Revisions - BD Molecular Diagnostics
Contents
1. Table 20 Salmonella Site to Site Qualitative Reproducibility across sites with pooled days runs and replicates Total SITE 3 5 Correct Incorrect Correct Incorrect Correct Incorrect Correct Incorrect ANA N NA N N N a N Category Concentration TN Blank 30 100 0 0 0 13011000 01 o 13011000101 0 301100 0 01 0 HN 75CFUmL 10 33 3 20 66 7 16 53 3 141467 14 407 16 53 3 40 44 4 50 556 LP ry 30 100 0 0 o 1281933 2167 29 9671 11 33 87 96 7131 33 MP ai 30 100 0 0 o 30 100 0 0 o 129 96714133 89 98911 1 1 Table 21 Salmonella Site to Site Quantitative Reproducibility across sites days runs and within run Within Run Between Run Between Day Within Day Within Day Within Site Between Site Total Variable Category N Mean SD CV SD CV SD CV SD CV SD CV HN 50 36 4 0 92 2 5 0 00 0 0 0 00 0 0 043 1 2 1 01 2 8 Ct Score LP 87 346 0 99 2 9 0 00 0 0 0 00 0 0 0 61 18 1 16 3 4 MP 89 33 2 0 61 1 9 0 34 1 0 0 23 0 7 043 13 0 85 2 6 N N N N TN Blank 130 100 0 0 o 30 10001 01 o 30 1000 01 o 90 1000 01 0 HN 9CFUmL 12 40 0 18 60 0 13 43 3 17 567 12 40 0 18 600 371 41 1 53 589 LP Bis 29 96 7 1 33 30 1000 0 o 29 96711 33 881 978 2 22 mp 228455 Jay2. LoD CFU mL in stool 95 Cl 14 250 10 500 19 200 Campylobacter jejuni ATCC 43429 LoD CFU mL in SBT 95 CI 42 36 49 8 250 6 150 11 400 10 9 10 LoD CFU mL in stool 95 Cl 6 300 5 400 7 350 1 500 1 350 1 500 LoD CFU mL in SBT 95 CI 374 249 561 229 151 347 LoD CFU mL in stool 95 Cl 56 100 37 350 84 150 34 350 22 650 52 050 LoD CFU mL in SBT 95 CI 84 59 118 LoD CFU mL in stool 95 CI 12 600 8 850 17 700 18 600 10 050 34 350 LoD CFU mL in SBT 95 CI 255 195 332 LoD CFU mL in stool 95 Cl 38 202 29 259 49 865 33 495 25 026 44 817 LoD CFU mL in SBT 95 CI 910 550 1 505 LoD CFU mL in stool 95 Cl 136 500 82 500 225 750 97 950 57 600 166 650 LoD CFU mL in SBT 95 CI 722 519 1006 LoD CFU mL in stool 95 CI 108 300 77 850 150 900 89 850 43 650 184 650 Shigella flexneri ATCC 700930 Shigella sonnei ATCC 10523 124 67 229 E coli stx1 ATCC 43890 223 167 299 E coli stx1 stx2 BD ENF 10513 653 384 1111 E coli stx2 ATCC 43889 599 291 1231 Analytical Specificity The BD MAX Enteric Bacterial Panel was performed on samples containing phylogenetically related species and other organisms bacteria viruses parasites and yeast likely to be found in stool specimens Nine 9 out of 9 Campy
3. 4o9 0 0 o 130100010 o 30 1000 0 o 90 1000101 0 x LoD 20 Table 23 Shigella Site to Site Quantitative Reproducibility across sites days runs and within run Within Run Between Run Between Day Within Day Within Day Within Site Between Site Variable Category N Mean SD CV SD CV SD CV SD CV HN 53 34 8 0 99 2 8 0 57 1 6 0 52 1 5 0 29 0 8 1 29 3 7 Ct Score LP 88 33 1 0 79 2 4 0 35 1 1 0 23 0 7 0 47 1 4 1 01 3 1 MP 90 32 5 0 80 2 5 0 39 1 2 0 00 0 0 0 50 1 5 1 03 3 2 SITE 3 5 Correct Incorrect Correct Incorrect Correct Incorrect Correct Incorrect Total Category Concentration TN Blank 30 100 0 0 0 13011000101 0 13011000101 0 90 1000101 0 HN 400CFUMmL 16 53 3 14 467 15 50 0 15 500 14 467 116 533 1 45 500 45 500 LP eis 30 100 0 0 o 30 100 0 0 o 30100010 0 90 1000 0 0 MP it 30 100 0 0 o 30 1000 0 o 29 967 1 33 89 989 1 414 Table 25 Shiga toxin Site to Site Quantitative Reproducibility across sites days runs and within run Within Run Between Run Between Day Within Day Within Day Within Site Between Site Variable Category N Mean SD CV SD CV SD CV HN 45 35 9 1 78 5 0 0 00 0 0 0 00
4. Negative POS Positive or UNR Unresolved based on the amplification status of the target and of the SPC IND Indeterminate or INC Incomplete results are due to BD MAX System failure In the case of a partial UNR where one or more targets have a POS result and all other targets have a UNR result no targets will be called NEG Table 1 BD MAX Enteric Bacterial Panel Result Interpretation ASSAY RESULT REPORTED INTERPRETATION OF RESULT Shig POS Shigella spp EIEC DNA Detected 2 Shig NEG No Shigella spp EIEC DNA Detected STX POS Shiga toxin producing gene s Detected 3 STX NEG No Shiga toxin producing gene s Detected Campy POS Campylobacter spp jejuni or coli DNA Detected Campy NEG No Campylobacter spp jejuni and coli DNA Detected Salm POS Salmonella spp DNA Detected Salm NEG No Salmonella spp DNA Detected UNR Unresolved inhibitory sample or reagent failure no SPC amplification IND Indeterminate due to BD MAX System failure with Warning or Error Codes INC Incomplete run with Warning or Error Codes 1 Analytical studies have demonstrated that certain strains of Shigella dysenteriae may harbor both the jpaH and stx BD MAX Enteric Bacterial Panel targets Additionally there have been literature reports of S boydii strains presenting with both jpaH and stx On rare occasions it may be possible that more than one BD MAX Enteric Bacterial Panel target is positive from a singl
5. OR at 25 2 C for a maximum of 48 h after the sample has been added to the Sample Buffer Tubes When an Indeterminate IND Unresolved UNR or Incomplete INC result is obtained or when an External Control failure occurs a repeat test from the prepared Sample Buffer Tube must be performed within this timeframe see the 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 or federal regulations or accreditation organizations For general QC guidance the user may wish to refer to CLSI MM03 and C24 1415 The procedure described below may be employed if appropriate based on local policies and procedures t External Positive and Negative Controls are not used by the BD MAX System software for the purpose of sample test result interpretation External Controls are treated as if they were patient samples Refer to Table 1 for the interpretation of External Control assay results One 1 External Positive Control and one 1 External Negative Control should be run at least daily until adequate process validation is achieved on the BD MAX System in each laboratory setting Reduced frequency of control testing should be in accordance with applicable regulations The External Positive Control is intended to monitor for su
6. 12 tubes The panels used were the same as described under the Precision heading above Each site was asked to perform the study on five 5 distinct days consecutive or not wherein each day two 2 panels were tested one 1 for each of two 2 technologists The overall Site to Site Reproducibility percent agreement was 100 for the TN category for all targets and ranged from 41 1 to 77 8 96 7 to 100 and 98 9 to 100 for the HN LP and MP categories respectively Table 17 The qualitative and quantitative reproducibility across sites and by target is presented below in Tables 18 through 25 Ct Score is an internal criterion used to determine final assay results and was selected as an additional means of assessing assay reproducibility Overall mean Ct Score values with variance components SD and CV are shown in Tables 19 21 23 and 25 Table 17 Site to Site Reproducibility Study Results using one lot of the BD MAX Enteric Bacterial Panel Campylobacter Shiga toxins s s Salmonella spp Shigella spp Category coli and jejuni stx1 and stx2 n 95 Cl Lusan ie Ibs ee n 95 Cl i 100 0 90 90 100 0 90 90 100 0 90 90 100 0 90 90 95 9 100 0 95 9 100 0 95 9 100 0 95 9 100 0 77 8 70 90 44 4 40 90 41 1 37 90 50 0 45 90 HN 68 2 85 1 34 6 54 7 31 5 51 4 39 9 60 1 r 100 0 90 90 96 7 87 90 97 8 88 90 100 0 90 90 9
7. HN categories the expected assay result was deemed to be negative Therefore percent agreement was calculated for negative results 21 Carryover Cross Contamination A study was conducted to investigate within run carryover and between run carryover while processing specimens with high bacterial loads of Salmonella enterica Shigella sonnei Campylobacter jejuni and Shiga toxin producing Escherichia coli in the BD MAX Enteric Bacterial Panel A panel made of one high positive member containing the four target organisms and one negative member was used to prepare numerous samples Strains of Salmonella enterica SpaO ATCC 13076 Shigella sonnei jpaH ATCC 10523 Campylobacter jejuni tuf ATCC 29428 and Shiga toxin producing Escherichia coli stx1 and stx2 ENF 10513 were used for the high positive panel member 1 x 106 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 One 1 operator performed 16 consecutive runs with 15 runs containing 24 samples and 1 run containing 4 samples Carryover contamination was assessed for each target in the BD MAX Enteric Bacterial Panel A total of 167 SBTs each containing the 4 BD MAX Enteric Bacterial Panel targets were assessed in the carryover contamination study Of the 668 readings
8. J Syst Evol Microbiol 55 2013 25 18 CDC National Salmonella Surveillance Annual Summary 2009 Located at http www cdc gov ncezid dfwed edeb reports htm 1 ial Manufacturer Contains sufficient for lt n gt tests Use by YYYY MM DD YYYY MM MM end of month Cli Consult Instructions for Use Catalog number o s A Keep Dry Authorized Representative in the European Community Q Keep away from light In Vitro Diagnostic Medical Device Temperature limitation Perforation LOT Batch Code Lot oy ii AA 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 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 BD Technical Information In the United States contact BD Technical Service and Support at 1 800 638 8663 or www bd com ds ml GeneOhm Sciences Canada Inc 2555 Boul du Parc Technologique Qu bec QC G1P 4S5 Canada Made in Canada ATCC is a trademark of the American Type Culture Collection TaqMan is a registered trademark of Life Technologies Corporation Nalgene is a registered trademark of ThermoFisher Scientific BD BD Logo and
