Cdiff - BD Molecular Diagnostics
Contents
1. e Check reagent strips for proper liquid fills ensure that the liquids are at the bottom of the tubes see Figure 1 Do not remove desiccant from reagent pouches Do not use reagents if desiccant is not present or is broken inside reagent pouches Do not use reagents if the foil has been broken or damaged Do not mix reagents from different pouches and or kits and or lots Do not interchange or reuse caps as contamination may occur and compromise test results e Proceed with caution when using chemical solutions as Master Mix and Extraction tube barcode readability may be altered Do not use expired reagents and or materials To avoid contamination by amplicons do not break apart the BD MAX PCR Cartridges after use The seals of the BD MAX PCR Cartridges are designed to prevent contamination e Performing the BD MAX Cdiff Assay outside the recommended time ranges can produce invalid results e Additional controls may be tested according to guidelines or requirements of local state provincial and or federal regulations or accrediting organizations e In cases where open tube PCR tests are conducted in the laboratory care must be taken to ensure that the BD MAX Cdiff Assay any additional reagents required for testing and the BD MAX System are not contaminated Gloves must be changed before manipulating reagents and cartridges e Always handle specimens as if they are infectious and in accordance w2. or negative for toxigenic C difficile Note It is recommended that bacterial strains be freshly prepared in saline to a turbidity of 0 5 McFarland 1 0 X 10 CFU mL from isolated colonies and subsequently diluted with saline to obtain a final concentration of 3 3 x 10 CFUML 2 One 1 External Positive Control and one 1 External Negative Control should be run daily until adequate process validation is achieved on the BD MAX System Reduced frequency of control testing should be based on adequate data and determined by the individual laboratory 3 An External Negative Control that yields a positive test result is indicative of a specimen handling and or contamination problem Review the specimen handling technique to avoid mix up and or contamination An External Positive Control that yields a negative result is indicative of a specimen handling preparation problem Review the specimen handling preparation technique P0137 01 2013 04 4 An External Control that yields an Unresolved Indeterminate or Incomplete test result is indicative of a reagent ora BD MAX System failure Check the BD MAX System monitor for any error messages Refer to the System Error Summary section of the BD MAX System User s Manual for interpretation of warning and error codes If the problem persists use reagents from an unopened pouch or use a new BD MAX Cdiff Assay kit 5 Each BD MAX Cdiff Extraction Tube contains a Sample Processing Co
3. 5 distinct days wherein each day 2 panels were tested by 2 technologists at 3 clinical sites using 1 lot of reagents Site to Site One of these clinical sites participated in an extended study where 2 additional lots of reagents were tested Lot to Lot Results are shown for each specimen category For Site to Site Reproducibility the overall percent agreement was 100 for MP LP and Neg categories with 92 2 and 50 0 negative agreement for HN1 100 and HN1 10 categories respectively Table 8 For Lot to Lot Reproducibility the overall percent agreement was 100 for MP LP and Neg categories with 96 7 and 64 4 negative agreement for HN1 100 and HN1 10 categories respectively Table 9 second Derivative Peak Abscissa SDPA an internal criteria used to determine a final assay result was selected as an additional means of assessing assay reproducibility Overall mean SDPA values with variance components SD and CV are shown in Tables 8 and 9 Table 8 Site To Site Reproducibility Study Results using One Lot of the BD MAX Cdiff Assay SITE 1 Site 1 Site 2 Site 3 Overall Percent SUES Percent Percent Percent Agreement Overall as Agreement Agreement Agreement Mean SP CV Category 1For the Negative and High Negative categories SDPA values reported are for the SPC For other categories SDPA values reported are for the toxigenic C difficile target 2 For the High Negative categories the expected assay result was deemed
4. CR Cartridge are sealed by the system prior to initiating PCR to contain the amplification mixture thus preventing evaporation and contamination The amplified DNA targets are detected using hydrolysis TaqMan probes labeled at one end with a fluorescent reporter dye fluorophore and at the other end with a quencher moiety Probes labeled with different fluorophores are used to detect tcdB and SPC amplicons in two different optical channels of the BD MAX System When the probes are in their native state the fluorescence of the fluorophore is quenched due to its proximity to the quencher However in the presence of target DNA the probes hybridize to their complementary sequences and are hydrolyzed by the 5 3 exonuclease activity of the DNA polymerase as it synthesizes the nascent strand along the DNA template As a result the fluorophores are separated from the quencher molecules and fluorescence is emitted The amount of fluorescence detected in the two optical channels used for the BD MAX Cdiff Assay is directly proportional to the quantity of the corresponding probe that is hydrolyzed The BD MAX System monitors these signals at each cycle of the PCR and interprets the data at the end of the program to provide a final result REAGENTS AND MATERIALS Contents Quantity BD MAX Cdiff Master Mix Dried PCR Master Mix containing tcdB specific 24 tests molecular probe and primers along with Sample Processing Control specific mol
