VITEK MS Collaborative Study Outline - eBook

AOAC INTERNATIONAL Official Methods of Analysis SM Program

Identification and Confirmation of Bacterial and Fungal Organisms by the VITEK ® MS Microbial Identification System: Collaborative Study Protocol

June 2019 Version 1

Prepared by: Patrick M. Bird AOAC Research Institute

2275 Research Blvd, Ste. 300 Rockville, MD 20850-3250 USA

VITEK MS Collaborative Study

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Table of Contents

1. Introduction 1.1. Description of the VITEK ® MS Microbial Identification Method

1.2. Summary of the Precollaborative Studies 1.3. Study Directors for Collaborative Study 2. Collaborators 3. Collaborative Study Design 4. Test Sample Preparation 5. Method – VITEK ® MS Microbial Identification Method

6. Reporting Raw Data 7. Analyzing Raw Data 8. Appendices

8.1. Instructions to Collaborators 8.2. Study Materials and Apparatus 8.3. Study Flowchart 8.4. Collaborator Comment Form 8.5. Data Report Form 8.6. User Manual

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1.0 Introduction The goal of this collaborative study is to estimate the reproducibility of the bioMérieux VITEK ® MS Microbial Identification system for the identification and confirmation of bacteria (Gram-positive and Gram-negative, aerobic and anaerobic) and fungal isolates. Twelve (12) Gram-positive, 12 Gram- negative and 12 fungal isolates will be shipped to each collaborator for analysis on the VITEK MS Microbial Identification system. The total number of correct identification for each specific organism, for each group of organisms and for all organisms will be determined. 1.1 Description of the VITEK ® MS Microbial Identification workflow. The VITEK MS System is a bacterial and fungal identification system which uses MALDI-TOF (MALDI: Matrix-assisted laser desorption/ionization; TOF: Time-of-Flight) mass spectrometry technology. A portion of a colony from an agar plate is applied to a spot on a VITEK MS-DS target slide. A matrix solution is applied to the spot on the VITEK MS-DS target slide. The VITEK MS-DS target slide is dried and then loaded into the VITEK MS. The sample is submitted to multiple laser shots inside the VITEK MS mass spectrometer. The matrix absorbs the laser light and vaporizes, along with the sample, in the process gaining an electrical charge (ionization). Electric fields then guide the ions into the vacuum tube which separates them according to 'weight' (mass, the smaller molecules flying faster than the larger ones), and their time of flight, and displays the results as a series of lines or peaks (spectrum) which correspond to different fragments that have broken away from the original molecules in the sample. By analyzing the pattern of fragments, it is possible to deduce the structure of the molecules. The sample spectra are compared to a database of spectra developed from a number of bacterial species. The sample spectra are interpreted to provide organism identification results associated with a confidence level. 1.2 Summary of the Precollaborative Studies The VITEK MS was validated according to AOAC guidelines for Performance Tested Methods SM and Official Methods of Analysis SM precollaborative studies for the identification and confirmation of microorganisms. In the combined internal and independent laboratory studies, 359 different claimed organisms (representing 208 Gram-negative, 123 Gram-positive and 28 fungal isolates) were evaluated by the VITEK MS method. All test strains were cultured on appropriate agar medium and growth conditions before analyzing on the VITEK MS instrument. Overall percent correct identification in the combined Internal and Independent studies (6122/6413 VITEK MS results from the 359 different organisms) was 95.5%. For each specific group of microorganisms tested, the percent correct identification was 94.4% for Gram-negative organisms, 96.1% for Gram-positive organisms and 97.5% for fungal organisms. The VITEK MS reagents were stable over 12 month time studies and provided reproducible results when compared between lots. Assay ruggedness was demonstrated for three critical parameters, including the use of 1-µL loops from two different suppliers, the age of the culture on an agar

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plate before analysis and various suppliers of the agar media. Overall results indicate that the VITEK MS method is an acceptable automated method for the identification of microorganisms 1.3 Co-Study Directors for the Collaborative Study Ron Johnson and John Mills bioMérieux, Inc. 595 Anglum Road, Hazelwood, MO, 63042 Email: ron.johnson@biomerieux.com ; john.mills@biomerieux.com Phone: 314-452-3241 2 Collaborators A minimum of 10 valid data sets are needed for the successful completion of this study. Approximately 15 collaborators will be solicited to participate. Qualified analysts, familiar with the VITEK MS Microbial Identification System, will act as collaborators. All collaborators have been, or will be, thoroughly 3.2 The study will include a minimum of 5 test sites, but preferably 10-12, with no more than 3 collaborators at any individual site. The collaborators at each site will share the use of a VITEK MS Microbial Identification system. Multiple collaborators at any site will be required to work independently. 3.3 Thirty-six (36) strains will be used in the evaluation. Twelve (12) of the strains evaluated will be Gram-positive organisms , 12 of the strains evaluated will be Gram-negative organisms, and 12 of the strains will be fungal organisms. Testing will be separated into 3 separate panels, with each panel evaluated during a separate week. 3.4 Collaborators will streak isolates onto agars as listed in Table 1, and after incubation, evaluate an isolate from each plate (if growth is present) on the VITEK MS Microbial Identification System. All strains within a set will be randomized and blind coded so that the collaborators do not know the identity of the strain. The randomization will be different for each collaborator. trained prior to participation. 3 Collaborative Study Design

Table 1. Test scheme for each collaborator

Number of strains per Type

Types of agar to be evaluated with each strain Tryptic Soy Agar with 5% Sheep Blood (TSAB), Modified Oxford Agar (MOX) Agar Listeria according to Ottavani and Agosti (ALOA) BACARA CHROMID S. aureus Elite agar (SAIDE)

Week of Analysis

Targets

Gram-positive, Gram-negative

1

12 – Gram-positive

and fungal organisms

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TSAB, ASAP, Xylose Lysine Deoxycholate (XLD), CHROMID Salmonella , Enterobacter Selective Isolation Agar (ESIA) CHROMID EHEC

12 – Gram-negative

2

12 – fungal

Sabouraud Dextrose Agar (SDA)

