summary and explanation of the procedure
Contents
1. MRSA XT Sample Buffer Tube s in the BD MAX Rack s following the Turn on the BD MAX System and log in by entering lt user name gt and lt password gt Remove the required number of BD MAX MRSA XT Reagent Strips from the BD MAX MRSA XT Kit Gently tap each strip onto a hard surface to ensure that all the liquids are at the bottom of the tubes Remove the required number of BD MAX MRSA XT Extraction Tube s and BD MAX MRSA XT Master Mix tube s from their protective pouches Remove excess air and close pouches with the zip seal For each specimen and external control to be tested place one 1 BD MAX MRSA XT Reagent Strip on the BD MAX System Rack starting with Position 1 of Rack A and continuing sequentially Do not skip spaces Snap one 1 BD MAX MRSA XT Extraction Tube white foil into Position 1 of each BD MAX MRSA XT Reagent Strip see Figure 1 Snap one 1 BD MAX MRSA XT Master Mix tube green foil into Position 2 of each BD MAX MRSA XT Reagent Strip see Figure 1 jee 2 aS hire ASE l Buffers Extraction Tube Master Mix tube 2S ae Figure 1 Snap BD MAX MRSA XT Extraction tubes and Master Mix tubes into reagent strip On the BD MAX software select the lt Consumable info gt tab under the Run screen Enter the kit lot number for the BD MAX MRSA XT Assay for lot traceability by either scanning the barcode with the scanner or by manual entry NOTE Re2. 96 8 98 1 PPV 73 2 95 Cl 67 8 78 3 NPV 99 5 95 Cl 99 1 99 7 Further investigation was performed on specimens with discordant results between the Reference Method and the BD MAX MRSA XT Assay e 12 of 54 MRSA False Positive BD MAX MRSA XT specimens were also found to be positive after repeat of Reference Method e 5of 11 MRSA False Negative BD MAX MRSA XT specimens were also found to be negative after repeat of Reference Method Based on this analysis the BD MAX MRSA XT Assay Positive and Negative Percent Agreement with the Reference Method for MRSA are 96 4 and 98 1 respectively All Sites BD MAX MRSA XT Assay A total of 2451 specimens were enrolled in the study Of those 2397 nasal swab specimens were found compliant at the specimen Direct culture and PCR level and 6 nasal swab specimens gave non reportable results A total of 2391 specimen results were used to determine the positive and negative percent agreement of the BD MAX MRSA XT Assay Table 2 Compared to Direct culture the BD MAX MRSA XT Assay identified 96 5 of the MRSA positive specimens and 96 9 of the MRSA negative specimens Table 2 Table 2 Results Obtained for MRSA with the BD MAX MRSA XT Assay in Comparison to Direct Culture Atl Cite Direct Culture Positive Negative Total Positive 137 69 206 BD MAX MRSA XT Assay Negative 5 2180 2185 T
3. MRSA XT Assay is directly proportional to the quantity of the corresponding probe that is hydrolyzed The BD MAX System measures these signals at the end of each amplification cycle and interprets the data to provide a result REAGENTS BD MAX MRSA XT Master Mix C7 Dried PCR Master Mix containing polymerase nucleotides and specific molecular probes and primers along with Sample Processing Control specific molecular probe BD MAX MRSA XT Reagent Strip Unitized Reagent strip containing all liquid reagents and disposable pipette tips 24 tests 443461 necessary for specimen processing and DNA extraction BD MAX MRSA XT Extraction Tube B8 Dried extraction reagent containing DNA magnetic affinity beads 24 tests Achromopeptidase and Sample Processing Control BD MAX MRSA XT Sample Buffer Tube with 25 septum caps 24 tests 24 tests EQUIPMENT AND MATERIALS REQUIRED BUT NOT PROVIDED BBL CultureSwab Liquid Stuart single or double swab BD catalog no 220099 or 220109 Copan Venturi Transystem Liquid Stuart single or double swab Copan catalog no 141C or 139C VWR Multi Tube Vortexer VWR catalog no 58816 115 NALGENE Cryogenic Vial Holder VWR catalog no 66008 783 Disposable gloves powderless Sterile scissors optional Sterile Gauze Stopwatch or timer BD MAX PCR Cartridges BD catalog no 437519 WARNINGS AND PRECAUTIONS For in vitro diagnostic use Do
4. SCCmec types I Il Ill IV and XI The swabs were then eluted in simulated nasal matrix Each MRSA strain was tested in replicates of 24 per concentration by 2 different operators using 3 different production lots of the BD MAX MRSA XT Assay Analytical sensitivity LoD defined as the lowest concentration at which 95 of all replicates tested positive ranged from 64 to 343 CFU swab Table 4 for the detection of MRSA strains Table 4 Limit of Detection of MRSA Genotypes by the BD MAX MRSA XT Assa LoD Concentration 1 MRSA Strain MREJ Genotype SCCmec type ICFU swab 95 C12 1 Type i 84 49 142 2 Type ii II 103 64 167 3 Type iii III 160 93 278 4 Type iv Ill 68 42 109 5 Type v IV 128 73 225 6 Type vi ND 343 186 632 7 Type vii II 219 110 439 8 Type ix ND 144 82 255 9 Type xiii ND 64 36 114 10 Type xiv ND 78 48 127 11 Type xxi4 Xl 112 64 197 SCCmec type does not correlate to the MREJ type as these are two different typing methods 2Cl Confidence Intervals 3ND not determined 4mecC containing MRSA strains Also known as mecA eazs strain Analytical Inclusivity An analytical inclusivity study was performed using a variety of MRSA strains taking into account geographic origin MREJ genotype wild type and mutant SCCmec type Pulsed Field Gel Electrophoresis PFGE type temporal diversity and susceptibility pattern Seventy seven 77 MRSA