5. OCEDURE A liquid or soft stool specimen is collected and transported to the laboratory For testing a disposable 10uL inoculating loop is dipped into the stool material and the contents dispersed into a BD MAX Cdiff Sample Buffer Tube The Sample Buffer Tube is closed with a septum cap and vortexed A worklist is created and the Sample Buffer Tube the BD MAX Cdiff unitized reagent strip and the BD MAX PCR Cartridge are loaded onto the BD MAX System The BD MAX System automates sample preparation including target lysis DNA extraction and concentration reagent rehydration and target nucleic acid amplification and detection using real time PCR The BD MAX System performs results interpretation automatically The assay also includes a Sample Processing Control SPC that is present in the Extraction Tube The SPC monitors DNA extraction steps thermal cycling steps reagent integrity and the presence of inhibitory substances Following enzymatic cell lysis the released nucleic acids are captured on magnetic beads The beads with the bound nucleic acids are washed using Wash Buffer and the nucleic acids are eluted by heat in Elution Buffer Eluted DNA is neutralized using Neutralization Buffer and transferred to a Master Mix to rehydrate PCR reagents After reconstitution the BD MAX System dispenses a fixed volume of PCR ready solution containing extracted nucleic acids into the BD MAX PCR Cartridge Microvalves in the BD MAX P
6. Preparation section CULTURING OF CLINICAL SPECIMENS To perform species identification directly from stools clinical specimens may be cultured using hospital procedures LIMITATIONS OF THE PROCEDURE This product is intended for use only with liquid or soft stools performance characteristics of other clinical specimen types have not been established This product can only be used on the BD MAX System Incorrect test results may occur from improper specimen collection handling or storage technical error sample mix up or because the number of organisms in the specimen is below the analytical sensitivity of the test Careful compliance with the package insert instructions and the BD MAX system User s Manual are necessary to avoid erroneous results Good laboratory technique is essential to the proper performance of this assay Due to the high analytical sensitivity of this test extreme care should be taken to preserve the purity of all materials and reagents A BD MAX Cdiff positive assay result does not necessarily indicate the presence of viable organisms It does however indicate the presence of the tcdB gene and allows for presumptive detection of a C difficile toxigenic organism The BD MAX Cdiff Assay cannot be used for species identification as it does not contain primers and probes specific to C difficile As with all PCR based in vitro diagnostic tests extremely low levels of target below the limit of detection o
7. R at 29 C for a maximum of 5 hours after the end of the run If an Indeterminate IND Unresolved UNR or Incomplete INC result is obtained or if an External Control failure occurs a repeat test from the Sample Buffer Tube must be performed within this timeframe see Repeat Test Procedure section QUALITY CONTROL Quality control procedures monitor the performance of the assay Laboratories must establish the number type and frequency of testing control materials according to guidelines or requirements of local provincial state and country regulations or accreditation organizations For general QC guidance the user may wish to refer to CLSI MM03 and C2415 1 The External Positive Control is intended to monitor for substantial reagent failure while the External Negative Control is used to detect reagent or environmental contamination or carry over by tcdB amplicons External Control materials are not provided by BD Various types of External Controls are recommended to allow the user to select the most appropriate for their laboratory quality control program e Commercially available control materials e g ATCC 43255 a C difficile strain bearing the tcdB gene and ATCC 700057 a non toxigenic C difficile strain can be used as positive and negative controls respectively e Suspensions of bacterial strains characterized by the user as toxigenic or non toxigenic e Previously characterized specimens known to be positive
8. SBD MAX Cdiff BD MAX Cdiff Assay REF 443418 For In Vitro Diagnostic Use For use with the BD MAX System ye 9 Sr me d IVD ae a INTENDED USE The BD MAX Cdiff Assay performed on the BD MAX System is an automated in vitro diagnostic test for the direct qualitative detection of the Clostridium difficile toxin B gene tcdB in human liquid or soft stool specimens from patients suspected of having C difficile infection CDI The test performed directly on the specimen utilizes real time polymerase chain reaction PCR for the amplification of C difficile toxin B gene DNA and fluorogenic target specific hybridization probes for the detection of the amplified DNA The BD MAX Cdiff Assay is intended to aid in the diagnosis of CDI SUMMARY AND EXPLANATION OF THE PROCEDURE Clostridium difficile is an anaerobic gram positive bacillus that is the leading cause of antibiotic associated diarrhea and pseudomembranous colitis in health care facilities Incidence of CDI has been increasing and severe cases are becoming more common CDI disease symptoms range from mild diarrhea to severe colitis and even bowel perforation and death The most common risk factor is exposure to antibiotics The diagnosis of C difficile infection is based upon clinical signs and symptoms such as diarrhea as well as laboratory tests or pathologic finding consistent with toxigenic C difficile It appears that Toxin B is necessary for the devel