9. Ott A Keszty s B and AM Kooistra Smid 2010 Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach J Clin Microbiol 48 4140 6 Clinical and Laboratory Standards Institute 2005 Approved Guideline M29 A3 Protection of laboratory workers from occupationally acquired infections 3rd ed CLSI Wayne PA 12 Centers for Disease Control and Prevention 1993 Biosafety in microbiological and biomedical laboratories Richmond JY and RW McKinney eds HHS Publication number CDC 93 8395 13 BD MAX System User s Manual US IVD Version BD Diagnostics Sparks MD USA 14 Clinical and Laboratory Standards Institute 2006 Approved Guideline MM32 AZ Molecular Diagnostic Methods for Infectious Diseases 2nd ed CLSI Wayne PA 15 Clinical and Laboratory Standards Institute 2006 Approved Guideline C24 A3 Statistical Quality Control for Quantitative Measurements Principles and Definitions 3rd ed CLSI Wayne PA 16 Jiali Ochman H Groisman EA Boyd EF Solomon F Nelson K AND Selander RK 1995 Relationship between evolutionary rate and cellular location among the Inv Spa invasion proteins of Salmonella enterica Proc Nat Acad Sci USA 92 16 7252 6 17 Paradis S Boissinot M Paquette N Belanger SD Martel EA Boudreau DK Picard FJ Ouellette M Roy PH Bergeron MG 2005 Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F ATPase beta subunit Int
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11. and 98 and 100 of the retrospective positive and negative specimens respectively For the unpreserved specimen type the BD MAX Enteric Bacterial Panel identified 100 and 99 4 of the Shigella spp EIEC organisms prospective positive and negative specimens respectively and 100 and 100 of the retrospective positive and negative specimens respectively Table 5 Table 5 Shigella spp EIEC Overall Performance Specimen RM Specimen Type 5 rigin BD MAX p N Total l P 19 5 24 Cary Blair ing N 0 1804 1804 Total 19 1809 1828 PPA 95 Cl 100 83 2 100 NPA 95 Cl 99 7 99 4 99 9 P 50 0 50 Cary Blair B N r 187 188 Total 51 187 238 PPA 95 CI 98 89 7 99 7 NPA 95 CI 100 98 100 P 22 7 29 Unpreserved lea N 0 1212 1212 Total 22 1219 1241 PPA 95 Cl 100 85 1 100 NPA 95 Cl 99 4 98 8 99 7 P 41 0 41 Unpreserved gg a N 0 264 264 Total 41 264 305 PPA 95 CI 100 91 4 100 NPA 95 CI 100 98 6 100 1These five 5 specimens were also tested using an alternate PCR assay followed by bi directional sequencing all five 5 specimens gave a positive result 2 These seven 7 specimens were also tested using an alternate PCR assay followed by bi directional sequencing six 6 of seven 7 gave a positive result For the Cary Blair preserved specimen type the BD MAX Enteric Bacterial Panel identified 75 and 9
12. automated procedures occur The bacterial cells are lysed DNA is extracted on magnetic beads and concentrated and then an aliquot of the eluted DNA is added to PCR reagents which contain the target specific primers used to amplify the genetic targets if present The assay also includes a Sample Processing Control SPC The SPC is present in the Extraction Tube and undergoes the extraction concentration and amplification steps to monitor for inhibitory substances instrument or reagent failure No operator intervention is necessary once the clinical sample and reagent strip are loaded into the BD MAX System The BD MAX System automates sample lysis DNA extraction and concentration reagent rehydration nucleic acid amplification and detection of the target nucleic acid sequence using real time polymerase chain reaction PCR Amplified targets are detected with hydrolysis probes labeled with quenched fluorophores The amplification detection and interpretation of the signals are done automatically by the BD MAX System PRINCIPLES OF THE PROCEDURE Stool specimens are collected from patients and transported to the laboratory unpreserved in a clean container or preserved in Cary Blair transport media A 10 uL loop is inserted to the depth of the loop into the specimen and expressed via a swirling motion into a BD MAX Sample Buffer Tube SBT The SBT is closed with a septum cap and vortexed Once the work list is generated and the clinical samp
13. event that sample associated inhibition or reagent failure prevents proper target or SPC amplification If the SPC does not amplify the sample will be reported as UNR however any positive POS assay results will be reported and no targets will be called NEG The BD MAX System reports results for each target individually and a UNR result may be obtained for one or more BD MAX Enteric Bacterial Panel targets In the case of a complete UNR where all targets have a UNR result it is necessary to repeat the test In the case of a partial UNR when one or more targets have a POS result and all other targets have a UNR result it is recommended that the test be repeated as described above In rare cases discrepant results may be observed when a repeat test is run for those targets that were initially reported as POS Follow appropriate procedures in accordance with current laboratory procedures Sample s can be repeated from their corresponding SBT s within the timeframes defined above Vortex the sample s for one 1 min and restart from the BD MAX System Operation section The remaining stool sample may also be used for repeat testing with a new SBT within the timeframes defined above Restart from the Sample 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 SBT s within the timeframe defined above Vortex the sample s for
14. followed by bi directional sequencing and gave a negative result Table 10 below shows the co infections detected by the BD MAX Enteric Bacterial Panel during the prospective segment of the clinical trial Note that there were no co infections detected by the reference method during the prospective segment of the clinical trial Table 10 Co infections observed during the BD MAX Enteric Bacterial Panel prospective clinical trial Distinct Co infection Combinations Detected by Number of BD MAX Enteric Bacterial Assay Discrepant Discrepant Analyte s Analyte 1 Analyte 2 Co Infections Shigella stx 1 stx stx Campylobacter 1 stx stx Salmonella 2 stx 2 and Salmonella 1 4 Campylobacter Salmonella 2 Campylobacter 2 Salmonella 1 5 1 A discrepant co infection or discrepant analyte was defined as one that was detected by the BD MAX assay but not detected by the reference method 2 One 1 discrepant six was investigated using an alternate method bi directional sequence analysis identified the analyte in 0 1 cases 3 One 1 discrepant stx was investigated using an alternate method bi directional sequence analysis identified the analyte in 1 1 cases 4 Two 2 discrepant stx were investigated using an alternate method bi directional sequence analysis identified the analyte in 0 2 cases One 1 discrepant Salmonella was investigated using an alternate method bi directional sequence analysis identified the analyte in 1
15. 0 0 1 03 2 9 2 06 5 7 Ct Score LP 90 31 8 0 65 2 0 0 00 0 0 0 00 0 0 0 36 1 1 0 74 2 3 MP 89 31 3 0 62 2 0 0 22 0 7 0 07 0 2 0 24 0 8 0 70 2 2 For the Lot to Lot reproducibility study two users each completed a single run of 12 panel members on a single instrument for each of two lots of reagents over a 5 day period The panels used were the same as described under the Precision heading above Results from 5 days of the accuracy and precision study were used to comprise data for one lot of reagents for the Lot to Lot study The overall Lot to Lot reproducibility percent agreement was 100 for the TN category for all targets and ranged from 13 33 to 62 22 95 56 to 100 and 97 78 to 100 for the HN LP and MP categories respectively Table 26 Table 26 Lot to Lot Reproducibility Study Results using three lots of the BD MAX Enteric Bacterial Panel 95 Cl LowerCl UpperCl 100 00 95 91 100 00 30 00 21 51 40 13 98 89 93 97 99 80 100 00 95 91 100 00 100 00 95 91 100 00 62 22 51 90 71 54 100 00 95 91 100 00 Level Correct Total Correct 97 78 92 26 99 39 100 00 95 91 100 00 16 67 10 37 25 69 95 56 89 12 98 26 98 89 93 97 99 80 100 00 95 91 100 00 13 33 7 19 21 87 98 89 93 97 99 80 100 00 95 91 100 00 For the True Negative TN and High Negative