5. frequently encountered in healthcare settings and represent over 50 of hospital acquired S aureus isolates in some North American hospitals Risk factors for infection with MRSA in healthcare settings include prolonged hospital stay proximity to patients infected or colonized with MRSA colonization with other resistant organisms such as vancomycin resistant Enterococci VRE and Clostridium difficile exposure to multiple and or prolonged broad spectrum antibiotic treatments exposure to high MRSA prevalence areas within the healthcare facility and prior MRSA nasal infection or carriage Early identification of patients with MRSA nasal carriage can be part of an effective infection prevention program for MRSA Culture based detection of MRSA requires isolation of pure colonies followed by either oxacillin or cefoxitin susceptibility testing detection of the mecA gene or detection of the penicillin binding protein PBP 2a encoded by the mecA gene The culture based process takes a minimum of 24 hours with a median time to result closer to 48 hours in order to identify MRSA With the rapidity at which MRSA infections can spread especially in healthcare settings where carriers are common providing MRSA nasal carriage results on the same day that the specimen was collected represents an advantage for infection prevention programs Active surveillance using molecular tests for rapid detection of MRSA is a proven strategy to reduce transmission in health
6. DNA is extracted on magnetic beads and concentrated and then an aliquot of the eluted DNA is added to PCR reagents which contain the MRSA 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 as well as process inefficiency due to 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 The BD MAX System uses a combination of lytic and extraction reagents to perform cell lysis and DNA extraction Following enzymatic cell lysis at elevated temperature the released nucleic acids are captured by magnetic affinity beads The beads with the bound nucleic acids are washed and the nucleic acids are eluted by heat in Elution Buffer Eluted DNA is neutralized with Neutralization Buffer and transferred to the M
7. SBD MAX MRSA XT BD MAX MRSA XT Assay 443461 P0167 01 2013 09 For In Vitro Diagnostic Use For use with the BD MAX System 25 C 4 CE INTENDED USE The BD MAX MRSA XT Assay performed on the BD MAX System is an automated qualitative in vitro diagnostic test for the rapid detection of methicillin resistant Staphylococcus aureus MRSA DNA from nasal swabs in patients at risk for nasal colonization The BD MAX MRSA XT Assay is intended to aid in the prevention and control of MRSA infections in healthcare settings It is not intended to diagnose MRSA infections nor guide or monitor treatment for MRSA infections A negative result does not preclude nasal colonization Concomitant cultures are necessary to recover organisms for epidemiological typing or for further susceptibility testing SUMMARY AND EXPLANATION OF THE PROCEDURE MRSA is a major cause of healthcare acquired infections Most transmissions occur in healthcare institutions as a result of contamination of the hands of healthcare workers or from the healthcare environment which has been contaminated from patients carrying MRSA While MRSA may cause infection with clinical manifestations ranging from pustules to sepsis and death it can also be found in the nose or on the skin of individuals asymptomatic carriers Treatment of MRSA infections has become a real challenge due to the broad range of antimicrobial agents Methicillin resistant strains of S aureus are
8. aster Mix Tube to rehydrate the PCR reagents The reconstituted amplification reagent is dispensed into the BD MAX PCR Cartridge Microvalves in the BD MAX PCR Cartridge are sealed by the system prior to initiating PCR to prevent evaporation and amplicon 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 a specific amplicon in the SCCmec right extremity junction MREJ the genes for methicillin resistance mecA and mecC in three different optical channels of the BD MAX System SCCmec right extremity junction MREJ amplicons are detected in the 475 520 channel methicillin resistance gene mecA and mecC amplicons are detected in the 585 630 channel and SPC amplicons are detected in the 680 715 channel 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 three optical channels used for the BD MAX