9. acterial interference Toxigenic C difficile negative specimens and toxigenic C difficile positive specimens at 2 3X LoD were tested with the highest amount of each compound likely to be found in the specimens or with interfering organisms 1 X 108 CFU mL of each strain Potentially interfering substances include calcium carbonate Tums as well as magnesium and aluminum hydroxide Maalox liquid Results demonstrated no reportable interference with any other tested substance except for Mesalamine rectal suspension enema and Gynol II that both showed slight inhibition delay of Second Derivative Peak Abscissa in the BD MAX Cdiff Assay however expected assay results were still obtained Table 7 Table 7 Endogenous and Commercial Exogenous Substances tested with the BD MAX Cdiff Assay Weight or Weight or Brand Name or Description Volume Result Brand Name or Description Volume Result Tested SBT Tested SBT NI Nystatin 10 uL Pepto Bismol Hyderm Hydrocortisone cream 0 0246 g Glycerin Suppositories 0 0129 g Metronidazole lhle s Paste 0 0388 g id Anusol Plus 0 01239 Polysporin 0 0240 g I P me 10305 1 Trayeorsemmicoci 10m N Waal ga ow Mesalamine Rectal P Sipentonenena E t Fleet Mineral Oil Enema TE Gynol ETE o s ON Palmitic Acid Imodium AD 0 0062 g E coli non toxigenic C difficile 10 uL Interference with the BD MAX Cdiff Assay NI No reportable interference wi
10. aluation of a real time PCR for Clostridium difficile toxin A and B genes Eur J Clin Microbiol Infect Dis 31 2219 2225 11 Clinical and Laboratory Standards Institute Protection of laboratory workers from occupationally acquired infections Approved Guideline Document M29 Refer to the latest edition 12 Centers for Disease Control and Prevention Biosafety in microbiological and biomedical laboratories Richmond JY and McKinney RW eds 1993 HHS Publication number CDC 93 8395 13 BD MAX System User s Manual Refer to the latest version BD Diagnostics Sparks MD USA 14 Clinical and Laboratory Standards Institute Molecular Diagnostic Methods for Infectious Diseases Approved Guideline document MMO3 Refer to the latest edition 15 Clinical and Laboratory Standards Institute Statistical Quality Control for Quantitative Measurements Principles and Definitions Approved Guideline Document C24 Refer to the latest edition P0137 01 2013 04 16 Cohen SH et al 1998 Isolation of a Toxin B deficient mutant strain of Clostridium difficile in a case of recurrent C difficile Associated Diarrhea Clin Infect Dis 26 410 412 17 Cohen SH et al 2000 Analysis of the pathogenicity locus in Clostridium difficile strains J Infect Dis 181 659 63 18 MacCannell D et al 2006 Characterization of a novel tcdB deficient NPA1 variant strain of Clostridium difficile 46th Annual ICAAC San Francisco Sept 2006 19 McFa
11. any additional specimens for testing by repeating Steps 1 through 5 then proceed immediately to Step 7 7 Vortex all prepared samples simultaneously at maximum speed for one 1 minute with the Multi Tube Vortexer The BD MAX Cdiff Assay must be performed immediately after the vortexing step BD MAX System Operation Note Refer to the BD MAX System User s Manual for detailed instructions Operation section Note The BD MAX Cdiff Assay must be performed immediately after the vortexing step above Specimen Preparation Step 7 Note It is recommended to use the reagent tubes removed from their protective pouch unreconstituted within 3 hours 1 Power the System on if not already done and log on by entering lt user name gt and lt password gt 2 Remove the required number of BD MAX Cdiff Reagent Strips from the BD MAX Cdiff kit Gently tap each strip onto a hard surface to ensure that all the liquids are at the bottom of the tubes 3 Remove the required number of Cdiff Extraction Tube s and Cdiff Master Mix tube s from their protective pouches Remove excess air and close pouches with the zip seal 4 For each sample to be tested place one 1 BD MAX Cdiff Reagent Strip on the BD MAX System Rack starting with Position 1 of Rack A and continuing sequentially with no open spaces 5 Snap one 1 BD MAX Cdiff Extraction Tube white foil seal into Position 1 of each of the BD MAX Cdiff Strips as shown
12. at steps 9 to 12 for Rack B 14 Place the BD MAX Cdiff Sample Buffer Tube s in the BD MAX Rack s following the same order as entered in the worklist Note Place the tubes into the sample rack with the 1D barcode labels facing outward this makes scanning tubes easier during sample login 15 Place the required number of BD MAX PCR Cartridge s into the BD MAX System see Figure 2 e Each cartridge accommodates 2 runs of up to 12 samples for a total of 24 samples e The BD MAX System will automatically select the position and row on the PCR cartridge for each run e Cartridges are used on a per run AND rack basis 2 runs per cartridge and 1 cartridge per rack Figure 2 Load PCR Cartridges P0137 01 2013 04 16 Load rack s onto the BD MAX System Figure 3 Ensure that the placement of rack s left to right corresponds to the worklist created top to bottom side B Figure 3 Load Rack s into the BD MAX System 17 Close the BD MAX System lid and click the lt Start Run gt button to begin processing 18 At the end of the run check the results immediately or store the Sample Buffer Tubes at 2 8 C for a maximum of 5 days OR at 25 C for a maximum of 5 hours until the results are checked Note If a septum cap was damaged during the run replace it with a new one before storing the specimen Note BD MAX Cdiff Sample Buffer Tubes can be stored at 2 8 C for a maximum of 120 hours 5 days O