16. 1 cases 5 Two 2 discrepant Campylobacter were investigated using an alternate method bi directional sequence analysis identified the analyte in 0 2 cases One 1 discrepant Salmonella was investigated using an alternate method bi directional sequence analysis identified the analyte in 0 1 cases Of the 3183 prospective specimens initially evaluated with the BD MAX Enteric Bacterial Panel 4 0 of the Cary Blair preserved and 7 8 of the unpreserved specimens initially reported as Unresolved Following a valid repeat test 0 1 of the Cary Blair preserved and 1 0 of the unpreserved specimens remained Unresolved Of the 783 retrospective specimens initially evaluated with the BD MAX Enteric Bacterial Panel 2 2 of the Cary Blair preserved and 4 1 of the unpreserved specimens initially reported as Unresolved Following a valid repeat test 0 2 of the Cary Blair preserved and 0 6 of the unpreserved specimens remained Unresolved Table 11 The total numbers provided in Table 11 are based on compliant specimens and BD MAX Enteric Bacterial Panel results Table 11 Unresolved Rates Initial Unresolved Rates Unresolved Rates After Repeat Specimen Type Specimen Origin Percent 95 Cl Percent 95 Cl gp 4 0 77 1905 3 2 5 0 0 1 2 1897 0 0 0 4 Cary Blair py ict 2 2 10 464 1 2 3 9 0 2 1 463 0 0 1 2 Gai 7 8 100 1278 6 5 9 4 1 0 13 1251 0 6 1 8
17. 5 9 100 0 90 7 98 9 92 3 99 4 95 9 100 0 i 100 0 90 90 98 9 89 90 100 0 90 90 98 9 89 90 95 9 100 0 94 0 99 8 95 9 100 0 94 0 99 8 For the True Negative TN and High Negative HN categories the expected assay result was deemed to be negative Therefore percent agreement was calculated for negative results Table 18 Campylobacter Site to Site Qualitative Reproducibility across sites with pooled days runs and replicates SITE Total 2 3 5 Category Concentration Correct Incorrect Correct Incorrect Correct Incorrect Correct Incorrect N N N N TN Blank 30 100 0 0 0 30 100 0 0 o 30 100 0 0 o 90 100 0 0 0 HN 5CFU mL 22 73 3 8 26 7 24 80 0 6 20 0 124 80 0 6 20 0 70 77 8 20 222 LP n 301100 0 0 o 30 100 0 0 o 30 1000 0 o 90 1000 0 0 MP es 30 100010 o 30 1000 0 o 30 1000 0 o 9011000 0 0 Table 19 Campylobacter Site to Site Quantitative Reproducibility across sites days runs and within run Da Da e Betwee o ota able atego ea D D D D D HN 20 36 2 0 54 1 5 1 18 3 2 0 00 0 0 0 00 0 0 1 30 3 6 Ct Score LP 90 32 7 0 49 15 0 28 0 9 0 00 0 0 0 00 0 0 0 57 1 7 MP 90 32 2 0 60 18 0 14 0 4 0 00 0 0 0 00 0 0 0 61 1 9
18. 8 24 h in ambient conditions For Campylobacter jejuni inoculate onto Brucella Agar with 5 Sheep Blood Hemin and Vitamin K4 Incubate 2 3 days in a microaerophilic environment or until there is sufficient growth to prepare the McFarland dilution All plates must be prepared fresh daily For preparation of the External Positive Control re suspend individual isolates in saline solution to a turbidity of 0 5 McFarland 1 X 108 CFU mL Perform serial dilutions with saline to obtain a suspension of 1 0 X 108 CFU mL for Salmonella spp Shigella spp or E coli organisms or 1 0 X 105 CFU mL for Campylobacter spp Inoculate a SBT with a 10 uL loop of the bacterial suspension Process and test as a sample refer to the Sample Preparation and BD MAX System Operation sections All external controls should yield the expected results positive for External Positive Control negative for External Negative Control and no failed external controls Unresolved or Indeterminate results Alternate culture storage conditions should be validated by individual laboratories as appropriate An External Negative Control that yields a positive test result is indicative of a sample handling and or contamination problem Review the sample handling technique to avoid mix up and or contamination An External Positive Control that yields a negative result is indicative of a sample handling preparation problem Review the sample handling preparation technique A
19. 9 3 of the Shiga toxins stx1 stx2 prospective positive and negative specimens respectively and 100 and 100 of the retrospective positive and negative specimens respectively For the unpreserved specimen type the BD MAX Enteric Bacterial Panel identified 100 and 99 of the Shiga toxins stx1 and or stx2 prospective positive and negative specimens respectively and 100 and 100 of the retrospective positive and negative specimens respectively Table 6 Table 6 Shiga toxins stx1 stx2 Overall Performance Specimen RM Specimen Type T BD MAX p N Total i P 6 132 19 Cary Blair a N 21 1768 1770 Total 8 1781 1789 PPA 95 Cl 75 40 9 92 9 NPA 95 Cl 99 3 98 8 99 6 P 41 0 41 Cary Blair a N 0 79 79 Total 41 79 120 PPA 95 Cl 100 91 4 100 NPA 95 Cl 100 95 4 100 P 2 78 9 Unpreserved pri N 0 704 704 Total 2 711 713 PPA 95 CI 100 34 2 100 NPA 95 CI 99 98 99 5 P 25 0 25 Unpreserved sa ceca N 0 11 11 Total 25 11 36 PPA 95 Cl 100 86 7 100 NPA 95 Cl 100 74 1 100 1 These two 2 specimens were also tested using an alternate PCR assay followed by bi directional sequencing and gave a negative result 2 These thirteen 13 specimens were also tested using an alternate PCR assay followed by bi directional sequencing seven 7 of thirteen 13 gave a positive result 3 These seven 7 spe
20. EFERENCES 1 CDC Estimates of Foodborne Illness in the United States Located at http www cdc gov foodborneburden 201 1 foodborne estimates html 2 Kosek et al Bulletin of the World Health Organization 2003 87 197 204 NIH Bacterial Gastroenteritis Located at http www nlm nih gov medlineplus ency article 000254 htm 4 Petri WA Miller M Binder HJ Levine MM Dillingham R and RL Guerrant 2008 Enteric infections diarrhea and their impact on function and development J Clin Invest 118 1277 1290 5 Wong CS Jelacic S Habeeb RL Watkins SL and PI Tarr 2000 The risk of the hemolytic uremic syndrome after antibiotic treatment of Escherichia coli 0157 H7 infections N Engl J Med 342 1930 1936 6 CDC Campylobacter General Information Located at http www cdc gov nczved divisions dfomd diseases campylobacter 7 CDC What is Salmonellosis Located at http www cdc gov salmonella general index html 8 Grys TE Sloan LM Rosenblatt JE and R Patel 2009 Rapid and sensitive detection of Shiga toxin producing Escherichia coli from nonenriched stool specimens by real time PCR in comparison to enzyme immunoassay and culture J Clin Microbiol 47 2008 12 o9 9 Cunningham SA Sloan LM Nyre LM Vetter EA Mandrekar J and R Patel 2010 Three hour molecular detection of Campylobacter Salmonella Yersinia and Shigella species in feces with accuracy as high as that of culture J Clin Microbiol 48 2929 33 10 de Boer RF
21. ROCEDURE e This product can only be used on the BD MAX System e This product is intended for use only with unpreserved and Cary Blair preserved human stool samples Stool samples from rectal swabs or fixed stools have not been validated with the BD MAX Bacterial Panel e Erroneous results may occur from improper sample collection handling storage technical error sample mix up or because the number of organisms in the sample is below the analytical sensitivity of the test e If the BD MAX Enteric Bacterial Panel result is IND INC or UNR for one or more targets then the test should be repeated ABD MAX Enteric Bacterial Panel positive result does not necessarily indicate the presence of viable organisms It does however indicate the presence of the Campylobacter specific tuf gene sequence variants SpaO ipaH and stx1 stx2 genes and allows for identification of the Enteric Bacterial Panel organisms e Mutations or polymorphisms in primer or probe binding regions may affect detection of the genera Salmonella and Campylobacter jejuni and coli Shigella spp Enteroinvasive E coli EIEC as well as Shiga toxin producing E coli variants resulting in a false negative result with the BD MAX Enteric Bacterial Panel The BD MAX Enteric Bacterial Panel does not distinguish which Shiga toxin gene stx1 stx2 is present in a specimen e In rare instances Shiga toxin genes can be found in Enterobacteriaceae other than STEC or
22. Revisions BALTSO0191 Version 7 0 Template 4 NOTES 1 BD Catalog Number 443378 Blank Sheet Size Length 13 Width 8 5 Number of Pages 24 Number of Sheets 6 Page Size Length 13 Width 8 5 Final Folded Size 11 x 5 5 Ink Colors No of Colors 2 PMS 2755 Standard Black A Printed two sides Yes No booklet style on Style see illustrations below 5 11 x 8 5 paper ne L OO HEADER HEADER HEADER 1 2 3 gt aa HEADER DER a ac 7 8 See specification control no N A for material information 9 Graphics are approved by Becton Dickinson and Company Supplier has the responsibility for using the most current approved revision level Label Label Design ate Date COMPANY CONFIDENTAL THIS DOCUMENT IS Ne BD Becton Dickinson and Company THE PROPERTY OF BECTON DICKINSON AND Procter Date COMPANY AND IS NOT TO BE USED OUTSIDE 7 Loveton Circle THE COMPANY WITHOUT WRITTEN PERMISSION Sparks MD 21152 USA Checked By Date N A Category and Description Sheet 1 of 25 Package Insert Part Number A P0166 BD MAX Enteric Bacterial Panel US Kit Scale N A GBD MAX Enteric Bacterial Pandks BD MAX Enteric Bacterial Panel 443378 For In Vitro Diagnostic Use P0166 01 For use with the BD MAX System 2014 05 25 C A Rx Only INTENDED USE The BD MAX Enteric Bacterial Panel performed on the BD MAX System is an automated in vitro diagnostic tes