3

3.5 Study schedule 3.5.1 All media and reagents as outlined in Appendix 8.3 will be provided by the study sponsor. 3.5.2 Test strains will be shipped by overnight courier to each test site to arrive the week before testing. 3.5.3 All collaborators will begin analyses on the following Monday. 3.5.4 Data forms will be completed and emailed to the co-Study Director within 2 weeks of initiation of analyses. Table 2 provides an overview of the study schedule. Table 2. Study Schedule Collaborator Activity Receipt of Test Strain Set Initiation of Analyses Submission of Data Report Form Panel 1 Week 1, Wed-Friday Week 2, Monday Week 4, Monday

Week 2, Wed-Friday Week 3, Wed-Friday

Week 3, Monday Week 4, Monday

Week 5, Monday Week 6, Monday

Panel 2

Panel 3

4 Test Portion Preparation and Analysis 4.2 Test Portion Preparation 4.2.1 The Coordinating Laboratory will streak each strain for isolation on a non-selective agar. Well isolated colonies will be transferred to non-selective agar slants. Slants will be incubated to produce optimal growth of the organisms. Slants will be blind coded with a 3-digit identification number and then randomized. Each set of strains will be shipped to arrive the week prior to testing. Strains will be stored at refrigeration temperature (2-8 o C) until analysis is initiated the following Monday. A list of strains to be used in the validation study is provided in Table 3.

Table 3. Test Microorganisms Gram Positive (Panel 1 – Week 1)

Gram Negative (Panel 2 – Week 2)

Fungal (Panel 3 – Week 3)

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1 2 3 4 5 6 7 8 9

Aerococcus viridans

Citrobacter freundii Cronobacter sakazakii Enterobacter aerogenes Escherichia coli O157:H7 Escherichia coli O26 Klebsiella pneumonia Escherichia coli

Candida glabrata Candida tropicalis

Bacillus cereus

Enterococcus faecalis Enterococcus faecium

Cryptococcus neoformans Geotrichum capitatum

Listeria innocua Listeria ivanovii

Kodamaea ohmeri Malassezia furfur

Listeria monocytogenes Micrococcus luteus Staphylococcus aureus

Rhodotorula mucilaginosa Saccharomyces cerevisiae Trichosporon mucoides Aspergillus fumigatus Penicillium chrysogenum Fusarium proliferatum ,

Proteus mirabilis

Pseudomonas fluorescens

10 11 12

Staphylococcus haemolyticus Staphylococcus hominis Streptococcus pneumonia

Salmonella Newport

Salmonella Typhimurium

Vibrio vulnificus

4.3 Shipment of Test Portions 4.3.1 Test portions will be packaged in leak-proof insulated containers and shipped (according to the Dangerous Goods Regulations IATA for Infections Substances) by overnight. 5 VITEK MS Microbial Identification Method 5.1 Applicability Applicable for the identification and confirmation of Gram-positive and Gram-negative (aerobic and anaerobic) and fungal isolates 5.2 Test Kit Information 5.2.1 VITEK ® MS.—bioMérieux, Inc., REF 411071

5.3 Additional supplies and reagents required but not provided 5.3.1 VITEK MS-CHCA matrix.—bioMérieux, Inc., REF 411071 5.3.2 VITEK MS-FA.—bioMérieux, Inc., REF 411072 5.3.3 VITEK MS-DS target slides.—bioMérieux, Inc., REF 410893 5.3.4 VITEK MS Mould Kit.—bioMérieux, Inc., 415680 5.3.5 API suspension medium.—bioMérieux, Inc., REF 70700/70640/20150 5.3.6 Acetonitrile 5.3.7 Ethanol (99.8%, 96% or absolute)

5.4 Apparatus and equipment

5.4.1 Calibrated loops.― 1μL 5.4.2 Precision micropipette.―Capable of sampling and delivering 0.5-2μL 5.4.3 Sterile colorless pipette tips without filter.―Capable of sampling and delivering 0.5-2μL 5.4.4 Eppendorf tube.―Capable of sampling and delivering 2 mL

5.4.5 Sterile cotton swabs 5.4.6 Vortex-type mixer

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5.4.7 Vortex-type mixer with adaptor or bead beater-type homogenizer 5.4.8 Microcentrifuge able to reach 10,000 to 14,000 x g

5.5 Standard Reference Materials 5.5.1 Calibration strain: Escherichia coli ATCC 8739

5.7 Safety Precautions

All samples, microbial cultures and inoculated products should be considered infectious and handled appropriately. Aseptic technique and usual precautions for handling the bacterial group studied should be observed throughout this procedure. Refer to "CLSI M29-A, Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline - Current revision". For additional handling precautions, refer to "Biosafety in Microbiological and Biomedical Laboratories - CDC/NIH - Latest edition", or to the regulations currently in use in each country. Do not use VITEK MS-DS target slides after the expiration date. Before use, check that the packaging and components are intact. Allow the VITEK MS-DS target slides to come to room temperature before use. 5.8.1.1 Sample preparation is done in conjunction with the VITEK MS Prep Station. Review the VITEK MS Prep Station section of the User’s Manual before preparing the samples. 5.8.2 Quality Control 5.8.2.1 Perform quality control analysis as outlined in the VITEK MS user manual or according to your standard laboratory procedures. 5.8.3 Sample Preparation for Bacteria NOTES: Powder-free gloves should be used when handling VITEK MS-DS target slides. Avoid touching the spots on the slide. Handle the slide by the end of the slide. Do not write or mark on the slide. 5.8.3.1 Before adding samples to the VITEK MS-DS target slide, place the VITEK MS-DS target slide on a flat surface, such as a silicone mat, that will prevent it from sliding. 5.8.3.2 The single-use disposable VITEK MS-DS target slide with 48 sample positions is used with the VITEK MS system. The sample positions are divided into three sections. Each section is called an acquisition group. The three small spots in the

5.8 VITEK MS Microbial Identification Method 5.8.1 General Preparation

center of each acquisition group must not be used for identification of microorganisms. These three small spots are used for calibration.