13. ation of warning and error codes REPEAT TEST PROCEDURE Note 1 Sufficient volume is available for one repeat test from the Sample Buffer Tube on the BD MAX system For Sample Buffer Tubes stored at room temperature retesting must be performed within 5 hours after the end of the run Alternatively for Sample Buffer Tubes stored at 2 8 C retesting must be performed within 120 hours 5 days The remaining stool specimen may also be used for repeat testing within 5 days of collection if stored at 2 8 C or within 48h if stored at 2 25 C Note 2 New samples may be tested in the same run with repeat samples UNRESOLVED RESULT Unresolved results may be obtained in the event that specimen associated inhibition or reagent failure prevents proper target or SPC amplification Sample s can be repeated from their corresponding sample Buffer Tube s within the timeframe defined above Vortex the sample s for one 1 minute and restart from the BD MAX System Operation section The remaining stool specimen may also be used for repeat testing within the timeframe defined above Restart from the Specimen Preparation section INDETERMINATE RESULT Indeterminate results may be obtained in the event that a System failure occurs Sample s can be repeated from their corresponding Sample Buffer Tube s within the timeframe defined above Vortex the sample s for one 1 minute and restart from the BD MAX Operation section The rema
14. ecular probe BD MAX Cdiff Strips Reagent strips containing all liquid reagents and 24 tests 443418 disposable pipette tips necessary for specimen processing and DNA extraction BD MAX Cdiff Extraction Tube Freeze dried pellet containing DNA magnetic affinity beads Achromopeptidase and Sample Processing Control BD MAX Cdiff Sample Buffer Tube Septum Caps 24 tests EQUIPMENT AND MATERIALS REQUIRED BUT NOT PROVIDED BD MAX PCR Cartridges BD Diagnostic Systems catalog no 437519 e VWR Multi Tube Vortexer VWR catalog no 58816 115 Vortex Genie 2 VWR catalog no 58815 234 or equivalent to vortex stool specimens only P0137 01 2013 04 e NALGENE Cryogenic Vial holder VWR catalog no 66008 783 e Disposable inoculating loops 10 uL e Disposable gloves powderless Dry clean containers for the collection of liquid or soft stool specimens e f culture is performed for External Controls Pre reduced agar plate for anaerobes e g Brucella Agar with 5 sheep blood hemin and vitamin K1 plate BBL catalog no 297716 WARNINGS AND PRECAUTIONS The BD MAX Cdiff Assay is for In Vitro Diagnostic use Do not use the kit if the label that seals the outer box is broken Do not use reagents if the protective pouches are open or broken upon arrival e Close protective pouches of reagents promptly with the zip seal after each use Remove any excess air in the pouches prior to sealing
15. ed molecular assays demonstrated excellent overall agreement In comparison to one FDA cleared molecular test Table 5a the PPA and NPA of the BD MAX Cdiff Assay were 99 1 Cl 94 9 99 8 and 97 4 Cl 95 7 98 4 respectively In comparison to a second FDA cleared molecular test Table 5b the PPA and NPA of the BD MAX Cdiff Assay were 95 5 Cl 92 1 97 5 and 98 8 Cl 97 9 99 3 respectively P0137 01 2013 04 Table 5a Results Obtained with the BD MAX Cdiff Assay in Comparison to a Commercially Available FDA cleared Molecular Assay for C difficile FDA cleared Molecular Assay 1 T oa All Sites BD MAX Cdiff Assay Positive Percent Agreement 99 1 95 Cl 94 9 99 8 Negative Percent Agreement 97 4 95 Cl 95 7 98 4 Table 5b Results Obtained with the BD MAX Cdiff Assay in Comparison to a Second Commercially Available FDA cleared Molecular Assay for C difficile FDA cleared Molecular Assay 2 a Sea All Sites BD MAX Cdiff Assay Positive Percent Agreement 95 5 95 Cl 92 1 97 5 Negative Percent Agreement 98 8 95 Cl 97 9 99 3 Analytical Sensitivity The analytical sensitivity Limit of Detection or LoD for the BD MAX Cdiff Assay was determined as follows individual inoculating loops were dipped into a wide range of C difficile bacterial suspensions at different concentrations prepared and quantified from cultures of 4 C difficile strains represe
16. f the assay may be detected but results may not be reproducible Mesalamine rectal suspension enema and Gynol II may cause slight inhibition in the BD MAX Cdiff Assay refer to Interfering Substances section for further details Tums and Maalox liquid may inhibit the BD MAX Cdiff Assay refer to Interfering Substances section for further details False negative results may occur due to loss of nucleic acid from inadequate collection transport or storage of specimens or due to inadequate bacterial cell lysis The Sample Processing Control has been added to the test to aid in the identification of specimens that contain inhibitors to PCR amplification The Sample Processing Control does not indicate if nucleic acid has been lost due to inadequate collection transport or storage of specimens or whether bacterial cells have been adequately lysed BD MAX Cdiff Assay results may sometimes be Unresolved due to an invalid Sample Processing Control or be Indeterminate or Incomplete due to System failure and require retesting that can lead to a delay in obtaining final results P0137 01 2013 04 e Mutations or polymorphisms in primer or probe binding regions may affect detection of C difficile tcdB gene variants resulting in a false negative result with the BD MAX Cdiff Assay e Variant toxigenic C difficile without the tcdB gene or with a non functional Toxin B protein are very rare 19 The BD MAX Cdiff Assay