9. ay is designed to detect MREJ genotypes i ii iii iv v vi vii ix xiii xiv and xxi which represents most of mecA and mecC harboring MRSA strains belonging to different SCCmec MREJ types accounting for more than 98 of worldwide strains tested by BD to date Polymorphisms or mutations in regions detected by this assay may impair detection of new or unknown MRSA variants resulting in a false negative result e The BD MAX MRSA XT Assay performance in detecting modified S aureus MOD SA is not known as those strains have not been evaluated The mechanism of oxacillin resistance in MOD SA strains is due to changes in affinity of penicillin binding proteins for oxacillin MOD SA strains are rare in the United States e As with all PCR based in vitro diagnostic tests extremely low levels of target below the LoD of the assay may be detected but results may not be reproducible e Tobramycin may interfere with the BD MAX MRSA XT Assay refer to Interfering Substances section for further details 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 Sample Processing Control has been added to the test to aid in the identification of specimens that contain inhibitors to PCR amplification and as a control for reagent integrity and of the assay system as a whole The Sample Processing Control does not indicate if nucleic aci
10. bcultured onto Blood Agar BA Identification was confirmed with an agglutination test while methicillin resistance was confirmed by Cefoxitin disk 30 ug diffusion susceptibility testing Enrichment in Trypticase Soy Broth with 6 5 NaCl TSB 6 5 NaCl was completed in the event that MRSA was not confirmed by the initial direct culture method Turbid TSB 6 5 NaCl broth was used to inoculate additional chromogenic medium and BA plates MRSA confirmation was performed as described above A total of 2451 specimens were enrolled in the study Of those 94 specimens were regarded as noncompliant per protocol criteria and five 5 fully compliant specimens gave final non reportable PCR results A total of 2352 specimen results were used to determine the clinical performance of the BD MAX MRSA XT Assay Table 1 Compared to the Reference Method Direct Enriched Culture the BD MAX MRSA XT Assay identified 93 1 of the MRSA positive specimens and 97 5 of the MRSA negative specimens Table 1 For the population tested this resulted in a Negative Predictive Value NPV of 99 5 and a Positive Predictive Value PPV of 73 2 Table 1 Results Obtained for MRSA with the BD MAX MRSA XT Assay in Comparison to the Reference Method Reference Method MRSA Positive Negative Total Positive 149 541 203 Negative 111 2138 2149 Total 160 2192 2352 Sensitivity 93 1 149 160 95 CI 88 1 96 1 Specificity 97 5 2138 2192 95 CI
11. care settings and prevent infection in vulnerable patients Inaccurate detection can lead to uncontrolled transmission of MRSA and inappropriate use of healthcare resources With other commercial assays up to 12 9 of positive MRSA test results are incorrect because the mecA gene is absent commonly called dropout mutants These false positive results can lead to unnecessary and expensive isolation and treatment of patients Strains of MRSA with the newly discovered mecC gene account for 3 4 of all new MRSA cases but cannot be detected by assays that do not detect that gene These false negative results can lead to uncontrolled transmission of undetected strains of MRSA Assay design is critical to detect MRSA accurately and ensure that appropriate infection control interventions are applied The BD MAX MRSA XT assay uses eXTended detection technology to detect new strains of MRSA including strains with the mecC gene and decreasing false positives due to mecA mecC dropouts A nasal specimen is collected and transported to the laboratory using the recommended swab refer to Equipment and Materials Required But Not Provided section The swab is placed in a BD MAX mm DES P0167 01 1 MRSA XT Sample Buffer Tube The Sample Buffer Tube is vortexed to release cells from the swab into the buffer The Sample Buffer Tube is placed into the BD MAX System and the following automated procedures occur the bacterial cells are lysed
12. cus epidermidis MRSE strains tested at 210 CFU swab exhibited a MRSA NEG result Analytical Specificity The BD MAX XT Assay was performed on samples containing high levels of non target organisms using the BD MAX System to demonstrate the specificity of the assay for detection of MRSA e Fifty seven 57 out of 57 strains of various non staphylococcal species tested at a concentration of at least 2106 CFU mL except for Cryptococcus neoformans which was tested at 3x105 CFU swab produced MRSA NEG results e Forty five 45 coagulase negative staphylococcal strains CoNS and coagulase positive staphylococcal strains CoPS representing 28 species were tested at a concentration of 0 5 McFarland with the BD MAX XT Assay Forty five 45 of the 45 strains tested exhibited MRSA NEG results e Sixty five 65 MSSA strains including 15 empty cassette variant MSSA strains tested at high concentrations 2106 CFU swab produced MRSA NEG results e Seventeen 17 viruses representing 12 different viral species were tested at 105 PFU mL All 17 viruses produced MRSA NEG results Interfering Substances Twenty nine 29 microorganisms and chemical substances which might be used in the nares or found in nasal swab specimens were evaluated for potential interference with the BD MAX MRSA XT Assay Table 5 MRSA negative and MRSA positive samples at 2 3 x LoD were tested with the highest amount of each compound likely to be found at the sa