23. Shigella dysenterieae The BD MAX Enteric Bacterial Panel detects only Campylobacter jejuni and Campylobacter coli and does not differentiate between the species Other Campylobacter species are not detected by the assay e In silico analysis predicts that variant stx2f will not be detected by the BD MAX Enteric Bacterial Panel e The BD MAX Enteric Bacterial panel does not differentiate between Shigella spp and Enteroinvasive Escherichia coli EIEC e Not all serotypes of Salmonella were evaluated in analytical studies however all but one Salmonella enterica serotype Mississippi of the most prevalent serotypes recently circulating in the U S were evaluated 8 As with all PCR based in vitro diagnostic tests extremely low levels of target below the analytical sensitivity of the assay may be detected but results may not be reproducible e 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 SPC has been added to the test to aid in the identification of specimens that contain inhibitors to PCR amplification The SPC does not indicate if nucleic acid has been lost due to inadequate collection transport or storage of specimens or whether bacterial cells have been inadequately lysed e Results from the BD MAX Enteric Bacterial Panel should be used as an adjunct to clinical observations and other information available to the physici
24. Shigella sonnei and Campylobacter coli The following values were used as spike levels and tested in triplicate for the target organisms contained in each panel member e Moderate Positive MP 3x LOD e Low Positive LP 1 5x LOD e High Negative HN C20 39 LOD e True Negative TN No Target Each sample contained negative unpreserved stool matrix True Negative TN samples contained no target High Negative HN samples were spiked with target organisms below the analytical LOD of the assay however the HN samples were expected to yield a positive result in approximately 20 to 80 of the replicates due to the inherent sensitivity of PCR assays Results are summarized by target and concentration in Table 16 Table 16 Precision Study Results using one lot of the BD MAX Enteric Bacterial Panel Percent Agreement by Analyte Category E colistx1 Salmonella Shigella Campylobacter Expected Values TN 100 00 100 00 100 00 100 00 100 00 HN 27 18 25 00 30 56 54 17 20 to 80 LP 98 61 100 00 98 61 100 00 95 00 MP 100 00 100 00 98 61 98 61 100 00 1 For the True Negative TN and High Negative HN categories the expected assay result was deemed to be negative Therefore percent agreement was calculated for negative results Reproducibility For the Site to Site reproducibility study three 3 clinical sites were provided with a total of ten 10 panels each consisting of
25. UTIONS e State and local public health authorities have published guidelines for notification of reportable diseases in their jurisdictions including but not limited to Salmonella Shigella and Shiga toxin stx1 stx2 producing E coli STEC to determine necessary measures for verification of results to identify and trace outbreaks Laboratories are responsible for following their state or local regulations for submission of clinical material or isolates on positive specimens to their state public health laboratories The BD MAX Enteric Bacterial Panel is for in vitro Diagnostic Use This product can only be used on the BD MAX System 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 Close protective pouches of reagents promptly with the zip seal after each use Remove any excess air in the pouches prior to sealing Check reagent strips for proper liquid fills ensure that the liquids are at the bottom of the tubes see Figure 1 Check reagent strips to ensure that all pipette tips are present 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 tes
26. Unpreserved se cae 4 1 13 319 2 4 6 8 0 6 2 317 0 2 2 3 Of the 3183 prospective specimens initially evaluated with the BD MAX Enteric Bacterial Panel 1 7 of the Cary Blair preserved and 1 6 of the unpreserved specimens initially reported as Indeterminate Following a valid repeat test 0 of the Cary Blair preserved and 0 2 of the unpreserved specimens remained Indeterminate Of the 783 retrospective specimens initially evaluated with the BD MAX Enteric Bacterial Panel 1 5 of the Cary Blair preserved and 1 9 of the unpreserved specimens initially reported as Indeterminate Following a valid repeat test 0 of the Cary Blair preserved and 0 of the unpreserved specimens remained Indeterminate Table 12 The total numbers provided in Table 12 are based on compliant specimens and BD MAX Enteric Bacterial Panel results Table 12 Indeterminate Rates Initial Indeterminate Rates Final Indeterminate Rates After Repeat Specimen Type Specimen Origin Percent 95 Cl Percent 95 Cl to 1 7 33 1905 1 2 2 4 0 0 0 1897 0 0 0 2 Cary Blair Retrospective 4 5 7 464 0 7 3 1 0 0 01463 0 0 0 8 Frozen a 1 6 20 1278 1 0 2 4 0 2 2 1251 0 0 0 6 Unpreserved Retrospective 4 99 6 319 0 9 4 0 0 0 0 317 0 0 1 2 Frozen Of the 3183 prospective specimens initially evaluated with the BD MAX Enteric Bacterial Pane
27. aboratory procedures such as those described in the CLSI Document M2911 and in Biosafety in Microbiological and Biomedical Laboratories 12 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 Consult the BD MAX System User s Manual for additional warnings precautions and procedures STORAGE AND STABILITY Collected specimens either unpreserved stool or stool stored in 15 mL Cary Blair transport media should be kept between 2 C and 25 C during transport Protect against freezing or exposure to excessive heat Specimens can be stored for up to 5 days at 2 8 C or for up to 24 h at 2 25 C before testing BD MAX Enteric Bacterial Panel components are stable at 2 25 C through the stated expiration date Do not use expired components BD MAX Enteric Bacterial Panel 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 14 days at 2 25 C after initial opening and re sealing INSTRUCTIONS FOR USE Specimen Collection Transport In order to obtain an adequate sample the procedure for sample collection must be fol
28. across all targets one SBT was positive for all 4 panel targets Expected Values In the BD MAX Enteric Bacterial Panel clinical study reportable results from compliant specimens were obtained from 8 geographically diverse sites and compared to the reference methods The study population was grouped based on specimen type The number and percentage of positive cases by target as determined by the BD MAX Enteric Bacterial Panel during the prospective segment of the clinical trial are presented below in Table 27 Table 27 Prevalence Values Observed during the BD MAX Enteric Bacterial Panel Clinical Trial Prevalence Specimen Type Site Salmonella Shigella Campylobacter Shiga toxins 1 0 0 0 186 0 0 0 186 1 1 2 188 0 0 0 185 2 0 8 3 377 0 3 1 377 1 6 6 368 0 8 3 391 3 0 9 5 548 0 2 1 548 0 8 4 528 0 2 1 551 Cary Blair Preserved 4 3 9 6 152 11 2 17 152 2 0 3 152 0 0 0 135 5 0 3 1 339 0 0 0 339 1 5 5 340 0 3 1 320 6 1 4 6 431 0 0 0 431 1 9 8 431 0 7 3 411 Total 1 0 21 2033 0 9 19 2033 1 4 28 2007 0 4 8 1993 i 1 6 6 376 0 3 1 376 0 8 3 376 0 0 0 176 7 1 6 5 305 0 0 0 305 2 0 6 304 0 0 0 229 Unpreserved 8 1 4 4 284 0 0 0 284 1 1 3 284 0 4 1 265 4 2 9 9 314 6 7 21 314 3 5 11 314 0 4 1 266 Total 1 9 24 1279 1 7 22 1279 1 8 23 1278 0 2 2 936 R
29. an e As with all in vitro diagnostic tests positive and negative predictive values are highly dependent on prevalence The BD MAX Enteric Bacterial Panel performance may vary depending on the prevalence and population tested BD MAX Enteric Bacterial Panel results may or may not be affected by concurrent antimicrobial therapy which may reduce the amount of target present e The sample buffer tube has not been designed to support organism viability If culture is necessary it must be performed from the original specimen The performance of this test has not been established for monitoring treatment of Salmonella spp Shigella spp C jejuni C coli or STEC infections e This test is a qualitative test and does not provide quantitative values nor indicate the quantity of organisms present e The performance of this test has not been evaluated for immunocompromised individuals or for patients without symptoms of gastrointestinal infection e The effect of interfering substances has only been evaluated for those listed in this labeling Potential interference has not been evaluated for substances other than those described in the Interference section below Cross reactivity with organisms other than those listed in the Analytical Specificity section below have not been evaluated PERFORMANCE CHARACTERISTICS Performance characteristics of the BD MAX Enteric Bacterial Panel were determined in a multi site investigational st
30. bstantial reagent failure The External Negative Control is used to detect reagent or environmental contamination or carry over by target nucleic acids Control strains should be tested according to guidelines or requirements of local state provincial and or federal regulations or accreditation organizations in order to monitor the effectiveness of the entire analytical process Various types of external controls are recommended to allow the user to select the most appropriate for their laboratory quality control program Suspension s of QC bacterial strains from commercially available sources such as the ATCC can be used for QC purposes The External Control strains listed below or previously characterized samples known to be positive or negative for the gene targets found in the BD MAX Enteric Bacterial Panel can be used a External Positive Control prepare a suspension of the following ATCC strains e Salmonella enterica subsp enteric serovar Typhimurium ATCC 14028 containing the SpaO gene target e Shigella sonnei ATCC 9290 containing the ipaH gene target e E coli stx1 ATCC 43890 containing the stx1 gene target e Campylobacter jejuni subsp jejuni ATCC 33291 containing the Campylobacter specific tuf gene sequence variants b External Negative Control Express a 10 uL loop of saline in the SBT For Salmonella Shigella and E coli inoculate organisms onto Trypticase Soy Agar with 5 Sheep Blood plates Incubate 1