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5.8.3.3 The calibration strain, E. coli ATCC 8739, is deposited on the calibration well positions, depending on the number of samples tested (1 calibrator per acquisition group of 16 spots). Follow the directions for Preparation of bacteria isolates (beginning in 5.8.3.4) to prepare the E. coli ATCC 8739 calibration strain. 5.8.3.4.1 Make sure not to pick up any agar with the colony as this will have a negative impact on the spectrum. 5.8.3.4.2 Some microorganisms, such as Streptococci , grow as very small colonies. In such cases, several similar colonies may be selected and deposited on one spot. 5.8.3.5 Apply the sample to the center of the spot. Smear a thin layer of the sample on the spot, then discard the used loop. 5.8.3.6 Take special care not to apply too much organism. The appropriate amount of organism on a spot is shown in the picture below. If insufficient colony material is deposited, use a new loop and introduce additional organism to the spot. 5.8.3.4 Using a 1 μl loop, pick up part of a suitable colony.

5.8.3.7 Open the microtube containing the VITEK MS-CHCA matrix. Add precisely 1.0 μl of matrix to the center of the spot. Discard the pipette tip. The matrix/microorganism suspension is then left to dry completely. 5.8.3.8 It is strongly recommended to deposit the matrix as indicated in the VITEK MS Prep Station workflow for bacteria: 2 by 2 in duplicate mode or 1 by 1 in single mode. Note: The VITEK MS-CHCA matrix contains a high concentration of organic solvents; it is recommended to close the tube after dispensing to avoid evaporation. 5.8.3.9 Proceed likewise for each additional sample. 5.8.3.10 After five minutes, check for crystal formation on the spots. Use of a magnifying lens may be helpful. An accurate measurement requires that the matrix crystals

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have become visible as a yellowish film. Ideally, most of the spot’s surface will be coated with crystals.

5.8.3.11 When the VITEK MS-DS target slides have been prepared, they must be tested within 72 hours. Prepared slides should be stored at room temperature in its original packaging until they are tested. 5.8.4 Sample Preparation for Yeast Isolates 5.8.4.1 The preparation of yeast samples differs from the standard procedure in that VITEK MS-FA (formic acid treatment) is applied to the organism on the VITEK MS- DS prior to adding VITEK MS-CHCA. Care is taken not to add VITEK MS-CHCA until the VITEK MS-FA is completely dry, usually after 1 - 3 minutes. 5.8.4.2 The sample is applied to the VITEK MS-DS in the same manner as described in 5.8.3.4 to 5.8.3.6. 5.8.4.3 Add 0.5 μl of VITEK MS-FA to the sample, using a new pipette tip for each

sample. Allow for enough time (1-3 minutes) for evaporation of VITEK MS-FA before adding VITEK MS-CHCA. Once the slide is completely dry, 1.0 μl of the VITEK MS-CHCA is added to each sample.

5.8.4.4 When the VITEK MS-DS target slides have been prepared, they must be tested within 72 hours. Prepared slides should be stored at room temperature in its original packaging until they are tested. 5.8.5 Sample Preparation for Mold Isolates 5.8.5.1 Wet a sterile cotton swab using API suspension media or sterile deionized water. Depress the tip of the swab to remove excess fluid. 5.8.5.2 Collect approximately a 1-2 cm diameter of mold from the agar plate. 5.8.5.3 Suspend the collected material in a 2 mL round-bottom tube containing 900 µL of 70% ethanol. Mix using a vortex-type mixer. 5.8.5.4 Centrifuge the tube for 2 minutes at a speed between 10,000 and 14,000 x g. 5.8.5.5 Discard all of the supernatant using a pipette. Use caution to avoid dislodging the pellet. 5.8.5.6 Add 40 µL of 70% formic acid and mix using a vortex-type mixer. 5.8.5.7 Add 40 µL of acetonitrile and mix using a vortex-type mixer. 5.8.5.8 Centrifuge the tube for 2 minutes at a speed between 10,000 and 14,000 x g. 5.8.5.9 Immediately spot 1 µL of the supernatant on a VITEK MS-DS slide. Allow to dry

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completely. 5.8.5.10 Once the spot is completely dry, 1.0 μl of the VITEK MS-CHCA is added to each spot. 5.8.5.11 When the VITEK MS-DS target slides have been prepared, they must be tested within 72 hours. Prepared slides should be stored at room temperature in its original packaging until they are tested. 5.8.6 Analysis on the VITEK MS Instrument 5.8.6.1 Follow VITEK MS package insert instructions for running the instrument. 5.8.6.2 Log onto the software, verify your VITEK MS-DS is dry and open the instrument drawer using the computer software. 5.8.6.3 Place the VITEK MS-DS into the adapter and scan the barcode. The application will display the spot positions of the test isolates. 5.8.6.4 Place the adaptor into the carrier, face up with angled corners to the front, barcodes on the left side. 5.8.6.5 Slide the adapter into the carrier until it cannot go any further and start the application. 5.8.6.6 When acquisition is complete, remove the VITEK MS-DS target slide. 5.8.7 Interpretation and Test Result Report 5.8.7.1 The VITEK MS system identifies organisms by comparing the characteristics of the obtained spectrum to the VITEK MS knowledge base. A percent agreement calculation is provided to the end user. 5.8.7.2 A quantitative value, the confidence value, is calculated. and expressed the similarity between the unknown organism and every organism or organism group of the knowledge base. Thresholds are applied in order to retain only the more significant organisms or organism groups. 5.8.7.2.1 A single identification is displayed with confidence value between 60 to 99.9 when one significant organism or organism group are identified. 5.8.7.2.2 Low Discrimination identifications are displayed when more than one significant organism or organism group are identified, but not more than 4 in total. In this case, the sum of confidence values is equal to 100.

5.8.7.2.3

When more than 4 organisms or organism groups are identified, the organism is considered as non-identified. In this case, a list of possible organisms is displayed, and the sum of confidence values is less than 100. When no match is found, the organism is considered as non- identified.