13. d has been lost due to inadequate collection transport or storage of specimens or if bacterial cells have been adequately lysed e n a mixed culture the detection of MRSA is variable when extremely high concentrations of MRSE are present Competition from MRSE was observed at an MRSA MRSE ratio of 1 1x10 e BD MAX MRSA XT Assay results may sometimes be Unresolved due to an invalid Sample Processing Control or be Indeterminate or Incomplete due to instrument failure and require retesting that can lead to a delay obtaining final results e Mutations or polymorphisms in primer or probe binding regions may affect detection of new or unknown MRSA resulting in a false negative result with the BD MAX MRSA XT Assay e As with all in vitro diagnostic tests positive and negative predictive values are highly dependent on prevalence BD MAX MRSA XT Assay performance may vary depending on the prevalence and population tested e The BD MAX MRSA XT Assay requires use of three 3 optical channels from the BD MAX System 475 520 channel 585 630 channel and 680 715 channel Performance of the remaining optical channel has not been established with this assay EXPECTED VALUES In the BD MAX MRSA XT Assay clinical study a total of 2393 reportable results from specimens compliant at the specimen and PCR levels were obtained from 3 geographically diverse sites and compared with Direct and Enriched culture The study population was grouped i
14. e 3 Percent Percent Percent Percent Cl 95 Agreement Corni Agreement Corni Agreement Corti HN MRSA 60 0 18 30 66 7 20 30 63 3 19 30 63 3 53 0 72 6 LP MRSA 95 0 57 60 98 3 59 60 96 7 58 60 96 7 92 9 98 5 MP MRSA 100 0 30 30 100 0 30 30 100 0 30 30 100 0 95 9 100 0 TN 100 0 30 30 100 0 30 30 100 0 30 30 100 0 95 9 100 0 1Percent Agreement correlates to the percent of negative results 2Confidence Interval For Lot to Lot Reproducibility the overall percent agreement was 100 for MRSA MP and TN 96 7 for MRSA LP and 61 1 for MRSA HN Table 7 Table 7 Lot To Lot Reproducibility Study Results using LOT Three Lots of the BD MAX MRSA XT Assay Overall Percent Agreement Category Lot1 Lot2 Lot3 Percent Percent Percent Percent CP 95 Agreement count Agreement count Agreement HN MRSA 63 3 19 30 63 3 19 30 56 7 17 30 61 1 50 8 70 5 LP MRSA 96 7 58 60 96 7 58 60 96 7 58 60 96 7 92 9 98 5 MP MRSA 100 0 30 30 100 0 30 30 100 0 30 30 100 0 95 9 100 0 TN 100 0 30 30 100 0 30 30 100 0 30 30 100 0 95 9 100 0 1Percent Agreement correlates to the percent of negative results 2Confidence Interval 14 Carry Over Cross Contamination A study was conducted to investigate the potential for cross contamination between high MRSA 2107 CFU swab specimens and negative
15. e 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 e Good laboratory technique is essential for 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 e Screening determines the colonization status at a given time Colonization may vary depending upon patient treatment e g decolonization regime patient status e g transient MRSA colonization or exposure to high risk environments e g contact with MRSA carrier and or prolonged hospitalization Colonization status should be monitored according to institutional policies e ABD MAX MRSA XT positive result does not necessarily indicate eradication treatment failure since DNA presence may persist A negative result following a previously positive test result may indicate eradication treatment success or may occur due to intermittent colonization e A positive test result does not necessarily indicate the presence of viable organisms A positive result is indicative of the presence of MRSA DNA The BD MAX MRSA XT Assay simultaneously detects the mecA mecC gene carried within the SCCmec cassette and a S aureus specific sequence located within the junction of the SCCmec cassette and the orfX gene MREv e The BD MAX MRSA XT Ass
16. ed on a per run AND rack basis 2 runs per cartridge and 1 cartridge per rack Figure 2 Load PCR Cartridges 16 Load rack s into 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 A 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 results immediately or store Sample Buffer Tubes at 2 8 C until the results are checked NOTE If a septum was damaged during the run replace it with a new one before storing the specimen NOTE Sample Buffer Tubes can be stored at 2 25 C for a maximum of 36 hours or at 2 8 C for a maximum of 120 hours 5 days after the run has been started When an Indeterminate IND Unresolved UNR or Incomplete INC result is obtained or when 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 MM3 and EP127 1 An External P
17. efer to the BD MAX System User s 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 they should be repeated from their Sample Buffer Tubes along with freshly prepared External Controls within the timeframes defined above Vortex the samples for one 1 minute and restart following the BD MAX System Operation section CULTURING OF CLINICAL SPECIMENS In order to perform antimicrobial susceptibility testing or epidemiological typing clinical specimens may be cultured from the collection device swab prior to performing the Specimen Preparation procedure using a Streak Plate method or after the Specimen Preparation procedure using an Enrichment Broth method Immediately after the end of the initial PCR run swabs may be stored at 2 8 C for up to 36 hours in Sample Buffer Tubes before culturing following hospital procedures LIMITATIONS OF THE PROCEDURE e This product is intended for use with nasal swab specimens collected using specimen collection and transport devices listed in the Equipment and Materials Required But Not Provided section e This product should only be used with the BD MAX System e Incorrect test results may occur from improper specimen collection handling or storage technical error sample mix up or because the number of organisms in th