31. cimens were also tested using an alternate PCR assay followed by bi directional sequencing three 3 of seven 7 gave a positive result Performance of the BD MAX Enteric Bacterial Panel by species toxin type as observed during the clinical trial is presented below in Tables 7 through 9 The species identification was obtained either from the culture and identification portion of the reference method testing or from sequencing performed for the confirmation of retrospective specimen historical results and on discrepant prospective specimens While the BD MAX Enteric Bacterial Panel is designed to detect the species and toxin types described below the panel does not report results to the species or toxin level Table 7 Campylobacter performance per species observed during the clinical trial Campylobacter PPA 95 Cl Specimen Type Specimen Origin Species Estimate Pi spect jejuni 95 8 23 24 79 8 99 3 Cary Blair Fresh Untyped 100 0 2 2 34 2 100 0 Preserved Retospeciye coli 100 0 2 2 34 2 100 0 Frozen jejuni 96 9 62 64 89 3 99 1 jejuni 100 0 19 19 83 2 100 0 ie jejuni or coli 100 0 1 4 20 7 100 0 Unpreserved Untyped 100 0 2 2 34 2 100 0 Reliospedte coli 100 0 5 5 56 6 100 0 Frozen jejuni 96 8 60 62 89 0 99 1 1 Of these specimens one 1 prospective specimen was also tested usin
32. e organism that harbors two or more genes detected by the assay The presence of more than one positive BD MAX Enteric Bacterial Panel target may also be indicative of a dual infection 2A positive BD MAX Enteric Bacterial Panel result for Shigella spp may be indicative of the presence of Shigella spp or Enteroinvasive E coli DNA 3A positive BD MAX Enteric Bacterial Panel result for Shiga toxin stx1 or 2 may be indicative of the presence of Shiga toxin producing E coli Shigella dysenteriae or other Enterobacteriaceae that rarely carry Shiga toxin genes BD MAX Enteric Bacterial Panel results may be used to guide the level of precautions in accordance with institutional programs and practices Refer to the Troubleshooting section of the BD MAX System User s Manual for interpretation of warning and error codes REPEAT TEST PROCEDURE NOTE Due to available sample volume one repeat test may be performed on the BD MAX System from the SBT For SBTs stored at room temperature retesting must be performed within 48 h following the initial SBT inoculation with the sample Alternatively for SBTs stored at 2 8 C retesting must be performed within 120 h 5 days The remaining stool sample may also be used for repeat testing within 5 days of collection if stored at 2 8 C or within 24 h if stored at 2 25 C NOTE New samples may be tested in the same run with repeat samples Unresolved Result Unresolved results may be obtained in the
33. e sole or definitive cause of patient illness Negative results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non infectious causes such as ulcerative colitis irritable bowel syndrome or Crohn s disease SUMMARY AND EXPLANATION OF THE PROCEDURE Organisms that cause enteric diseases represent a significant cause of morbidity and mortality worldwide Enteric infections enter the body through the gastrointestinal tract and typically are spread via contaminated food and water or contact with vomit or feces CDC estimates there are 48 million cases of foodborne illness in the United States each year resulting in 128 000 hospitalization and 3 000 deaths In the developing world these illnesses cause approximately 2 million deaths annually in young children 2 Each of the causative agents may result in slightly different symptomology including abdominal cramps or pain loss of appetite nausea or vomiting however all result in diarrhea Repeated bouts of diarrhea and persistent diarrheal disease disrupt intestinal function and absorption potentially leading to childhood malnutrition and growth retardation Although the most common gram negative enteric bacterial agents are easily cultivated on standard selective and differential media with toxin detection by antibody mediated lateral flow isolation and identification are time consuming Diagnosis may take s
34. everal days which places patients at risk for an untreated infection as well as the spread of the infection to others Alternatively empirical antimicrobial therapy may have severe consequences for some enteric bacterial infections such as those caused by Shiga toxin producing E coli STEC potentially leading to fatal complications known as hemolytic uremic syndrome in children In persons with compromised immune systems Campylobacter and Salmonella infections occasionally spread to the bloodstream and cause a serious life threatening infection Oo P0166 01 The BD MAX Enteric Bacterial Panel procedure can be performed in approximately 3 hours as compared to traditional culture methods which can take 48 to 96 hours The BD MAX Enteric Bacterial Panel simultaneously detects the pathogens responsible for gastreoenteritis due to Salmonella spp Campylobacter spp jejuni and colli Shigella spp EIEC and stx1 stx2 found in Shiga toxin producing E coli The assay includes an internal Sample Processing Control SPC The BD MAX Enteric Bacterial Panel automates the testing process and minimizes operator intervention from the time the sample is placed onto the BD MAX System until results are available A soft to diarrheal stool is collected and transported to the laboratory homogenized and looped into a BD MAX Enteric Bacterial Panel Sample Buffer Tube SBT The Sample Buffer Tube is placed into the BD MAX System and the following
35. g a validated PCR assay followed by bi directional sequencing and gave a negative result Table 8 Shigella performance per species type observed during the clinical trial Shigella Specimen Type Specimen Origin Species Estimate 95 Cl Prospective flexneri 100 0 1 1 20 7 100 0 Cary Blair Fresh sonnei 100 0 18 18 82 4 100 0 Preserved Ret i e sonnei 98 0 50 51 89 7 99 7 Prospective flexneri 100 0 2 2 34 2 100 0 Fresh sonnei 100 0 20 20 83 9 100 0 Unpreserved Retrospective flexneri 100 0 1 1 20 7 100 0 Frozen sonnei 100 0 40 40 91 2 100 0 Table 9 Shiga toxins performance per toxin type observed during the clinical trial Shiga toxins PPA Specimen Type Specimen Origin Toxin Type Estimate 95 Cl std 100 0 4 4 51 0 100 0 ae stx2 100 0 1 1 20 7 100 0 Cary Blair stx1 and stx2 33 3 1 3 6 1 79 2 Preserved stx 100 0 28 28 87 9 100 0 ae stx2 100 0 6 6 61 0 100 0 stx1 and stx2 100 0 7 7 64 6 100 0 paepealia std 100 0 1 1 20 7 100 0 Fresh std and stx2 100 0 1 1 20 7 100 0 Unpreserved stx1 100 0 5 5 56 6 100 0 ei stx2 100 0 6 6 61 0 100 0 stx1 and stx2 100 0 14 14 78 5 100 0 1 Two 2 prospective specimens were also tested using a validated PCR assay
36. he other end with a quencher moiety Probes labeled with different fluorophores are used to detect amplicons for enteric bacterial targets Campylobacter specific tuf gene sequence variants the SpaO gene for specific detection of Salmonella spp the jpaH gene for specific detection of Shigella spp or Enteroinvasive E coli EIEC the stx1 amp stx2 genes associated with production of Shiga toxins in STEC and S dysenteriae and the SPC in five 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 BD MAX System monitors these signals at each cycle and interprets the data at the end of the program to report the final results REAGENTS AND MATERIALS REF Contents Quantity BD MAX Enteric Bacterial Panel Master Mix B5 Oven dried PCR Master Mix containing TaqMan specific molecular probe and primers along with Sample Processing Control specific TaqMan probe and primers BD MAX Enteric Bacterial Panel Reagent Strips Unitized reagent strip containing all the liquid reagents and disposab
37. ixture which consisted of a combination of 8 different antibiotics tested simultaneously with each antibiotic at a concentration that may be excreted in a stool sample Vagisil was identified as a potentially interfering substance at a concentration of 9 2 Vagisil in a stool sample or 0 92 mg mL of SBT Nystatin cream and spermicidal lubricant both demonstrated potential interference at a concentration of 50 5 0 mg mL of interferent in the SBT The BD MAX Enteric Bacterial Panel demonstrated acceptable performance with nystatin cream at a concentration of 31 3 1 mg mL of nystatin cream in the SBT and spermicidal lubricant at 34 3 4 mg mL of spermicidal lubricant in the SBT Results demonstrated no reportable interference with any other substance tested Table 15 Table 15 Endogenous and Commercial Exogenous Substances tested with the BD MAX Enteric Bacterial Panel Brand Name or Description Result Brand Name or Description Fecal Fat NI Spermicidal Lubricant P Human DNA NI Diaper Rash Cream NI Mucus NI Vagisil l Whole human blood NI Laxatives NI Hydrocortisone Cream NI Anti Diarrheal liquid NI Antiseptic Towelettes NI Anti Diarrheal pill NI Enema NI Antibiotics Mixture NI Hemorrhoidal Gel NI Antacids NI Nystatin Cream P Non Steroidal Anti Inflammatory NSAID NI Topical Antibiotic NI Interference with the BD MAX Enteric Bacterial Panel P Potential interference with the BD MAX Ente