5.8.7.2.4

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6 Reporting Raw Data 6.1 Report data using the data report form in Appendix 8.2.

6.2 Upon completion the laboratory will email the completed data form to the Study Director. 6.3 Copies of all related test results (data sheets and confirmation results) should be retained by the collaborators for a minimum of one year. 7 Analyzing Raw Data 7.1 Analyze the data for correct identification, misidentification and unidentified organisms by laboratory. 7.2 The data are reported as number of species/strains correctly identified by laboratory. 7.2.1 For example, “Of the N specific inclusivity strains tested, N-2 were correctly identified and 2 were misidentified in Laboratory 1. Those strains misidentified were the following: …” 7.3 The study report should include a table which lists all species/strains tested, their source, origin and essential characteristics plus testing outcome by laboratory. 8 Appendices

8.1 Instructions to Collaborators 8.2 Test Portion Data Report Forms 8.3 Study Materials 8.4 Flow Diagram 8.5 Collaborator Comment Form 8.6 User Manual

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Appendices Appendix 8.1

Instructions to Collaborators VITEK MS Microbial Identification System Workflow Collaborative Study

General Instructions about Collaborative Studies Introduction The purpose of this document is to provide detailed instructions for performing the collaborative study for the VITEK MS Microbial Identification System. The study will be conducted by co-Study Directors Ron Johnson and John Mills. The Study Directors are responsible for providing method-specific training, providing test portions, providing select supplies as outlined in Appendix 8.3, clarifying procedures, collating results, and submitting a report to AOAC INTERNATIONAL. Should any questions arise before or during the course of the study, please direct them immediately to one of the co-Study Directors: Ron Johnson and John Mills bioMérieux, Inc. 595 Anglum Road, Hazelwood, MO, 63042 Email: ron.johnson@biomerieux.com ; john.mills@biomerieux.com Phone: 314-452-3241 Important Information 1. Read the methods carefully. If you have any questions, contact one of the co-Study Directors. 2. It is advised to make at least one practice run before the trial using your own materials so that you can minimize errors in manipulations. Check to make sure all supplies are on hand. 3. Begin the analyses on the specified date. Store the strains at refrigerated temperatures. It is essential for the validity of the trial that all collaborators commence the analysis on the designated day. Immediately upon receiving the shipment, confirm the contents with the Study Director by faxing or emailing the form provided in the shipment. 4. THIS IS A STUDY OF THE METHOD, NOT OF THE LABORATORY. THE METHOD MUST BE FOLLOWED AS CLOSELY AS PRACTICABLE, AND ANY DEVIATIONS FROM THE METHOD AS DESCRIBED, NO MATTER HOW TRIVIAL THEY MAY SEEM, MUST BE NOTED ON THE REPORT FORM. 5. Report all of your results as soon as analyses are completed. Do not do more or less than indicated in the instructions. For example, do not do duplicate analyses and report the best or average result. More or fewer results complicate the statistical analyses and may invalidate your results. Data sheets are provided with instructions and indicate which results to report. A space is provided on the Data Report Form to indicate any deviations to the methods or the instructions to collaborators. Please include any criticisms, suggested improvements, or general comments about the products on

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the Collaborators’ Comments Form provided. Any results that were derived from modified protocols should be included but must be clearly indicated. Results and comments should be returned to the Study Director immediately upon completion of the study. Information about this Collaborative Study Shipment Schedule and Receipt of Study Materials and Test Strains All test kits, study materials, media and reagents as outlined in Appendix 8.3 should be received 1 week prior to testing. Collaborators shall inventory all test kits, study materials, media and reagents against the packing lists upon arrival to ensure all materials have arrived. Contact the co-Study Directors if any test kits or study materials have not arrived by the Wednesday prior to analysis. Strains will be shipped by overnight courier on Wednesday of Week 1 to arrive on Thursday. Each package shall be examined for potential damage. The collaborator must alert the co-Study Directors if any packages appear compromised or if the contents are not cold to the touch. Each package should contain 12 strains with 3 digit random codes on non-selective agar slants. Inventory the strains to be sure all are present and alert the co-Study Directors if any are missing. The strains should be stored at refrigerated temperature until initiation of the study on the following Monday.

Table 1. Study Schedule (Specific Dates TBD)

Receipt of Test Kits and Materials Week 1, Wed-Friday Week 2, Wed-Friday Week 3, Wed-Friday

Receipt of Test Portions Set Week 1, Wed-Friday Week 2, Wed-Friday Week 3, Wed-Friday

Submission of Data Report Form

Collaborator Activity

Initiation of Analyses

Week 2, Monday Week 3, Monday Week 4, Monday

Week 4, Monday Week 5, Monday Week 6, Monday

Panel 1

Panel 2

Panel 3

Safety Precautions All samples, microbial cultures and inoculated products should be considered infectious and handled appropriately. Aseptic technique and usual precautions for handling the bacterial group studied should be observed throughout this procedure. Refer to "CLSI M29-A, Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline - Current revision". For additional handling precautions, refer to "Biosafety in Microbiological and Biomedical Laboratories - CDC/NIH - Latest edition", or to the regulations currently in use in each country.

Do not use VITEK MS-DS target slides after the expiration date. Before use, check that the

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packaging and components are intact. Allow the VITEK MS-DS target slides to come to room temperature before use.