17. gistered trademark of the American Type Culture Collection BD BD logo and all other trademarks are property of Becton Dickinson and Company 2013 BD P0137 01 2013 04
18. in Figure 1 6 Snap one 1 BD MAX Cdiff Master Mix Tube green foil seal into Position 2 of each of the BD MAX Cdiff Strips as shown in Figure 1 P0137 01 2013 04 Waste Pipette Tips Extraction Tube Master Mix tube aa Of Figure 1 Snap BD MAX Cdiff Extraction tubes and Master Mix tubes into reagent strips 7 On the BD MAX software select the lt Consumable info gt tab under the Run screen 8 Enter the kit lot number for the BD MAX Cdiff Assay for lot traceability by either scanning the 1D barcode with the scanner or by manual entry Note Repeat Steps 7 and 8 for each new kit lot number 9 Select the lt Work List gt tab click on the lt Assay gt field and using the pull down menu select lt BD MAX Cdiff gt This will automatically populate the remaining assay fields for Rack A with BD MAX Cdiff 10 Enter the BD MAX Cdiff Sample Buffer Tube ID Patient ID and Accession Number if applicable for Position 1 of Rack A either by scanning the 1 D barcode with the scanner or by manual entry 11 Click on the lt Lot Numbers field and using the pull down menu select the appropriate kit lot number on the outer box This will automatically populate the remaining lot number fields for Rack A with the same lot number Note Step 11 must be repeated for each new kit lot number 12 Enter the information for Position 2 of Rack A and continue for all remaining Sample Buffer Tubes in the rack 13 Repe
19. ining stool specimen may also be used for repeat testing within the timeframe defined above Restart from the P0137 01 2013 04 Specimen Preparation section For the interpretation of warning or error code messages refer to the BD MAX User s Manual Troubleshooting section INCOMPLETE RESULT Incomplete results may be obtained in the event that the Sample Preparation or the PCR did not reach its expected time points Sample s can be repeated from their corresponding Sample Buffer Tube s within the allowed timeframe defined above Vortex the sample s for one 1 minute and restart from BD MAX Operation section The remaining stool specimen may also be used for repeat testing within the timeframe defined above Restart from the Specimen Preparation section For the interpretation of warning or error code messages refer to the BD MAX Users Manual Troubleshooting section EXTERNAL CONTROL FAILURE External Controls should yield expected results when tested If specimens have to be repeated due to an incorrect External Control result the specimens should be repeated from their Sample Buffer Tube along with freshly prepared External Controls within the timeframe defined above Vortex the samples for one 1 minute and restart from the BD MAX Operation section The remaining stool specimen may also be used for repeat testing within the timeframe defined above Restart from the Specimen
20. ith safe laboratory procedures such as those described in the CLS Document M2911 and in Biosafety in Microbiological and Biomedical Laboratories e Wear protective clothing and disposable gloves while handling all reagents e Wash hands thoroughly after performing the test Do not pipet by mouth Do not smoke drink chew or eat in areas where specimens or kit reagents are being handled e Dispose of unused reagents and waste in accordance with local state provincial and or federal regulations e Consult the BD MAX System User s Manual for additional warnings precautions and procedures STORAGE AND STABILITY Collected specimens should be kept between 2 C and 25 C during transport Protect against freezing or exposure to excessive heat Specimens can be stored at 2 25 C for a maximum of 48 hours or at 2 8 C for a maximum of 120 hours 5 days before testing BD MAX Cdiff Assay components are stable at 2 25 C through the stated expiration date Do not use expired components P0137 01 2013 04 BD MAX Cdiff Master Mix and Extraction Tubes are provided in sealed pouches To protect product from humidity immediately re seal after opening Reagent tubes are stable for up to 7 days at 2 25 C after initial opening and re sealing INSTRUCTIONS FOR USE Specimen Collection Transport In order to obtain an adequate specimen the procedure for specimen collection must be followed closely Using a dry clean containe
21. itive results due to carry over contamination REFERENCES 1 Dubberke ER Wertheimer Al Review of current literature on the economic burden of Clostridium difficile infection Infect Control Hosp Epidemiol 2009 30 57 66 2 Poutanen SM Simor AE Clostridium difficile associated diarrhea in adults Can Med Assoc J 2004 171 51 8 3 Redelings MD Sorvillo F Mascola L Increase in Clostridium difficile related mortality rates United States 1999 2004 Emerg Infect Dis 2007 13 1417 9 4 McDonald LC Owing M Jernigan DB Clostridium difficile Infection in Patients Discharged from US Short Stay hospitals 1996 2003 Emerging Infectious Diseases 2006 12 3 409 415 5 Pepin J Valiquettte L Alary ME Clostridium difficile associated diarrhea in a region of Quebec from 1991 to 2003 a changing pattern of disease severity CMAJ 2004 171 466 72 6 Bignardi GE Risk factors for Clostridium difficile infection J Hosp Infect 1998 40 1 15 7 Lyras D O Connor JR Howarth PM et al Toxin B is essential for virulence of Clostridium difficile Nature 2009 458 1176 9 8 Peterson LR Robicsek A Does my patient have Clostridium difficile infection Ann Intern Med 2009 151 176 9 9 Peterson L R et al 2007 Detection of toxigenic Clostridium difficile in stool samples by real time polymerase chain reaction for the diagnosis of C difficile associated diarrhea Clin Infect Dis 45 1152 60 10 de Jong et al 2012 Clinical and laboratory ev