38. l 1 3 of the Cary Blair preserved and 2 0 of the unpreserved specimens initially reported as Incomplete Following a valid repeat test 0 of the Cary Blair preserved and 0 of the unpreserved specimens remained Incomplete Of the 783 retrospective specimens initially evaluated with the BD MAX Enteric Bacterial Panel 1 3 of the Cary Blair preserved and 0 of the unpreserved specimens initially reported as Unresolved Following a valid repeat test 0 of the Cary Blair preserved specimens remained Incomplete Table 13 The total numbers provided in Table 13 are based on compliant specimens and BD MAX Enteric Bacterial Panel results Table 13 Incomplete Rates Initial Incomplete Rates Final Incomplete Rates After Repeat Specimen Type Specimen Origin Percent 95 Cl Percent 95 Cl pati 1 3 24 1905 0 8 1 9 0 0 0 1897 0 0 0 2 Cary Blair Retrospective 4 3 6 464 0 6 2 8 0 0 0 463 0 0 0 8 Frozen gel 2 0 26 1278 1 4 3 0 0 0 0 1251 0 0 0 3 Unpreserved Retrospective Oo 0 319 0 0 1 2 0 0 0 317 0 0 1 2 Frozen Analytical Inclusivity A variety of BD MAX Enteric Bacterial Panel assay target strains were included in this study Strain selection criteria included prevalence serotype and motility where appropriate One hundred twenty one 121 strains were tested including strains from public collections and well charac
39. le 24 tests 24 tests 443378 pipette tips necessary for DNA Extraction BD MAX Enteric Bacterial Panel Extraction Tubes B2 Oven dried pellet containing DNA magnetic affinity beads protease 24 tests reagents and Sample Processing Control BD MAX Enteric Bacterial Panel Sample Buffer Tubes 24 tests Septum Caps 25 EQUIPMENT AND MATERIALS REQUIRED BUT NOT PROVIDED BD MAX PCR Cartridges BD Cat No 437519 e VWR Multi Tube Vortex Mixer VWR Cat No 58816 115 Vortex Genie 2 VWR Cat No 58815 234 or equivalent e Nalgene Cryogenic Vial Holder VWR Cat No 66008 783 e Rack compatible with a multi tube vortex mixer e g Cryogenic Vial Holder or equivalent e Disposable 10 uL inoculating loops BD Cat No 220216 e Lab coat and powderless disposable gloves For Unpreserved Stool Specimen types Dry clean containers for the collection of liquid or soft stool samples For Preserved Stool Specimen types e Cary Blair transport media 15 mL Suggested Media for Cultivation of Control Isolates see Quality Control Section e Trypticase Soy Agar with 5 Sheep Blood For Salmonella Shigella and E coll e g BBL Trypticase Soy Agar with 5 Sheep Blood TSA II BD Cat No 221292 e Brucella Agar with 5 Sheep Blood Hemin amp Vitamin K For Campylobacter jejuni e g BBL Brucella Agar with 5 Sheep Blood Hemin and Vitamin K4 BD Cat No 297848 WARNINGS AND PRECA
40. le is loaded on the BD MAX instrument along with a BD MAX Enteric Bacterial Panel Unitized Reagent Strip and PCR Cartridge the run is started and no further operator intervention is required The BD MAX System automates sample preparation including target organism lysis DNA extraction and concentration reagent rehydration target nucleic acid sequence amplification and detection using real time PCR The interpretation of the signal is performed automatically by the BD MAX System The assay also includes an SPC that is provided in the Extraction Tube and subjected to extraction concentration and amplification steps The SPC monitors for the presence of potential inhibitory substances as well as system or reagent failures Following enzymatic cell lysis at an elevated temperature the released nucleic acids are captured on magnetic affinity beads The beads with the bound nucleic acids are washed and the nucleic acids are eluted Eluted DNA is neutralized and transferred to the Master Mix Tube to rehydrate the PCR reagents After rehydration the BD MAX System dispenses a fixed volume of PCR ready solution into the BD MAX PCR Cartridge Microvalves in the BD MAX PCR 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 t
41. lled in the clinical evaluation Table 2 describes the number of compliant specimens enrolled by patient age and specimen type A total of 104 retrospective specimens were not included in the performance calculations below as the historical results were not confirmed by an alternate PCR and bi directional sequencing Tables 3 through 6 describe the performance characteristics of the BD MAX Enteric Bacterial Panel that were observed during the clinical trial Table 2 Compliant clinical trial enrollment summary by age group and specimen type Cary Blair Age Group Preserved Unpreserved Combined lt 1 110 43 153 1 4 302 128 430 5 12 210 209 479 13 18 271 168 439 19 65 1222 799 2021 Over 65 388 249 637 Unknown 3 2 5 Total 2566 1598 4164 For the Cary Blair preserved specimen type the BD MAX Enteric Bacterial Panel identified 96 2 and 98 7 of the Campylobacter spp prospective positive and negative specimens respectively and 97 and 100 of the retrospective positive and negative specimens respectively For the unpreserved specimen type the BD MAX Enteric Bacterial Panel identified 100 and 97 5 of the Campylobacter spp prospective positive and negative specimens respectively and 97 and 99 1 of the retrospective positive and negative specimens respectively Table 3 Table 3 Campylobacter spp Overall Performance Specimen RM Specime
42. lobacter strains Campylobacter species other than C jejuni or C colli with undetectable tuf gene sequences tested at a concentration 2 1 x 108 CFU mL in the SBT produced negative results with the BD MAX Enteric Bacterial Panel Six 6 out of 6 E coli strains other than Shiga toxin producing strains tested at a concentration 2 1 x 106 CFU mL of SBT produced negative results with the BD MAX Enteric Bacterial Panel Ninety eight 98 out of 99 other bacterial strains including 53 species and subspecies tested at a concentration 1 x 106 CFU mL of SBT or 1 x 108 genomic DNA cp mL or 1 x 108 elementary bodies mL of SBT produced negative results with the BD MAX Enteric Bacterial Panel S boydii ATCC 12028 produced 1 replicate out of 3 as positive for the presence of stx Fifteen 15 out of 15 viruses tested at a concentration 1 x 104 PFU mL of SBT produced negative results with the BD MAX Enteric Bacterial Panel Three 3 out of 3 ova and parasites tested at a concentration 1 x 105 cysts mL of SBT produced negative results with the BD MAX Enteric Bacterial Panel Two 2 out of 2 Candida species tested at a concentration 2 1 x 10 organisms mL of SBT produced negative results with the BD MAX Enteric Bacterial Panel Sixteen 16 Enteric organisms representing each target of the BD MAX Enteric Bacterial Panel were tested with results as follows o Three 3 of 3 Campylobacter spp one C coli one C jejuni subsp doylei a
43. lowed closely Using a dry clean container liquid or soft stool samples are collected according to the following procedure 1 Unpreserved specimens Transfer liquid or soft stool samples to a dry clean container Avoid contamination with water or urine Label the container and transport to the laboratory according to institutional standard operating procedures Refer to the Storage and Stability section Avoid mixing toilet paper water or soap with the sample 2 Cary Blair preserved specimens Transfer liquid or soft stool samples to a 15 mL transport device according to the manufacturer s instructions Avoid contamination with water or urine and avoid mixing toilet paper or soap with the sample Label the container and transport to the laboratory according to institutional standard operating procedures Refer to the Storage and Stability section Sample Preparation NOTE One 1 Sample Buffer Tube SBT one 1 Septum Cap one 1 Master Mix one 1 Extraction Tube and one 1 Unitized Reagent Strip are required for each sample and each External Control to be tested Set aside the required number of materials from their protective pouches or boxes To store opened Master Mix or Extraction Tube pouches remove excess air and close using the zip seal 1 Label a bar coded BD MAX SBT clear cap with the appropriate sample identification Do not obscure write or label over the 2D barcode 2 Vortex unpreserved or Cary Blair preserved samples a
44. n External Control that yields an Unresolved Indeterminate or Incomplete test result is indicative of a reagent or a 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 Enteric Bacterial Panel Each BD MAX Enteric Bacterial Extraction Tube contains a Sample Processing Control 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 sample will be reported as Unresolved however any positive POS assay results will be reported and no targets will be called NEG An Unresolved result is indicative of sample associated inhibition or reagent failure Repeat any sample reported as Unresolved according to the Repeat Test Procedure section below RESULTS INTERPRETATION Results are available on the lt Results gt tab in the lt Results gt window on the BD MAX System monitor The BD MAX System software automatically interprets test results Results are reported for each of the analytes and for the Sample Processing Control A test result may be called NEG