Test Portion Analysis For each panel, all collaborators shall initiate the analysis according to the collaborator outline. Refer to the Study Flowchart in Appendix 8.4 for an overview of the method. 1. Culture Preparation 1.1 For each isolate, use a sterile loop to streak growth onto the appropriate agar. See Table 1 for a list of agars and incubation time periods for each panel. Observe plates each day for growth. Testing can be initiated when growth on the plates is observed. Note * - Growth may or may not be present on each agar plate during the incubation time period. In instances where growth appears on multiple agar plates for the same isolate, testing should be performed from each plate. If no growth is present, mark as no growth on the data sheet. Table 1: Panel Agar Plates and Incubation Condition Panel Agar Incubation Conditions

Tryptic Soy Agar with 5% Sheep Blood (TSAB), Modified Oxford Agar (MOX) Agar Listeria according to Ottavani and Agosti (ALOA) BACARA CHROMID S. aureus Elite agar (SAIDE)

18-72 h 35 o C

Panel 1

TSAB, ASAP, Xylose Lysine Deoxycholate (XLD), CHROMID Salmonella, Enterobacter Selective Isolation Agar (ESIA) CHROMID EHEC

18-72 h 35 o C

Panel 2

2 – 7 days 25 o C

Panel 3

Sabouraud Dextrose Agar (SDA)

2. VITEK MS Microbial Identification Method 2.1 General Preparation

2.1.1 Sample preparation is done in conjunction with the VITEK MS Prep Station. Review the VITEK MS Prep Station section of the User’s Manual before preparing the samples. Scan each VITEK MS-DS and use the keyboard to fill in the slide information. 2.2 Quality Control 2.2.1 Perform quality control analysis as outlined in the VITEK MS user manual or according to your standard laboratory procedures. 2.2.2 A negative control check, VITEK MS-CCHA with no bacteria, should be analyzed with each set of testing. 2.3 Sample Preparation for Bacteria NOTES: Powder-free gloves should be used when handling VITEK MS-DS target slides. Avoid touching the spots on the slide. Handle the slide by the end of the slide. Do not write or mark on the slide.

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2.3.1 Before adding samples to the VITEK MS-DS target slide, place the VITEK MS-DS target slide on a flat surface, such as a silicone mat, that will prevent it from sliding. 2.3.2 The single-use disposable VITEK MS-DS target slide with 48 sample positions is used with the VITEK MS system. The sample positions are divided into three sections. Each section is called an acquisition group. The three small spots in the center of each acquisition group must not be used for identification of microorganisms. These three small spots are used for calibration.

2.3.3 The calibration strain, E. coli ATCC 8739, is deposited on the calibration well positions, depending on the number of samples tested (1 calibrator per acquisition group of 16 spots). Follow the directions for Preparation of bacteria isolates (beginning in 5.8.2.4) to prepare the E. coli ATCC 8739 calibration strain. 2.3.4.1 Make sure not to pick up any agar with the colony as this will have a negative impact on the spectrum. 2.3.4.2 Some microorganisms, such as Streptococci , grow as very small colonies. In such cases, several similar colonies may be selected and deposited on one spot. 2.3.5 Apply the sample to the center of the spot. Smear a thin layer of the sample on the spot, then discard the used loop. 2.3.6 Take special care not to apply too much organism. The appropriate amount of organism on a spot is shown in the picture below. If insufficient colony material is deposited, use a new loop and introduce additional organism to the spot. 2.3.4 Using a 1 μl loop, pick up part of a suitable colony.

2.3.7 Open the microtube containing the VITEK MS-CHCA matrix. Add precisely 1.0 μl of matrix to the center of the spot. Discard the pipette tip. The matrix/microorganism suspension is then left to dry completely.

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2.3.8 It is strongly recommended to deposit the matrix as indicated in the VITEK MS Prep Station workflow for bacteria: 2 by 2 in duplicate mode Note: The VITEK MS-CHCA matrix contains a high concentration of organic solvents; it is recommended to close the tube after dispensing to avoid evaporation. 2.3.9 Proceed likewise for each additional sample. 2.3.10 After five minutes, check for crystal formation on the spots. Use of a magnifying lens may be helpful. An accurate measurement requires that the matrix crystals have become visible as a yellowish film. Ideally, most of the spot’s surface will be coated with crystals. 2.3.11 When the VITEK MS-DS target slides have been prepared, they must be tested within 72 hours. Prepared slides should be stored at room temperature in its original packaging until they are tested. 2.4 Sample Preparation for Yeast Isolates 2.4.1 The preparation of yeast isolates differs from the standard procedure in that VITEK MS-FA (formic acid treatment) is applied to the organism on the VITEK MS-DS prior to adding VITEK MS-CHCA. Care is taken not to add VITEK MS-CHCA until the VITEK MS-FA is completely dry, usually after 1 - 3 minutes. 2.4.2 The sample is applied to the VITEK MS-DS in the same manner as described in 5.8.2.4 to 5.8.2.6. 2.4.3 Add 0.5 μl of VITEK MS-FA to the sample, using a new pipette tip for each sample. Allow for enough time (1-3 minutes) for evaporation of VITEK MS-FA before adding VITEK MS-CHCA. Once the spot is completely dry, 1.0 μl of the VITEK MS-CHCA is added to each spot. 2.4.4 When the VITEK MS-DS target slides have been prepared, they must be tested within 72 hours. Prepared slides should be stored at room temperature in its original packaging until they are tested.

2.5

Sample Preparation for Mold Isolates 2.5.1 Wet a sterile cotton swab using API suspension media or sterile deionized water. Depress the tip of the swab to remove excess fluid. 2.5.2 Collect approximately a 1-2 cm diameter of mold from the agar plate. 2.5.3 Suspend the collected material in a 2 mL round-bottom tube containing 900 µL

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of 70% ethanol. Mix using a vortex-type mixer. 2.5.4 Centrifuge the tube for 2 minutes at a speed between 10,000 and 14,000 x g. 2.5.5 Discard all of the supernatant using a pipette. Use caution to avoid dislodging the pellet. 2.5.6 Add 40 µL of 70% formic acid and mix using a vortex-type mixer. 2.5.7 Add 40 µL of acetonitrile and mix using a vortex-type mixer. 2.5.8 Centrifuge the tube for 2 minutes at a speed between 10,000 and 14,000 x g. 2.5.9 Immediately spot 1 µL of the supernatant on a VITEK MS-DS slide. Allow to dry completely. 2.5.10 Once the spot is completely dry, 1.0 μl of the VITEK MS-CHCA is added to each spot. 2.5.11 When the VITEK MS-DS target slides have been prepared, they must be tested within 72 hours. Prepared slides should be stored at room temperature in its original packaging until they are tested. 2.6 Analysis on the VITEK MS Instrument 2.6.1 Follow VITEK MS package insert instructions for running the instrument. 2.6.2 Log onto the software, verify your VITEK MS-DS is dry and open the instrument drawer using the computer software. 2.6.3 Place the VITEK MS-DS into the adapter and scan the barcode. The application will display the spot positions of the test isolates. 2.6.4 Place the adaptor into the carrier, face up with angled corners to the front, barcodes on the left side. 2.6.5 Slide the adapter into the carrier until it cannot go any further and start the application. 2.6.6 When acquisition is complete, remove the VITEK MS-DS target slide. 2.7 Interpretation and Test Result Report 2.7.1 The VITEK MS system identifies organisms by comparing the characteristics of the obtained spectrum to the VITEK MS knowledge base. A percent agreement calculation is provided to the end user. 2.7.2 A quantitative value, the confidence value, is calculated and expressed the