18. for the BD MAX MRSA XT Assay at one 1 site The Precision panel consisted of 4 sample categories near the LoD Each specimen contained simulated nasal matrix MRSA strains were tested as follows e Moderate Positive MP 2 and lt 5 x LoD e Low Positive LP 2 1 and lt 2 x LoD e High Negative HN lt 1 x LoD e True negative TN Negative samples no target Testing was performed in duplicate over 12 days with 2 runs per day by 2 different technologists Precision study results for TN MP LP and HN MRSA samples demonstrated 100 100 97 9 and 60 4 agreement respectively Reproducibility The reproducibility study was performed using the same sample categories as defined above for the Precision Study Samples in each category were tested in triplicate on 5 distinct days wherein each day 2 panels were tested by 2 different technologists at 3 clinical sites using 1 lot of reagents Site to Site One 1 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 sample category with the data from MRSA strains For Site to Site Reproducibility the overall percent agreement was 100 for MRSA MP and TN categories 96 7 for MRSA LP and 63 3 for MRSA HN Table 6 Table 6 Site To Site Reproducibility Study Results Using One Lot of the BD MAX MRSA XT Assay Overall Percent SITE I Agreement Category Site 1 Site 2 Sit
19. ification overrides this control MRSA NEG MRSA DNA not detected e Fluorescence signal is not detected by the BD MAX MRSA XT Assay for any target mecA mecC and MREJ targets and fluorescence signal is detected for the SPC or e Fluorescence signal is detected for the mecA mecC gene only the mecA and mecC genes are not unique to S aureus species and can be found in other bacterial genera e g S epidermidis MRSA UNR Unresolved result e Fluorescence signal not detected for mecA mecC or MREJ targets and e Fluorescence signal not detected for the SPC Inhibitory specimen or reagent failure IND Indeterminate result e BD MAX System failure with Warning or Error Codes Refer to the Troubleshooting section of the BD MAX System User s Manual for interpretation of warning and error codes INC Incomplete run e BD MAX System failure with Warning or Error Codes Refer to the Troubleshooting section of the BD MAX System User s Manual for interpretation 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 2 25 C retesting must be performed within 36 hours of the steps covered in the Specimen Preparation section above Alternatively for Sample Buffer Tubes stored at 2 8 C retesting may be performed within 120 hours 5 days of the steps covered in the Spec
20. imen Preparation section above 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 an inhibitory substance or a reagent failure prevents proper target or SPC amplification Sample s can be repeated from their corresponding Sample Buffer Tube s within the timeframes defined above Vortex the sample s for one 1 minute and restart following the BD MAX System Operation 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 timeframes defined above Vortex the sample s for one 1 minute and restart following the BD MAX System Operation section For the interpretation of warning or error code messages refer to the BD MAX User s Manuals Troubleshooting section Incomplete Result Incomplete results may be obtained in the event that a terminating warning or error code occurs or in the event that the Sample Preparation or the PCR did not reach it s expected time points The Incomplete results may apply to a run or a lane Sample s can be repeated from their corresponding Sample Buffer Tube s within the timeframes defined above Vortex the sample s for one 1 minute and restart following BD MAX System Operation section For the interpretation of warning or error code messages r
21. ion during PCR analysis If the SPC result fails to meet the acceptance criteria for a negative specimen the result 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 MRSA NEG negative MRSA POS positive or MRSA 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 decision algorithm ASSAY RESULT REPORTED _ INTERPRETATION OF RESULT MRSA POS MRSA DNA detected MRSA NEG MRSA DNA not detected No target amplification no SPC KSA UNR amplification Indeterminate due to BD MAX IND 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 Users Manual for interpretation of warning and error codes MRSA POS MRSA DNA detected e Fluorescence signal is detected for both MREJ S aureus specific and mecA mecC targets and e The SPC is ignored since MRSA target ampl