22. nting 3 toxinotypes 0 III VIII Each loop was then transferred to a SBT already containing fecal matrix negative for toxigenic C difficile Each C difficile strain was tested in replicates of 24 per concentration by 2 different operators using 3 different production lots of the BD MAX Cdiff Assay Analytical sensitivity LoD defined as the lowest concentration at which 95 of all replicates tested positive ranged from 125 to 265 CFU per loop Table 6 Table 6 Limit of Detection of the BD MAX Cdiff Assay C difficile Strain Toxinotype LoD in CFU per loop ATCC 43255 265 95 Cl 140 502 ATCC 9689 156 95 CI 82 298 ATCC BAA 1805 205 95 Cl 102 412 ATCC 43598 125 95 Cl 66 235 Analytical Inclusivity A variety of toxigenic Clostridium difficile strains were included in this study taking into account geographic Origin toxinotype NAP1 outbreaks and temporal diversity Sixty four 64 strains including 23 toxinotypes 22 and representing 21 countries were tested including strains from public collections and well characterized clinical isolates The assay correctly identified 62 of the 64 toxigenic C difficile strains which were tested at 3xLoD Two 2 strains strains IS25 toxinotype XII and R9385 toxinotype XV produced low signal results and were false negative in 1 out of 5 replicates P0137 01 2013 04 Analytical Specificity The BD MAX Cdiff Assay was performed on samples containing ph
23. ntrol SPC which is a plasmid containing a synthetic target DNA sequence The SPC monitors the efficiency of DNA capture washing and elution during the sample processing steps as well as the efficiency of DNA amplification and detection during PCR analysis If the SPC result fails to meet the acceptance criteria the result of the specimen will be reported as Unresolved An Unresolved result is indicative of specimen associated inhibition or reagent failure Repeat any specimen reported as Unresolved according to the Repeat Test Procedure section below RESULTS INTERPRETATION Results are available on the Results tab in the Results window on the BD MAX System monitor The BD MAX System software automatically interprets test results A test result may be called as NEG negative POS positive or UNR unresolved based on the amplification status of the target and of the sample Processing Control IND indeterminate or INC incomplete results are due to BD MAX System failure Results are based on the following decisional algorithm ASSAY RESULT REPORTED INTERPRETATION OF RESULT tcdB gene DNA detected NEG No tcdB gene DNA detected UR Unresolved inhibitory specimen or reagent failure Indeterminate due to BD MAX System failure with Warning or Error Codes INC Incomplete Run with Warning or Error Codes Refer to the Troubleshooting section of the BD MAX System User s Manual for interpret
24. on for which diagnostic tests were indicated and ordered Only soft or liquid stools and only one specimen per patient were included The Comparative Reference Method consisted of direct culture complemented by enriched culture Enriched culture analysis was completed for all specimens that were negative for toxigenic C difficile by direct culture The anaerobic culture was used to isolate C difficile if present This was followed by confirmation of the isolate identification and a Tissue Culture Cytotoxicity Assay to determine the toxigenicity of the isolate Of the 2071 soft or liquid stool specimens compliant with the Reference Method Direct and Enriched Culture 1819 gave compliant and reportable results with the BD MAX Cdiff Assay In comparison to the Reference Method the BD MAX Cdiff Assay identified 87 7 of the toxigenic C difficile positive specimens and 96 8 of the toxigenic C difficile negative specimens Tables 1 and 2 P0137 01 2013 04 Table 1 Results Obtained with the BD MAX Cdiff Assay in Comparison to the Reference Method Reference Method All Sites BD MAX Cdiff Assay Sensitivity 87 7 265 302 95 Cl 83 6 91 0 Specificity 96 8 1469 1517 95 Cl 95 8 97 6 PPV 83 5 95 Cl 79 4 87 1 NPV 97 7 95 Cl 97 0 98 4 1 Further investigation was performed on specimens with discordant results between the Reference Method and the BD MAX Cdiff Assay e 2 of 48 False Positi
25. opment of CDI Laboratory diagnosis of toxigenic C difficile includes anaerobic culture followed by detection of toxin by tissue culture cytotoxicity testing or by detection of toxin gene s by PCR testing While culture for toxigenic C difficile followed by tissue culture cytotoxicity is highly sensitive and specific it is also highly technical time consuming and has very slow time to result 48 to 96 hours making this method impractical in the management of patients Enzyme immunoassay EIA used for the detection of toxin A and toxin B and glutamate dehydrogenase GDH an enzyme found in all C difficile strains are currently used in many clinical laboratories because results are available the same day are easy to perform and are relatively inexpensive However the sensitivity is low especially for the toxin EIAs which can lead to missed cases of CDI PCR methods for the detection of toxin A and or toxin B have been developed and have demonstrated high sensitivity and specificity as compared to toxigenic culture cell cytotoxicity and immunoassays 9 Additionally these tests can be performed in approximately 2 hours The combination of a highly sensitive and specific assay with rapidly available results may allow for prompt targeted treatment of CDI patients and thus potential improvement in patient outcomes reduced recovery times and improved infection control practices tes 1 F Ofpkt P0137 01 2013 04 PRINCIPLES OF THE PR