similarity between the unknown organism and every organism or organism group of the knowledge base. Thresholds are applied in order to retain only the more significant organisms or organism groups. 2.7.2.1 A single identification is displayed with confidence value between 60 to 99.9 when one significant organism or organism group are identified. 2.7.2.2 Low Discrimination identifications are displayed when more than one significant organism or organism group are identified, but not more than 4 in total. In this case, the sum of confidence values is equal to 100. 2.7.2.3 When more than 4 organisms or organism groups are identified, the organism is considered as non-identified. In this case, a list of possible organisms is displayed, and the sum of confidence values is less than 100. 2.7.2.4 When no match is found, the organism is considered as non-identified.

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Reporting Results Complete the Data Report Form (included in your packet as Excel file Appendix 8.2) for each isolate noting any deviations. Once analyses are complete, finalize the Data Report Form, and submit it by email to co-Study Directors.

Collaborator Comment Form At the completion of the study, please fill out the Collaborator Comment Form (Appendix 8.5) to submit any comments you may have regarding the VITEK MS Microbial Identification System or the conduct of the study. Completed forms should be returned by email to the co-Study directors.

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VITEK MS Collaborative Study

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Appendix 8.3

Study Materials and Apparatus VITEK MS Microbial Identification System Collaborative Study

Materials provided by collaborator :

a. VITEK® MS.—bioMérieux, Inc., REF 411071 b. Vortex mixer c. Vortex-type mixer with adaptor or bead beater-type homogenizer d. Sterile cotton swabs e. Microcentrifuge able to reach 10,000 to 14,000 x g f. Incubator.—Capable of maintaining 25 ± 2 C and 35 ± 2 C. g. Acetonitrile h. Ethanol (99.8%, 96% or absolute) i. Calibrated loops.― 1μL j. Precision micropipette.―Capable of sampling and delivering 0.5-2μL k. Sterile colorless pipette tips without filter.―Capable of sampling and delivering 0.5-2μL l. Eppendorf tube.―Capable of sampling and delivering 2 mL m. Calibration strain: Escherichia coli ATCC 8739 n. Gram-stain reagents

Materials provided by Study Sponsor:

a. VITEK MS-CHCA matrix.—bioMérieux, Inc., REF 411071 b. VITEK MS-FA.—bioMérieux, Inc., REF 411072 c. VITEK MS-DS target slides.—bioMérieux, Inc., REF 410893 d. VITEK MS Mould Kit.—bioMérieux, Inc., 415680 e. API suspension medium.—bioMérieux, Inc., REF 70700/70640/20150 f. TSAB Plates.—bioMérieux, Inc., Ref 43001 g. Modified Oxford (MOX) Plates h. ALOA Plates .—bioMérieux, Inc., Ref AEB520080 i. BACARA.—bioMérieux, Inc., Ref AEB120102 j. CHROMID S. aureus ELITE Agar.—bioMérieux, Inc., Ref 419042 k. XLD Plates.—bioMérieux, Inc., Ref 43563.

l. ASAP Plates.—bioMérieux, Inc., Ref AEB520090US m. CHROMID Salmonella .—bioMérieux, Inc., Ref 43621 n. ESIA.—bioMérieux, Inc., Ref AEB520010 o. CHROMID EHEC.—bioMérieux, Inc., Ref 413697 p. Sabouraud Dextrose Agar (SDA) Plates

19

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VITEK MS Collaborative Study June 2019

Appendix 8.4: Collaborative Study Flowchart – VITEK MS Microbial Identification System – Panel 1

21

VITEK MS Collaborative Study June 2019

Appendix 8.4: Collaborative Study Flowchart – VITEK MS Microbial Identification System – Panel 2

22

VITEK MS Collaborative Study June 2019

Appendix 8.4: Collaborative Study Flowchart – VITEK MS Microbial Identification System – Panel 3

VITEK MS Collaborative Study

June 2019

Appendix 8.5 – Collaborator Comment Form

As part of the AOAC OMA First and Final Action Processes, feedback on the performance of alternative methods is sought. Please include any positive or critical feedback, suggested improvements, or general comments about the test method:

Collaborator:

Site:

Comments about the Method:

Comments about the Conduct of the Study:

23

℠  011501

VITEK ® MS Method for the Rapid Identification of Microorganisms 

1  2  3 

AOAC Performance Tested Method

Abstract   4  VITEK ®  MS was validated according to AOAC guidelines for Performance Tested Methods and Official  5  Methods precollaborative studies for the identification of microorganisms. In the combined internal and  6  independent laboratory studies 359 different claimed organisms (representing 208 Gram negative, 123  7  Gram positive and 28 yeast) were evaluated by the VITEK MS method. All test strains were cultured on  8  appropriate agar medium and growth conditions before analyzing on the VITEK MS instrument. Overall  9  percent correct identification in the combined Internal and Independent studies (6122/6413 VITEK MS  10  results from the 359 different organisms) was 95.5%. For each specific group of microorganisms tested  11  the percent correct identification was 94.4% for Gram‐negative organisms, 96.1% for Gram‐positive  12  organisms and 97.5% for yeast. The VITEK MS reagents were stable over 12 month time studies and  13  provided reproducible results when compared between lots.  Assay ruggedness was demonstrated for  14  three critical parameters, including the use of 1‐µL loops from two different suppliers, the age of the  15  culture on an agar plate before analysis and various suppliers of the agar media. Overall results indicate  16  that the VITEK MS method is an acceptable automated method for the identification of microorganisms.   