22. ions or accrediting organizations In cases where other PCR tests are conducted in the same general area of the laboratory care must be taken to ensure that the BD MAX MRSA XT Assay any additional reagents required for testing and the BD MAX System are not contaminated Gloves must be changed before manipulating reagents and cartridges Always handle specimens as if they are infectious and in accordance with safe laboratory procedures such as those described in CLS Document M29 and in Biosafety in Microbiological and Biomedical Laboratories Wear protective clothing and disposable gloves while handling kit reagents Wash hands thoroughly after performing the test Do not pipette by mouth Do not smoke drink or eat in areas where specimens or kit reagents are being handled Dispose of unused reagents and waste in accordance with country federal provincial state and local regulations 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 MRSA XT Assay reagents and components are stable at 2 25 C through the stated expiration date Do not use expired components BD MAX MRSA XT Master Mix and Extraction Tubes are provided in sealed pouches To protect product from humidity immediatel
23. line Document MM3 Refer to the latest edition Clinical and Laboratory Standards Institute User Protocol for Evaluation of Qualitative Test Performance Approved Guideline Document EP12 Refer to the latest edition BD MAX System User s Manual refer to the latest version BD Diagnostics Sparks MD USA 15 Temperature limitation Authorized Representative in the European Community Manufacturer Perforation Ea E Catalog number Batch Code Lot lt o In Vitro Diagnostic Medical Device Use by YYYY MM DD YYYY MM MM end of month Keep away from light Contains sufficient for lt n gt tests Keep Dry Consult Instructions for Use lt A al e HEES R 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 BD Diagnostics Technical Service 1 800 638 8663 tal GeneOhm Sciences Canada Inc Benex Limited 2555 Boul du Parc Technologique Rineanna House Qu bec QC G1P 4S5 Canada Shannon Free Zone Shannon County Clare Ireland Made in Canada Brands are trademarks of their res
24. mpling site or on the nasal swab sample Results demonstrated no reportable interference with any microorganisms or chemical substance except for Tobramycin which showed inhibition in the BD MAX MRSA XT Assay when tested at a concentration of 4 5 x 10 3 g swab Table 5 Endogenous and Exogenous Substances Tested with the BD MAX MRSA XT Assay Substance Result Substance Result Mucin from bovine submaxillary glands NI Rhinocort aqua NI Dexamethasone Sodium Phosphate Ophtalmic Solution NI Zicam No Drip Liquid Nasal NI USP 0 1 Dexamethasone Phosphate Equivalent Gel Extreme Congestion Relief Chloraseptic NI Fluticasone Propionate NI Taro Mupirocin Mupirocin Ointment USP 2 NI Luffeel NI Long Lasting Dristan Nasal Mist NI Staphylococcus epidermidis NI Neo Synephrine NI Micrococcus luteus NI Equate Nasal Spray Decongestant NI Enterococcus faecium NI Beconase AQ NI Enterococcus faecalis NI Flunisolide Nasal Solution USP 0 025 NI Escherichia coli NI Nasacort AQ NI Corynebacterium flavescens NI Nasonex NI Moraxella catarrhalis NI k Staphylococcus hominis subsp NI Relenza NI hominis Tobramycin Haemophilus influenzae NI Blood NI Streptococcus pneumoniae NI Flumist NI INI No reportable interference with the BD MAX MRSA XT Assay I Reportable interference with the BD MAX MRSA XT Assay 13 Precision Within laboratory precision was evaluated
25. 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 torn upon arrival Close reagent protective pouches promptly with the zip seal after each use Remove any excess air in the pouches prior to sealing Do not remove desiccant from reagent pouches 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 use reagents if desiccant is not present or broken inside reagent pouches Do not use reagents if the foil has been opened or damaged Do not mix reagents from different pouches and or kits and or lots Do not use expired reagents and or materials Do not mix caps between tubes or re use caps as contamination may occur and compromise test results Proceed with caution when using chemical solutions as Master Mix and Extraction tube barcode readability may be altered To avoid contamination of the environment with MRSA amplicons do not break apart the BD MAX PCR Cartridge after use The seals in the BD MAX PCR Cartridges prevent contamination Performing the assay outside of the recommended time ranges may produce invalid results Assays not performed within specified time ranges should be repeated Additional controls may be tested according to guidelines or requirements of local state provincial and or federal regulat
26. nto in patient out patient categories The number and percentage of positive cases as determined by the BD MAX MRSA XT Assay are presented in the table below BD MAX MRSA XT Assay Group Number of Number of MRSA Positive pa Specimens In patient 1683 178 Pa Out patient 710 28 pate Total 2393 as pin Total specimens based on compliant PCR results PERFORMANCE CHARACTERISTICS Clinical Performance Clinical performance characteristics of the BD MAX MRSA XT Assay were determined in a multi site prospective investigational study Three 3 investigational centers participated in the study To be enrolled in the study patients had to be eligible for MRSA testing according to institutional policies Eligibility requirements for targeted screening as per Clinical site policies included but were not limited to patients admitted into the particular healthcare system patients admitted to the Intensive Care Unit patients transferred to the Intensive Care Unit pre elective surgery patients and patients being admitted from long term care facilities Specimens from patients previously enrolled in the study were excluded The Comparative Reference Method consisted of direct culture complemented by enriched culture Enriched culture analysis was completed for all specimens that were negative for MRSA by direct culture Presumptive S aureus colonies observed on selective S aureus chromogenic medium were su