45. n Type Origin 7 Total P 25 232 48 Cary Blair j N 1 1751 1752 Total 26 1774 1800 PPA 95 Cl 96 2 81 1 99 3 NPA 95 Cl 98 7 98 1 99 1 l P 64 0 64 Cary Blair gael N 2 151 153 Total 66 151 217 PPA 95 Cl 97 89 6 99 2 NPA 95 Cl 100 97 5 100 P 22 313 53 Unpreserved jr N 0 1185 1185 Total 22 1216 1238 PPA 95 CI 100 85 1 100 NPA 95 CI 97 5 96 4 98 2 P 65 2 67 Unpreserved go N 2 221 223 Total 67 223 290 PPA 95 CI 97 89 8 99 2 NPA 95 CI 99 1 96 8 99 8 1 This specimen was also tested using an alternate PCR assay followed by bi directional sequencing and gave a negative result 2 These twenty three 23 specimens were also tested using an alternate PCR assay followed by bi directional sequencing ten 10 of twenty three 23 gave a positive result 3 These thirty one 31 specimens were also tested using an alternate PCR assay followed by bi directional sequencing fourteen 14 of thirty one 31 gave a positive result For the Cary Blair preserved specimen type the BD MAX Enteric Bacterial Panel identified 85 and 99 1 of the Salmonella spp prospective positive and negative specimens respectively and 99 1 and 100 of the retrospective positive and negative specimens respectively For the unpreserved specimen type the BD MAX Enteric Bacterial Panel identified 91 7 and 98 9 of the Salmonella spp prospective positive and negative specimens respectivel
46. nclusion in the relevant Target Mix Individual inoculating loops were dipped into each of the two Target Mixes and each inoculating loop was then transferred to a SBT already containing fecal matrix preserved or unpreserved that was pre determined to be negative for all the targets detected by the BD MAX Enteric Bacterial Panel Each Target Mix was tested in replicates of 30 per sample type preserved or unpreserved by a single operator using 3 different production lots of the BD MAX Enteric Bacterial Panel Analytical sensitivity LoD defined as the lowest concentration at which greater than 95 of all replicates are expected to test positive with 95 confidence ranged from 10 to 653 CFU mL in SBT and 1 500 to 97 950 CFU mL in stool for preserved specimens and 42 to 910 CFU mL in SBT and 6 300 to 136 500 CFU mL in stool for unpreserved specimens Table 14 Table 14 BD MAX Enteric Bacterial Panel Limit of Detection Unpreserved Cary Blair Preserved Salmonella typhimurium ATCC 14028 LoD CFU mL in SBT 95 CI 296 233 376 193 142 263 LoD CFU mL in stool 95 Cl 44 400 34 950 56 400 Salmonella enteriditis ATCC 13076 LoD CFU mL in SBT 95 Cl 620 403 954 28 950 21 300 39 450 502 345 729 LoD CFU mL in stool 95 Cl 93 000 60 450 143 100 Campylobacter coli ATCC 43134 LoD CFU mL in SBT 95 CI 95 70 128 75 300 51 750 109 350 55 41 76
47. nd oneC jejuni subsp jejuni bearing the tuf gene tested at a concentration 2 1 x 10 CFU mL of SBT produced positive results for Campylobacter and negative results for all other targets with the BD MAX Enteric Bacterial Panel o Four 4 of 4 E coli two 0157 and two non O0157 strains bearing the stx gene tested at a concentration 1 x 106 CFU mL of SBT produced positive results for E coli and negative results for all other targets with the BD MAX Enteric Bacterial Panel o Five 5 of 5 Salmonella spp bearing the spaO gene tested at a concentration 1 x 10 CFU mL of SBT produced positive results for Salmonella and negative results for all other targets with the BD MAX Enteric Bacterial Panel o Three 3 of 4 Shigella spp one S sonnei one S boydii one S flexneri and S dysentariae bearing the ipaH gene tested at a concentration 1 x 106 CFU mL of SBT produced positive results for joaH and negative results for all other targets with the BD MAX Enteric Bacterial Panel Initial testing of S boydii ATCC 12028 produced 1 replicate out of 3 as positive for the presence of stx Subsequent testing of this strain produced positive results with 8 out of 20 replicates for the presence of stx Interfering Substances Nineteen 19 biological and chemical substances occasionally used or found in stool specimens were evaluated for potential interference with the BD MAX Enteric Bacterial Panel Included in this study was an Antibiotics M
48. ng the barcode with the scanner or by manual entry Select the appropriate kit lot number found on the outer box from the pull down menu Repeat steps 9 to 11 for all remaining SBTs Place the SBTs in the BD MAX System Rack s corresponding to the Unitized Reagent Strips assembled in steps 5 to 7 NOTE Place the tubes in the sample rack s with the 1D barcode labels facing outward this makes scanning sample tubes easier during sample login Place the required number of BD MAX PCR Cartridge s into the BD MAX System see Figure 2 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 PCR Cartridges are used on a per run AND rack basis 2 runs per cartridge and 1 cartridge per rack Figure 2 Load BD MAX PCR Cartridges Load rack s onto the BD MAX System Figure 3 ou 1a ORG 4 Side A Side B Figure 3 Load Rack s onto the BD MAX System Close the BD MAX System lid and click lt Start gt to begin processing At the end of the run check results immediately or store SBTs at 2 8 C for up to 5 days OR at 25 2 C fora maximum of 48 h until the results are checked NOTE If a septum cap was damaged during the run replace it with a new one before storing the sample NOTE Prepared BD MAX Enteric Bacterial Panel Sample Buffer Tubes can be stored at 2 8 C for a maximum of 120 h 5 days
49. one 1 min and restart from the BD MAX Operation section The remaining stool sample with a new SBT may also be used for repeat testing within the timeframe defined above Restart from the Sample Preparation section For the interpretation of warning or error code messages refer to the BD MAX System User s Manual Troubleshooting section Incomplete Result Incomplete results may be obtained in the event that the Sample Preparation or the PCR failed to complete Sample s can be repeated from their corresponding SBT s within the allowed timeframes defined above Vortex the sample s for one 1 min and restart from BD MAX Operation section The remaining stool sample may also be used for repeat testing with a new SBT within the timeframes defined above Restart from the Sample Preparation section For the interpretation of warning or error code messages refer to the BD MAX System User s Manual Troubleshooting section External Control Failure External Controls should yield expected results when tested If samples have to be repeated due to an incorrect External Control result they should be repeated from their SBT along with freshly prepared External Controls within the allowed timeframes defined above Vortex the samples for one 1 min and restart from the BD MAX Operation section CULTURING OF SAMPLES Culture and identification of organisms from positive samples should be performed per laboratory procedures LIMITATIONS OF THE P
50. ric Bacterial Panel at high concentrations NI No reportable interference with the BD MAX Enteric Bacterial Panel Mixed Infection Competitive Interference The mixed infection competitive interference study was designed to evaluate the ability of the BD MAX Enteric Bacterial Panel to detect low positive results in the presence of other targets at high concentrations Four 4 organisms Salmonella typhimurium Campylobacter coli Shigella sonnei and E coli 0157 H7 were individually prepared at 1 5X their respective LoD to serve as a low target in the BD MAX Enteric Bacterial Panel SBT A high target mix comprised of the organisms representative of the other three BD MAX Enteric Bacterial Panel analytes at a concentration of gt 1x106 CFU mL in the SBT was spiked into the SBT along with 10 uL of unpreserved stool and tested to simulate mixed infections All four low target organisms were successfully detected by the BD MAX Enteric Bacterial Panel when combined with their respective simulated high target concentration mixed infection preparations Precision Within laboratory precision was evaluated for the BD MAX Enteric Bacterial Panel at one 1 site Testing was performed over 12 days with 2 runs per day one each by 2 technologists for a total of 24 runs Four specific target organisms at different concentrations were used to create the panel members for this study The panel members contained Escherichia coli stx 1 Salmonella typhimurium