17  18  19  20  21  22  23  24  25 

Method Authors  

Ronald Johnson, John Mills, Pat Rule, Tom Bortz 

bioMérieux, Inc., 595 Anglum Road, Hazelwood, MO 63042 

Submitting Company 

bioMérieux, Inc., 595 Anglum Road, Hazelwood, MO 63042 

Independent Laboratory   26  Laboratory Services Division, University of Guelph, 95 Stone Road West, Guelph, Ontario H1H 8J7  27  Canada 

28  29  30  31 

Reviewers

Jim Agin, 1400 Harrison Avenue, Cincinnati, OH 45214  Wayne Ziemer, 1301 Kristen Lane, Loganville, GA 30052  32  Thomas Hammack, Microbial Methods Development Branch, Division of Microbiology, Office of  33  Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5100  34  Paint Branch Parkway, Rm 3E‐021, College Park, MD 20740 

35  36  37 

Scope of method  

( a ) Target organisms. – 359 Gram‐negative and Gram‐positive, aerobic and anaerobic  38

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1 

bacteria and yeast. See Table 1 for complete list.

( b ) Summary of Validated Performance Claims .– The VITEK MS was validated for the detection of  2  Gram‐negative and Gram‐positive, aerobic and anaerobic bacteria, and yeast. The method  3  correctly identified 95.5% of the microorganisms correctly: 94.4% Gram negative organisms,  4  96.1% Gram positive organisms and 97.5% yeast organisms using the combined internal and  5  independent study data. 

6  7  8 

Definitions  

( a ) Percent agreement.— The percentage of time the organism was correctly identified by the VITEK  9  MS method. 

10  11 

Principle of the Method  12  The VITEK MS is a mass spectrometer based on MALDI‐TOF (MALDI: Matrix‐assisted laser  13  desorption/ionization; TOF: Time‐of‐Flight) technology. A portion of a colony from an agar plate is  14  applied to a spot on a VITEK MS‐ DS target slide. A matrix solution is applied to the spot on the VITEK  15  MS‐DS target slide. The VITEK MS‐DS target slide is dried and then loaded into the VITEK MS. The sample  16  is submitted to multiple laser shots inside the VITEK MS mass spectrometer. The matrix absorbs the  17  laser light and vaporizes, along with the sample, in the process gaining an electrical charge (ionization).  18  Electric fields then guide the ions into the vacuum tube which separates them according to 'weight'  19  (mass, the smaller molecules flying faster than the larger ones), and their time of flight, and displays the  20  results as a series of lines or peaks (spectrum) which correspond to different fragments that have  21  broken away from the original molecules in the sample. By analyzing the pattern of fragments it is  22  possible to deduce the structure of the molecules. The sample spectra are compared to a database of  23  spectra developed from a number of bacterial species. The sample spectra are interpreted to provide  24  organism identification results associated with a confidence level. 

25  26  27  28  29  30  31  32  33  34  35  36  37  38

Materials and Methods 

Test Kit Information  

( a )   Kit Name .—VITEK ® MS ( b )  Catalog Number .—411071 

( c )    Ordering Information

USA – (800) 634 7656, prompt 1   Europe –  +33 4 78 87 77 51  Asia/Pacific – +33 4 78 87 77 51 

Additional supplies and reagents required but not provided 

(a) VITEK MS‐CHCA matrix (Ref. 411071)  (b) VITEK MS‐FA (formic acid, Ref. 411072)  

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1  2  3  4  5  6  7  8  9 

(c) VITEK MS‐DS target slides (Ref. 410893)  (d) API suspension medium (Ref. 70700/70640/20150)  

(e) Acetonitrile 

(f) Ethanol (99.8%, 96% or absolute) 

Apparatus 

(a) Calibrated loops (1μL) 

(b) Precision micropipette (0.5‐2μL) 

(c) Sterile colorless pipette tips without filter (0.5‐2μL) 

10  11  12  13  14 

(d) Eppendorf tube (2 mL) 

Standard Reference Materials 

(a) Calibration strain: Escherichia coli ATCC 8739 

Safety Precautions   15  All samples, microbial cultures and inoculated products should be considered infectious and handled  16  appropriately. Aseptic technique and usual precautions for handling the bacterial group studied should  17  be observed throughout this procedure. Refer to "CLSI M29‐A, Protection of Laboratory Workers From  18  Occupationally Acquired Infections; Approved Guideline ‐ Current revision". For additional handling  19  precautions, refer to "Biosafety in Microbiological and Biomedical Laboratories ‐ CDC/NIH ‐ Latest  20  edition", or to the regulations currently in use in each country.  21  Do not use VITEK MS‐DS target slides after the expiration date. Before use, check that the packaging and  22  components are intact. Allow the VITEK MS‐DS target slides to come to room temperature before use. 

23  24 

General Preparation  25  Sample preparation is done in conjunction with the VITEK MS Prep Station. Review the VITEK MS Prep  26  Station section of the User’s Manual before preparing the samples.  