27. o performing the BD MAX MRSA XT Assay refer to Culturing of Clinical Specimens section 1 Obtain the number of Sample Buffer Tubes corresponding to the number of specimens and external controls to be run 2 Label each Sample Buffer Tube with the appropriate patient identification making sure not to obscure write or label over the barcodes Remove the cap from the Sample Buffer Tube 4 Remove the swab from the sample transport tube and place the swab in the corresponding Sample Buffer Tube 5 Hold the swab by the stem near the rim of the tube use sterile gauze to minimize risk of contamination Lift the swab approximately one 1 cm from the bottom of the Sample Buffer Tube and bend the stem against the edge of the tube to break it Alternative method use sterile scissors to cut the stem 6 Close the Sample Buffer Tube with a septum cap 7 Place Sample Buffer Tube in a NALGENE Cryogenic Vial Holder and vortex at maximum speed for one 1 minute with the Multi Tube Vortexer Up to 24 samples can be processed simultaneously with the Multi Tube Vortexer wo BD MAX System Operation NOTE Refer to the BD MAX System User s Manual for detailed instructions Operation section NOTE The BD MAX MRSA XT Assay must be performed immediately after the vortexing step above Specimen Preparation Step 7 If retesting is necessary re vortex sample s i 2 10 1 12 13 Place the BD MAX
28. ositive Control is intended to monitor for substantial reagent failure while an External Negative Control is used to detect reagent or environmental contamination or carry over from other specimens or MRSA 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 control for their laboratory quality control program e Commercially available control materials e g a reference MRSA strain ATCC 43300 can be used as positive controls Staphylococcus epidermidis strain e g ATCC 12228 can be used as negative control e Previously characterized specimens known to be positive or negative for MRSA NOTE It is recommended that bacterial strains be freshly prepared in saline to a turbidity of 0 5 McFarland 1 0 x 108 CFU mL from isolated colonies and subsequently diluted with saline to obtain a final concentration of 1 0 x 104 CFU mL Dip a swab into the diluted bacterial suspension express the excess liquid place the swab in a corresponding Sample Buffer Tube and follow instructions described in step 5 of the Specimen Preparation section 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 a protocol and data as determined by the individual laboratory 3 An Ex
29. otal 142 2249 2391 Positive Percent Agreement 96 5 137 142 95 CI 92 0 98 5 Negative Percent Agreement 96 9 2180 2249 95 CI 96 1 97 6 Out of 2399 nasal swab specimens compliant at the specimen and PCR level tested with the BD MAX MRSA XT Assay 16 0 7 were reported as Unresolved after initial testing The Unresolved Rate after repeat testing was 0 1 2 2398 Table 3 Table 3 Unresolved Rates Initial Unresolved Rates 0 7 16 2399 95 Cl 0 4 1 1 Unresolved Rates After Repeat 0 1 2 2398 95 Cl 0 0 3 Total number based on compliant specimens and BD MAX MRSA XT Assay results Out of 2399 nasal specimens tested with the BD MAX MRSA XT Assay 14 0 6 were initially reported as Indeterminate No result remained Indeterminate upon repeat two specimens were not retested Out of 2399 nasal specimens tested with the BD MAX MRSA XT Assay 8 0 3 were initially reported as Incomplete No result remained Incomplete upon repeat one specimen was not retested Analytical Sensitivity The analytical sensitivity Limit of Detection or LoD for the BD MAX MRSA XT Assay was determined as follows positive specimens were prepared by soaking swabs in a wide range of MRSA bacterial suspensions prepared and quantified from cultures The tested strains included 11 MRSA strains representing 11 MREJ genotypes i ii iii iv v vi vii ix xiii xiv and xxi corresponding to 5
30. peat steps 7 and 8 for each new kit lot number Select the lt Work List gt tab click on the lt Assay gt field and using the pull down menu select lt BD MAX MRSA XT gt This will automatically populate the remaining assay fields for Rack A with BD MAX MRSA XT Enter the BD MAX MRSA XT Sample Buffer Tube ID Patient ID and Accession Number if applicable for Position 1 of Rack A either by scanning the 1D barcode with the scanner or by manual entry Click on the lt Lot Numbers gt 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 Awith the same lot number Enter the information for the next position in the Rack and continue for all remaining Sample Buffer Tubes in the rack NOTE Steps 11 and 12 must be repeated for each new kit lot number Repeat steps 9 to 12 for Rack B same order as entered in the worklist Do not skip or leave empty positions between tubes NOTE Place the tubes into the sample rack with 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 specimens e The BD MAX System will automatically select the position and row on the PCR cartridge for each run e Cartridges are us