26. r liquid or soft stool specimens are collected according to the following procedure 1 Transfer liquid or soft stool but not urine into the container Avoid mixing toilet paper water or soap with the sample 2 Label the container 3 Ship the container to the laboratory according to hospital standard operating procedures Refer to Storage and Stability section Specimen Preparation Note One 1 Sample Buffer Tube one 1 Septum Cap one 1 Master Mix one 1 Extraction Tube and one 1 Strip are required for each specimen and each External Control to be tested Remove the required number of tubes strios from their protective pouches or boxes Remove the excess air and close the pouches with the zip seal 1 Label a Sample Buffer Tube clear cap with the appropriate specimen identification making sure not to obscure write or label over the barcodes on the Sample Buffer Tube 2 Vortex the specimen at high speed for 15 seconds and dip a 10 uL inoculating loop into the liquid or soft stool for testing For soft stool specimens remove any excess stool present on the outside of the loop in order to obtain approximately 10 uL 3 Remove the cap from the Sample Buffer Tube then place the loop into the liquid Roll the shaft of the inoculating loop between your fingers in order to release the specimen in the tube 4 Seal the tube with a Septum Cap 5 Place the Sample Buffer Tube in a NALGENE Cryogenic Vial holder 6 Prepare
27. rland L V et al Implications of the changing face of Clostridium difficile disease for health care practitioners Am J Infect Control 2007 35 4 p 237 53 20 Rupnik M et al A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates J Clin Microbiol 1998 36 8 2240 2247 21 Rupnik M et al Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes Microbiology 2001 147 439 447 22 Rupnik M et al New types of Toxin A Negative Toxin B Positive strains among Clostridium difficile isolates from Asia J Clin Microbiol 2003 41 3 1118 1125 Definition Temperature limitations In Vitro Diagnostic Medical Device Authorized Representative Reseal pouch after use The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences for providing human in vitro diagnostics No general patent or other license of any kind other than this specific right of use from purchase is granted hereby This product is sold under license and purchase of this product does not include rights to use for certain blood and tissue screening applications nor for certain industrial applications Mm v BD BD Diagnostics Technical Service 1 800 638 8663 GeneOhm Sciences Canada Inc 2555 Boul du Parc Technologique Qu bec QC G1P 4S5 Canada Made in Canada ATCC is a re
28. s tested with the BD MAX Cdiff Assay 58 3 1 were initially reported as unresolved 42 of those were repeated and 32 were resolved upon repeat testing Overall 0 5 remained unresolved after repeat Table 4 Table 4 Unresolved Rate Initial Unresolved Rate Unresolved Rate After Repeat 3 1 58 1860 95 Cl 2 4 4 0 0 5 10 1844 95 Cl 0 3 1 0 Total number based on compliant specimens and BD MAX Cdiff Assay results Out of 1916 soft or liquid stool specimens tested with the BD MAX Cdiff Assay 21 1 1 were initially reported as indeterminate Out of a total of 1844 compliant results no result remained Indeterminate upon repeat considering valid results and compliant repeats Out of 1916 soft or liquid stool specimens tested with the BD MAX Cdiff Assay 28 1 5 were initially reported as incomplete Out of a total of 1844 compliant results no result remained Incomplete upon valid repeat considering valid results and compliant repeats Comparison Studies In addition to the multi site investigational study a comparison study was performed using the BD MAX Cdiff assay and two 2 commercially available FDA cleared molecular assays for the detection of the C difficile tcdB gene Testing was performed on 2013 specimens at two external sites The Positive Percent Agreement PPA and Negative Percent Agreement NPA of the BD MAX Cdiff assay in comparison to the two commercially available FDA clear
29. targets the tcdB gene and it is unknown whether it would detect Toxin At Toxin B variant strains e An excess amount of stool may inhibit the BD MAX Cdiff Assay e As with all in vitro diagnostic tests positive and negative predictive values are highly dependent on prevalence BD MAX Cdiff Assay performance may vary depending on the prevalence and population tested EXPECTED VALUES In the BD MAX Cdiff Assay clinical study a total of 1834 reportable results from specimens compliant at the specimen and PCR levels were obtained from 6 geographically diverse sites The study population was grouped into in patient out patient and unknown categories The number and percentage of positive cases as determined by the BD MAX Cdiff Assay are presented in the table below BD MAX Cdiff Assay Group bee Number of Number Number Positive Percentage pecimens Positive Negative In patient 1249 184 1065 14 7 184 1249 Out patient 457 114 343 24 9 114 457 Unknown 128 17 111 13 3 17 128 Total 1834 315 1519 17 2 315 1834 1 Total specimens based on compliant PCR results PERFORMANCE CHARACTERISTICS Clinical performance characteristics of the BD MAX Cdiff Assay were determined in a multi site prospective investigational study Six 6 investigational centers participated in the study To be enrolled in the study specimens had to be from patients suspected of having C difficile infecti