27  28 

Sample Preparation for Bacteria  29  NOTES: Powder‐free gloves should be used when handling VITEK MS‐DS target slides. Avoid touching the  30  spots on the slide. Handle the slide by the end of the slide. Do not write or mark on the slide.  31  Before adding samples to the VITEK MS‐DS target slide, place the VITEK MS‐DS target slide on a flat  32  surface, such as a silicone mat, that will prevent it from sliding.  33  The VITEK MS‐DS target slide used for the VITEK MS is the disposable VITEK MS‐DS target slide with 48  34  sample positions. This VITEK MS‐DS target slide is for single‐use only. The sample positions are divided  35  into three sections. Each section is called an acquisition group. The three small spots in the center of  36  each acquisition group must not be used for identification of microorganisms. These three small spots  37  are used for calibration 38

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1  2 

Calibration Wells

3  4 

 Acquisition  

  Acquisition             Acquisition  

Group   5  6  The E. coli ATCC 8739 strain must be deposited on calibration well positions, depending on the number  7  of samples tested (1 calibrator per acquisition group of 16 spots). Follow the directions for Preparation  8  of Bacteria to prepare the E. coli ATCC 8739 calibration strain.  9  Using a 1 μl loop, pick up part of a suitable colony.  10  Make sure not to pick up any agar with the colony as this would have a negative impact on the  11  spectrum.  12  Some microorganisms, such as Streptococci, grow as very small colonies. In such cases, pick up several  13  similar colonies and deposit them on one spot.  14  NOTE: Do not deposit more than 1 (Single Deposit Mode) or 2 (Duplicate Deposit Mode) samples at a  15  time before adding matrix to the target spots.  16  Apply the sample to the center of the spot. Take special care not to apply too much organism. The  17  appropriate amount on a spot is shown in the picture below. If insufficient colony material was  18  deposited, take a new loop and introduce additional organism.  19  21  22  Smear a thin layer of the sample on the spot, then discard the used loop.  23  Open the microtube containing the VITEK MS‐CHCA matrix. Add precisely 1.0 μl of matrix to the center  24  of the spot. Discard the pipette tip. The matrix/microorganism suspension is then left to dry completely.  25  It is strongly recommended to deposit the matrix as indicated in the VITEK MS Prep Station workflow for  26  bacteria: 2 by 2 in duplicate mode or 1 by 1 in single mode.  27  Note: The VITEK MS‐CHCA matrix contains a high concentration of organic solvents; it is recommended  28  to close the tube after dispensing to avoid evaporation.  29      Group           Group  20     Too much     Too little          OK                OK 

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Proceed likewise for a second sample, etc.  1  After about five minutes check for crystal formation on the spots. Inexperienced users may find using a  2  magnifying glass helpful. Measurement will succeed only if matrix crystals have become visible as a  3  yellowish film. Ideally, most of the spot’s surface will be coated with crystals. 

4  5 

6  When the VITEK MS‐DS target slides have been prepared, they must be tested within 48 hours.  7  Prepared slides should be stored at room temperature until they are tested.  10  The preparation of yeast samples differed from the standard procedure in that VITEK MS‐FA (formic acid  11  treatment) was applied to the organism on the VITEK MS‐DS prior to adding VITEK MS‐CHCA. Care was  12  taken not to add VITEK MS‐CHCA until the VITEK MS‐FA was completely dry, usually after 1 ‐ 3 minutes.  13  The sample was applied to the VITEK MS‐DS as in the standard procedure. Then 0.5 μl of VITEK MS‐FA  14  was added to the sample, using a new pipette tip for each sample. Enough time was allowed for  15  evaporation of VITEK MS‐FA before adding VITEK MS‐CHCA. The cell suspension formed a thin, moist  16  film on the target position. This usually took between 1 ‐ 3 minutes. Once the slide was completely dry,  17  1.0 μl of the VITEK MS‐CHCA was added to each sample as in the standard procedure.  18  Follow VITEK MS package insert instructions for running the instrument.  19  Note: The E. coli calibrator was not treated with VITEK MS‐FA.  20  8  9  Sample Preparation for Yeast 

21  22 

Interpretation and Test Result Report  23  The VITEK MS system identified blind‐coded organisms (previously characterized) by comparing the  24  characteristics of the obtained spectrum to the VITEK MS knowledge base. A percent agreement  25  calculation was performed.  26  A quantitative value, the confidence value, was calculated and expressed the similarity between the  27

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unknown organism and every organism or organism groups of the knowledge base. Then thresholds  1  were applied in order to retain only the more significant organisms or organism groups.  2  (a) A single identification is displayed with confidence value between 60 to 99.9 when one significant  3  organism or organism group are retained.   4  (b) Low Discrimination identifications are displayed when more than one significant organism or  5  organism group are retained, but not more than 4. In this case, the sum of confidence values is  6  equal to 100.   7  (c) When more than 4 organisms or organism groups are found, the organism is considered as non‐ 8  identified. In this case, a list of possible organisms is displayed and the sum of confidence values  9  is less than 100.   10  (d) When no match is found, the organism is considered as non‐identified  13  This validation study was conducted under the AOAC Research Institute Performance Tested Method SM 14  program and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of  15  Microbiological Methods for Food and Environmental Surfaces  (1). Two studies were conducted to  16  comprise the Internal Validation portion of the PTM study:  a Food Drug and Administration (FDA)  17  Clinical Trial (2, 3) and a study at bioMerieux. University of Guelph Laboratory Services Division  18  performed the Independent laboratory study. The VITEK MS system identified blind‐coded organisms,  19  either unknown clinical isolates or previously characterized organisms by comparing the characteristics  20  of the obtained spectrum to the VITEK MS knowledge base. A percent agreement calculation was  21  performed.  24  The majority of the claimed organism data included in this study was collected in a FDA Clinical Trial to  25  evaluate the ability of the VITEK MS to identify microorganisms. There were 359 different organisms  26  tested with multiple strains of each organism resulting in a total of 6344 VITEK MS results. The clinical  27  trial data was augmented with 193 blind coded organisms tested at bioMérieux in St. Louis, MO. These  28  two internal studies generated 6413 VITEK MS results.  31  An FDA‐approved protocol was provided to five independent clinical laboratories. Each laboratory  32  tested any unknown clinical isolates using the VITEK MS and DNA sequencing. If there was a shortage of  33  strains for a specific claimed organism, bioMérieux provided stock isolates to each laboratory for  34  analysis.   35  Unknown clinical isolates stored on an agar plate or slant, never frozen. Frozen stock isolates, provided  36  by bioMérieux , were subcultured twice prior to testing.  For this validation study report, organisms from  37  the FDA Clinical Trial that will be claimed for industry use are presented.  There were others tested but  38  only claimed for clinical use.  39 11  12  Validation Study   22  23  Internal Validation Study 29  30  FDA Clinical Trial  

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