ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1619

( g ) Add 25 μL of 70% formic acid and pipette up and down to resuspend the pellet. Vortex thoroughly and allow to dry at room temperature (20–25°C). ( h ) Add 25 μL of 100% acetonitrile and mix by pipetting up and down 2–3 times. ( i ) Centrifuge 2 min at 15871–21130 g, equivalent to 13000 to 15000 rpm for Eppendorf tube centrifuged with a 5424R rotor. ( j ) Dispense 1 μL of sample onto the reusable steel target or the disposable Biotarget. ( k ) As a valid BTS control is mandatory per target and run, it is advised to use two BTS control positions. Select the BTS control positions on a target to inoculate with BTS solution. ( l ) Pipette 1 μL of reconstituted BTS solution onto the two selected positions and dry at room temperature (20–25°C). Note: After samples and BTS have dried, HCAA Matrix portioned solution must be added within 30 min, or the target must be cleaned and the inoculation of samples BTS must be repeated. ( m ) Overlay each sample position and BTS control positions with 1 μL HCCA Matrix. Use a new pipet tip to add HCCA Matrix to each inoculated sample position. ( n ) Dry the inoculated target plate at room temperature (20–25°C). ( o ) The inoculated target plate is now ready for use. Note: An inoculated target plate must be processed within 24 h of preparation, or the target must be cleaned and the inoculation of samples and BTS must be performed again.

to produce a correct identification rate of 99.9%. Some of the participants were dropped from some of the agars because of no growth, and two participants did not receive OXA. One isolate, L. monocytogenes Ad273, was identified as Staphylococcus hominis (one of the exclusivity organisms) by one collaborator. The Bruker MALDI Biotyper methodology correctly identified and confirmed 985 out of 990 isolates to the species level to produce a correct species identification and confirmation rate of 99.5%. In most instances in which the organismwas not identified or confirmed to species level, the collaborator did not completely follow the Bruker MALDI Biotyper method completely (eDT, EXT) to obtain the most accurate result. Confirmatory reference method results.— When the L. monocytogenes strains were analyzed following the confirmatory reference methods from the nonselective agar (TSA/YE) only, 256 out of 256 isolates were correctly confirmed and identified to the genus level to produce a correct confirmation and identification rate of 100.0%. When analyzed to the species level, 223 out of 256 isolates were correctly identified to produce a correct species identification rate of 87.1%. BrukerMALDI Biotyper results.— For the non- monocytogenes Listeria species target organisms analyzed from four different agar types, 742 out of 742 isolates were correctly confirmed and identified to the genus level, producing a correct identification rate of 100.0%. There were two instances in which no growth was obtained, and two participants did not receive OXA. When analyzed to the species level, 726 out of 742 isolates were correctly confirmed and identified to produce a correct species identification rate of 97.8%. In most instances in which the organism was not identified or confirmed to species level, the collaborator did not completely follow the Bruker MALDI Biotyper method completely (eDT, EXT) to obtain the most accurate result. Confirmatory reference methods results.— When the Listeria species strains isolated onto TSA/YE agar plate were analyzed following the reference methods, 192 out of 192 isolates were correctly confirmed and identified to the genus level, producing a correct identification rate of 100.0%. When analyzed to the species level, 166 out of 192 isolates were correctly identified to produce a correct species identification rate of 86.5%. Bruker MALDI Biotyper.— For the Gram-positive non- Listeria (exclusivity) organisms, 263 out of 265 organisms were correctly confirmed as non- Listeria species, as well as correctly identified to the genus level, to produce a correct identification rate of 99.3%. A total of 247 possible results were not interpreted because no growth was observed on 231 agar plates (OXA, O&A, and RLM), and two participants (16 results) did not receive OXA. When analyzed to the species level, 262 out of 265 isolates were correctly identified to produce a correct species, or group, identification rate of 98.9%. The MALDI Biotyper method identified Bacillus thuringiensis as a member of the B. cereus group without any claim to a possible identification to the species level, as described in the ISO 7932 standard and the FDA/BAM Chapter 14 method (11, 12); this Listeria Species Gram-Positive Non-Listeria Organisms (Exclusivity)

K. Generating a Run Result Report

( a ) After the acquisition of the target plate has been completed, click View > Results to generate a PDF report. ( b ) The PDF report header contains the Run Identifier and Run Creation Date/Time from the Run Info section. ( c ) The PDF report footer contains the report creation date/ time, page count, and the appropriate area of application.

Results of Collaborative Study

The collaborative study involved a comparison evaluation of the BrukerMALDI Biotyper method to the confirmation protocol listed in the FDA/BAM, USDA/FSIS-MLG, and ISO reference methods for L. monocytogenes , Listeria species, and closely related non- Listeria spp. Gram-positive organisms. A total of 16 laboratories throughout Europe participated in this study, and the study was organized by ADRIA Développement (France). All 16 laboratories had one participating analyst. Each collaborator analyzed 36 blind-coded organisms: 16 target L. monocytogenes organisms, 12 target Listeria species organisms, and 8 non- Listeria Gram-positive organisms (exclusivity panel). Tables 2017.10A , 2017.10B , and 2017.10F summarize the collaborative results. Detailed results for each laboratory are presented in Tables 2017.10C , 2017.10D , and 2017.10G . The individual collaborator sample results are presented inTables 1–3 of the Supplemental Information.

L. monocytogenes

Bruker MALDI Biotyper results.— For the L. monocytogenes target organisms analyzed from four different agar types, 989 out of 990 isolates were correctly identified and confirmed to the genus level following the Bruker MALDI Biotyper methodology