51. t and lt password gt 2 Gloves must be changed before manipulating reagents and cartridges 3 Remove the required number of Unitized Reagent Strips from the BD MAX Enteric Bacterial Panel kit Gently tap each strip onto a hard surface to ensure that all liquids are at the bottom of the tubes 4 Remove the required number of Extraction Tube s and Master Mix Tube s from their protective pouches Remove excess air and close pouches with the zip seal 5 For each specimen to be tested place one 1 Unitized Reagent Strip on the BD MAX System Rack starting with Position 1 of Rack A 6 Snap one 1 Extraction Tube white foil into each Unitized Reagent Strip in Position 1 as shown in Figure 1 7 Snap one 1 Master Mix Tube green foil into each Unitized Reagent Strip in Position 2 as shown in Figure 1 Pipette Tips Buffers Extraction Tube Master Mix Tube Figure 1 Snap BD MAX Enteric Bacterial Extraction Tubes and Master Mix Tubes into reagent strips 8 Click on the Run icon and enter the kit lot number for the BD MAX Enteric Bacterial Panel for lot traceability by either scanning the barcode with the scanner or by manual entry NOTE Repeat step 8 each time a new kit lot is used 11 12 13 14 15 17 Navigate to the Worklist Using the pull down menu select lt BD MAX Ent Bac gt Enter the Sample Buffer Tube ID Patient ID and Accession Number if applicable into the Worklist either by scanni
52. t for the direct qualitative detection and differentiation of enteric bacterial pathogens The BD MAX Enteric Bacterial Panel detects nucleic acids from Salmonella spp Campylobacter spp jejuni and coli Shigella spp Enteroinvasive E coli EIEC Shiga toxin 1 stx1 Shiga toxin 2 stx2 genes found in Shiga toxin producing E coli STEC as well as Shigella dysenteriae which can possess a Shiga toxin gene stx that is identical to the stx1 gene of STEC Testing is performed on unpreserved soft to diarrheal stool specimens or Cary Blair preserved stool specimens from symptomatic patients with suspected acute gastroenteritis enteritis or colitis The test is performed directly on the specimen utilizing real time polymerase chain reaction PCR for the amplification of SpaO a Campylobacter specific tufgene sequence jpaH and stx1 stx2 The test utilizes fluorogenic sequence specific hybridization probes for detection of the amplified DNA This test is intended for use in conjunction with clinical presentation laboratory findings and epidemiological information as an aid in the differential diagnosis of Salmonella Shigella EIEC Campylobacter and Shiga toxin producing E coli STEC infections Results of this test should not be used as the sole basis for diagnosis treatment or other patient management decisions Positive results do not rule out co infection with other organisms that are not detected by this test and may not be th
53. t high speed for 15 seconds 3 Remove the clear cap from the SBT and inoculate as follows a Insert a 10 uL disposable inoculation loop until the entire loop portion is submerged in the sample Do not insert beyond the loop as any additional stool on the shaft can overload the PCR reaction b Insert the loaded loop into the SBT and express the sample using a swirling motion NOTE Removal of the entire sample from the loop is not necessary The resultant SBT solution should be tea stained in color 4 Recap the inoculated SBT using a Septum Cap 5 Place the SBT in a rack compatible with a multi tube vortex mixer if available e g cryogenic vial holder or equivalent 6 Prepare any additional samples for testing by repeating Steps 1 through 5 ensuring gloves are clean prior to handling additional specimens 7 Vortex all prepared samples simultaneously at maximum speed for one 1 min with the multi tube vortex mixer 8 Proceed to BD MAX System Operation section to perform testing of the BD MAX Enteric Bacterial Panel on the BD MAX System BD MAX System Operation NOTE Refer to the BD MAX System User s Manual for detailed instructions Operation section NOTE Testing of the BD MAX Enteric Bacterial Panel must be performed immediately after the vortexing step above Sample Preparation Step 7 If retesting is necessary re vortex sample s 1 Power on the BD MAX System if not already done and log in by entering lt user name g
54. t 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 e 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 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 BD MAX PCR Cartridges may be used for up to two runs e Performing the BD MAX Enteric Bacterial Panel outside the recommended time ranges can produce invalid results Assays not performed within the specified time ranges should be repeated with a new specimen e Additional controls may be tested according to guidelines or requirements of local state provincial and or federal regulations or accrediting organizations In cases where culture or other PCR tests are conducted in the laboratory care must be taken to ensure that the BD MAX Enteric Bacterial Panel any additional reagents required for testing and the BD MAX System are not contaminated Avoid microbial and deoxyribonuclease DNase contamination of reagents at all times Gloves must be changed before manipulating reagents and cartridges e Always handle specimens as if they are infectious and in accordance with safe l
55. terized clinical isolates Inclusivity testing included 30 strains of Campylobacter spp jejuni and coli 30 strains of Salmonella spp enterica and bongori 31 strains of Shigella spp Enteroinvasive E coli EIEC and 35 strains found to be positive for Shiga toxin types 1 or 2 including 30 E coli strains of which 20 were non O157 and 5 S dysenteriae strains The strains were tested as target pools containing three or four assay targets each at the LOD for the assay in unpreserved stool matrix The assay correctly identified 120 of the 121 strains tested at the LOD One strain of Shigella sonnei ENF 15987 demonstrated 79 17 positivity at a concentration of 56 1 CFU mL The isolate was further evaluated and yielded 100 positivity at a concentration of 405 CFU mL Seven 7 other strains of Shigella sonnei were evaluated during the analytical inclusivity study and met the study acceptance criteria at a concentration of 56 1 CFU mL Analytical Sensitivity The analytical sensitivity Limit of Detection or LoD for the BD MAX Enteric Bacterial Panel was determined as follows Two 2 individual Target Mixes were prepared each of which contained a bacterial suspension that was comprised of a representative strain for each of the target organisms detected by the BD MAX Enteric Bacterial Panel including one strain bearing a variation of a gene coding for a Shiga like toxin Each target organism was prepared and quantified from culture prior to i
56. udy The study involved a total of eight 8 geographically diverse clinical centers where specimens were collected as part of routine patient care enrolled into the trial and tested with the BD MAX Enteric Bacterial Panel An additional four 4 collection centers enrolled specimens to be evaluated at a central location Specimens were obtained from pediatric or adult patients suspected of acute bacterial gastroenteritis enteritis or colitis for which stool culture had been ordered by a healthcare provider For prospective fresh specimens clinical centers performed their standard culture and identification method for Salmonella Shigella Campylobacter and E coli 0157 with a reference center performing culture and identification for three 3 sites The reference method for Shiga toxin 1 and 2 detection was via broth enriched enzyme immunoassay Reference method testing was performed in accordance with each product s respective package insert For retrospective frozen specimens the historical culture results were recorded at the collection site and the specimens were not re cultured The historical culture results were confirmed using an alternate PCR assay and bi directional sequencing as part of the composite reference method in order to confirm the presence of target DNA A total of 3457 prospective specimens 2112 Cary Blair preserved and 1345 unpreserved and 785 retrospective specimens 464 Cary Blair preserved and 321 unpreserved were enro
57. y and 100 and 99 6 of the retrospective positive and negative specimens respectively Table 4 Table 4 Salmonella spp Overall Performance i RM Specimen Type one BD MAX l P 17 172 34 Cary Blair a N 31 1791 1794 Total 20 1808 1828 PPA 95 Cl 85 64 94 8 NPA 95 Cl 99 1 98 5 99 4 P 105 0 105 Cary Blair pe el N 1 213 214 Total 106 213 319 PPA 95 CI 99 1 94 8 99 8 NPA 95 CI 100 98 2 100 l P 22 133 35 Unpreserved jr N 21 1202 1204 Total 24 1215 1239 PPA 95 CI 91 7 74 2 97 7 NPA 95 Cl 98 9 98 2 99 4 l P 61 1 62 Unpreserved pe u N 0 237 237 Total 61 238 299 PPA 95 CI 100 94 1 100 NPA 95 CI 99 6 97 7 99 9 1 These three 3 specimens were also tested using an alternate PCR assay followed by bi directional sequencing and gave a negative result 2 These seventeen 17 specimens were also tested using an alternate PCR assay followed by bi directional sequencing eleven 11 of seventeen 17 gave a positive result 3 These thirteen 13 specimens were also tested using an alternate PCR assay followed by bi directional sequencing eleven 11 of thirteen 13 gave a positive result For the Cary Blair preserved specimen type the BD MAX Enteric Bacterial Panel identified 100 and 99 7 of the Shigella spp EIEC organisms prospective positive and negative specimens respectively
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