate a test result The Amplification Trays are contained within a carrier labeled as CDF 20 011 022 MATERIALS NEEDED BUT NOT PROVIDED Instruments and Equipment e Verigene Reader Catalog number 10 0000 02 Verigene Processor SP Catalog number 10 0000 07 2 8 C Refrigerator lt 20 C Freezer Micro pipettors amp filtered tips Vortex Microfuge Sterile paddles Decontamination Wipes Spray or comparable sanitizer REAGENT STORAGE HANDLING STABILITY CDF Test Component Storage Conditions Stool Prep Buffer SPB Tubes Room Temperature Do not freeze amp Swabs ea one
42. s were not included in the performance characteristics above Page 20 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re 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 NAN022 Performance vs Enriched Culture and Bi Directional Sequencing Relative to enriched culture with Bi Directional Sequencing CDF demonstrated a sensitivity and specificity for toxigenic C difficile of 91 9 and 92 5 respectively CDF also demonstrated a 93 7 positive agreement and 98 7 negative agreement for 027 by sequencing The results are summarized in Table 8 Table 8 Verigene CDF test Performance vs Enriched Culture amp Sequencing Enriched Culture amp Sequencing Tox C difficile Tox C difficile Pe anes e nec e Tox C difficile gge w o om leo Tox C difficile Verigene CDF Test Toxigenic C difficile Toxigenic C difficile PCR ribotype 027 Sensitivity 91 9 251 273 Pos Agreement 93 7 59 63 88 1 94 9 84 5 98 2 Specificity 92 5 1480 1600 Neg Agreement 98 7 1787 1810 91 1 93 7 98 1 99 2 Accuracy 92 4 1731 1873 Total Agreement 98 6 1846 1873 91 1 93 6 97 9 99 1 PPV 67 7 251 371 PPV 72 0 59 82 62 6 72 4 60 9 81 3 NPV 98 5 1480 1
43. tive 82 8 99 9 82 8 99 9 88 4 100 92 2 99 7 Mutant C Positive difficile LP High 20 80 36 70 40 00 o 11 30 12 30 32 90 ositive 19 9 56 1 22 7 59 4 14 7 49 4 25 7 46 4 Negative HN 95 Two sided Exact Binomial Confidence Interval calculation C Analytical Sensitivity Limit of Detection Analytical sensitivity LoD testing of seven strains of C difficile representing all major toxinotypes found in North America ranged from 63 to 1250 CFU ml of stool The study established the overall limit of detection of CDF to be 1250 CFU ml of organism present in stool based upon demonstration that at this concentration the test produces a positive result greater than 95 of the time The results of the LOD study are provided in Table 12 Table 11 Analytical Sensitivity Study Results Calculated LoD adan Pesonanon Toxinotype CFU mL Stool ee PEDE Confirmation Source ID Test at LoD at LoD Results ATCC BAA 1805 oo o mo 820 20 ATCC 43255 VPI 10463 0 68 15 f 20 Oo vy x 0 2 V CDC 2009087 0 150 235 2020 CDC 2009292 m 5 235 2020 Page 23 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 Customer Service or Technical Service N a N O 5 p h re In the U S Phone 1 888 837 4436 toll free OR E Mail productsupport nanosphere us Outside the
44. udes PCR ribotype 027 0 14 a Adolescent 212 lt 18 years old 0 0 14 0 0 14 23 1 204 883 6 2 55 883 19 8 371 1875 a Prevalence based on CDF results Page 16 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012 w Customer Service or Technical Service y N a nN O S p h re In the U S Phone 1 888 837 4436 toll free k OR E Mail productsupport nanosphere us Outside the U S Contact your local Nanosphere distributor i www e labeling eu NAN022 PERFORMANCE CHARACTERISTICS The results of 10 studies conducted to establish the performance characteristics of CDF are provided in the following sections A Clinical Performance A method comparison study n 1875 was conducted at five external geographically diverse clinical study sites to evaluate the performance of CDF by comparing CDF results to reference culture followed by cell cytotoxicity testing on the isolates and strain typing on the toxigenic strains by PCR ribotyping and bi directional sequencing methods Subjects included individuals whose routine care called for C difficile testing There were 1 877 evaluable specimens enrolled in the clinical trial 71 specimens 3 8 required repeat testing 46 specimens 2 4 had an initial No Call result due to assay internal control errors 17 specimens 1 0 had an initial Indeterminate call No Call IND and 8 specimens 0 4 had pre analytica
45. ults together with the patient s medical history clinical signs and symptoms and the results of other diagnostic tests e The detection of bacterial nucleic acids is dependent on proper specimen collection handling transport storage and preparation including extraction Failure to observe proper procedures in any of these steps could lead to incorrect results e There is a risk of false negative results due to sequence variants in the CDF targets of the assay e A negative result for Clostridium difficile should not be used as the sole basis for diagnosis treatment or patient management decisions e Performance characteristics were not established for patients lt 2 years of age e Because identification of the PCR ribotype 027 strain of C difficile is by detection of binary toxin cdf gene sequences and the single base pair deletion at nucleotide 117 in the tcdC gene calls identifying PCR ribotype 027 strains by CDF should be considered presumptive e CDC 2009048 toxinotype XIV XV non PCR ribotype 027 will be reported as Toxigenic C difficile Detected and PCR ribotype 027 Detected using CDF WARNINGS AND PRECAUTIONS GENERAL e CDF is for in vitro diagnostic use only e Federal law restricts this device to sale by or on the order of a physician or to a clinical laboratory its use is restricted to by or on the order of a physician e Never use any Tips Trays Tubes or Test Cartridges which have been broken cr
46. um difficile Colitis Gut 41 3 366 370 PMC 1891485 Stubbs S Brazier J O Neill G amp Duerden B 1999 PCR Targeted to the 16S 23S rRNA Gene Intergenic Spacer Region of Clostridium difficile and Construction of a Library Consisting of 116 Different PCR Ribotypes Journal of Clinical Microbiology 37 2 461 463 PMID 9889244 gt Chapin K C Dickenson R A Wu F amp Andrea S B 2011 Comparison of Five Assays for Detection of Clostridium difficile Toxin Journal of Molecular Diagnostics 13 4 395 400 10 1016 j jmoldx 2011 03 004 Tenover F Akerlund T Gerding D Goering R Bostrom T Jonsson A et al 2011 Comparison of Strain Typing Results for Clostridium difficile solates from North America Journal of Clinical Microbiology 49 5 1831 1837 PMID 21389155 Weiss K Boisvert A Chagnon M Duchesne C Habash S Lepage Y et al 2009 Multipronged Intervention Strategy to Control an Outbreak of Clostridium difficile Infection CDI and its Impact on the rates of CDI from 2002 to 2007 Infection Control and Hospital Epidemiology 30 2 156 162 PMID 19125681 Page 29 of 29 Verigene Clostridium difficile Nucleic Acid Test CDF 027 00035 01 Rev A December 2012
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