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32. specimens throughout the BD MAX MRSA XT workflow Overall from 210 reportable results 3 false positive results were obtained 1 4 due to carry over contamination REFERENCES fl Blanc et al High Proportion of Wrongly Identified Methicillin Resistant Staphylococcus aureus Carriers by Use of a Rapid Commercial PCR Assay Due to Presence of Staphylococcal Cassette Chromosome Element Lacking the mecA Gene J Clin Microbiol 201 1 49 722 724 Petersen et al Epidemiology of methicillin resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans Clin Microbiol Infect 2013 19 E16 E22 Shore et al Detection of Staphylococcal Cassette Chromosome mec Type XI Carrying Highly Divergent mecA mecl mecR 1 blaZ and ccr Genes in Human Clinical Isolates of Clonal Complex 130 Methicillin Resistant Staphylococcus aureus Antimicrob Agents and Chemother 2011 55 3765 3773 Clinical and Laboratory Standards Institute Protection of laboratory workers from occupationally acquired infections Approved Guideline Document M29 Refer to the latest edition Centers for Disease Control and Prevention and National Institutes of Health Biosafety in microbiological and biomedical laboratories Chosewood L C and Wislon D E eds 2009 HHS Publication No CDC 21 1112 Clinical and Laboratory Standards Institute Molecular Diagnostic Methods for Infectious Diseases Approved Guide
33. strains from 27 countries were tested in this study including strains from public collections and from well characterized clinical isolates including Vancomycin Resistant Staphylococcus aureus VRSA and Vancomycin Intermediate Staphylococcus aureus VISA strains The BD MAX MRSA XT Assay detected MREJ types i ii iii iv v vi vii ix xiii xiv and xxi when tested at low bacterial load 2 3 x LoD The BD MAX MRSA XT Assay detected MRSA SCCmec types Il Ill IV V VI VII VIII and XI as well as MRSA PFGE types USA 100 to 800 1000 and 1100 at 2 3 x LoD All MRSA strains displaying additional resistance to vancomycin VRSA and VISA were also detected Evaluation of a Well Characterized Challenge Strain Panel An additional analytical study was carried out to evaluate the analytical performance of the BD MAX MRSA XT Assay using a well characterized challenge strain panel e Seventeen 17 out of 17 MRSA strains with high and low oxacillin minimum inhibitory concentrations MICs including PFGE types USA 100 to 800 1000 PFGE type IV IBERIAN and mecC variant mecA containing S aureus strain LGA251 tested at a concentration of 2 3 x LoD exhibited MRSA POS results e Four 4 out of 4 BORSA strains Borderline oxacillin resistant S aureus tested at 2106 CFU swab exhibited MRSA NEG results e Five 5 out of 5 MSSA strains tested at 210 CFU swab exhibited MRSA NEG results e One 1 out of 1 methicillin resistant Staphylococ
34. ternal 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 4 An 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 MRSA XT Assay kit NOTE 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 5 Each BD MAX MRSA XT Assay Extraction Tube contains a Sample Processing Control SPC which is a plasmid containing a synthetic target DNA sequence The SPC will be extracted eluted and amplified along with any DNA present in the processed specimen ensuring the predictivity of the assay 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 detect
35. y re seal after opening e Reagent tubes are stable for up to 7 days at 2 25 C after initial opening and re sealing of the pouch e Unreconstituted Extraction and Master Mix reagent tubes are stable for up to 5 hours at 2 25 C after being removed from their protective pouch INSTRUCTIONS FOR USE Specimen Collection Transport Using a recommended swab transport device refer to Equipment and Materials Required But Not Provided section nasal specimens should be collected according to institutional and laboratory standard operating procedures and or the following Moisten the swab s with two drops approximately 50 uL of sterile physiological saline or use dry 2 Carefully insert the swab s into the patient s nostril a swab tip should be inserted up to 2 5 cm 1 inch from the edge of the nares Roll the swab s along the mucosa inside the nostril 5 times Insert the same swab s into the second nostril and repeat steps 2 and 3 Place the swab s in its transport tube Label the transport tube Transport the swab s to the laboratory according to institutional and laboratory standard operating procedures Refer to Storage and Stability section SS OF 08 Specimen Preparation NOTE One 1 Sample Buffer Tube one 1 Septum Cap one 1 Master Mix C7 one 1 Extraction Tube B8 and one 1 Reagent Strip are required for each specimen and each External Control to be tested NOTE For culturing clinical specimens prior t
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