30. th the BD Cdiff Assay Mesalamine rectal suspension enema and Gynol II with nonoxynol 9 showed slight inhibition delay of Second Derivative Peak Abscissa in the BD MAX Cdiff Assay however expected assay results were still obtained P0137 01 2013 04 Precision Within laboratory precision was evaluated for the BD MAX Cdiff Assay at one 1 site The Precision panel consisted of 5 specimen categories as follows Moderate Positive MP 2 5 x LoD Low Positive LP 1 2 x LoD High Negative 1 10 HN1 10 10 fold dilution of 1 x LoD High Negative 1 100 HN1 100 100 fold dilution of 1 x LoD True Negative Neg Escherichia coli ATCC 25922 was added to every tube in the panel C difficile strains were tested in each of the specimen categories with the exception of the negative Neg specimen category Testing was performed in duplicate over 12 days with 2 runs per day by 2 technologists Precision study results for Neg LP and MP samples demonstrated 100 agreement Precision study results for HN1 100 and HN1 10 demonstrated agreement of 95 8 and 58 3 respectively Reproducibility The Reproducibility panel consisted of 5 specimen categories as follows Moderate Positive MP 2 5 x LoD Low Positive LP 1 2 x LoD High Negative 1 10 HN1 10 10 fold dilution of 1 x LoD High Negative 1 100 HN1 100 100 fold dilution of 1 x LoD True Negative Neg Specimens in each category were tested in triplicate on
31. to be negative Therefore percent agreement was calculated for negative results P0137 01 2013 04 Table 9 Lot to Lot Reproducibility Study Results using Three Lots of the BD MAX Cdiff Assay LOT Lot 1 Lot 2 Lot 3 Overall Percent SDPA Values Percent Percent Percent Agreement Overall Agreement Agreement Agreement Mean Category SD CV 1For the Negative and High Negative categories SDPA values reported are for the SPC For other categories SDPA values reported are for the toxigenic C difficile target 2 For the High Negative categories the expected assay result was deemed to be negative Therefore percent agreement was calculated for negative results Carryover Cross Contamination A study was conducted to investigate within run carryover and between run carryover while processing specimens with high bacterial load of toxigenic C difficile in the BD MAX Cdiff assay A panel made of one high positive member and one negative member was used to prepare numerous samples A Clostridium difficile strain Tox 0 ATCC 9689 was used for the high positive C difficile panel member 3x108 CFU mL The negative member did not contain any target analyte Twelve 12 replicates of the high positive panel member and 12 replicates of the negative panel member were tested in each run by alternating negative and positive samples Three 3 operators performed 3 consecutive runs for a total of 9 runs of 24 samples There were no false pos
32. ve BD MAX Cdiff specimens were also found to be positive using another commercially available FDA cleared RT PCR assay targeting the C difficile tcdB gene e 32 of 37 False Negative BD MAX Cdiff specimens were also found to be negative using another commercially available FDA cleared RT PCR assay targeting the C difficile tcdB gene Table 2 Results Obtained by Site using the BD MAX Cdiff Assay in Comparison to the Reference Method Sensitivity Specificity 90 0 18 20 95 3 202 212 69 9 97 2 91 5 97 4 84 4 38 45 95 3 205 215 71 2 92 3 91 7 97 5 94 7 36 38 97 9 275 281 82 7 98 5 95 4 99 96 1 49 51 98 4 186 189 86 8 98 9 95 4 99 5 83 8 93 111 98 2 279 284 75 8 89 5 95 9 99 2 Numbers in parentheses express the 95 confidence interval boundaries 83 8 31 37 95 8 322 336 68 9 92 3 93 1 97 5 In comparison to direct culture the BD MAX Cdiff Assay identified 96 5 of the toxigenic C difficile positive specimens and 92 7 of the toxigenic C difficile negative specimens Table 3 P0137 01 2013 04 Table 3 Results Obtained with the BD MAX Cdiff Assay in Comparison to Direct Culture Direct Culture All Sites BD MAX Cdiff Assay Positive Percent Agreement 96 5 194 201 95 Cl 93 0 98 3 Negative Percent Agreement 92 7 1507 1625 95 Cl 91 4 93 9 Out of 1860 soft or liquid stool specimen
33. ylogenetically related species Clostridium other than toxigenic C difficile and other organisms bacteria viruses likely to be found in stool specimens e Six 6 out of 6 C difficile strains not bearing the tcdB gene tested at a concentration 2 1 X 108 CFU mL produced negative results with the BD MAX Cdiff Assay e Thirty 30 out of 30 Clostridium strains other than C difficile including 4 strains of C sordellii tested at a concentration 2 1 X 108 CFU mL produced negative results with the BD MAX Cdiff Assay e Ninety five 95 out of 98 other bacterial strains including 93 species and subspecies were tested at a concentration 2 1 X 108 CFU mL or 1 X 108 genomic DNA cp mL or 1 X 108 elementary bodies mL and produced negative results with the BD MAX Cdiff Assay An investigation was conducted and determined the false positive results were due to contamination New suspensions of 3 strains were tested and generated the expected negative results e Seven 7 out of 7 viruses tested at a concentration 2 1 X 10 PFU mL produced negative results with the BD MAX Cdiff Assay Interfering Substances Twenty five 25 biological and chemical substances occasionally used or found in perianal rectal and or stool specimens were evaluated for potential interference with the BD MAX Cdiff Assay Two 2 organisms E coli ATCC 25922 and non toxigenic C difficile ATCC 700057 were also tested at high loads in order to assess b
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