Multilaboratory Collaborative Study Research Articles

Determination and Identification of Nicarbazin, Measured as 4,4'-Dinitrocarbanilide (DNC), in Chicken Tissues by Liquid Chromatography With Tandem Mass Spectrometry: Final Action 2013.07.

BackgroundAOAC Method 2013.07 was adopted as First Action in 2013. Since then, the method has been used in numerous residue depletion studies with favorable comments from analysts.ObjectiveTo analyze data from residue depletion studies to support Final Action status.MethodTen residue depletion studies were conducted during May 2014 through May 2019. For each study, harvested incurred tissues were analyzed for nicarbazin using AOAC Method 2013.07 in 1 of 4 laboratories. Each analytical run included one or more fortified quality control test portions. The data from these known fortified matrix test portions were analyzed for reproducibility and repeatability.ResultsFor muscle tissues, relative recovery was 90.4% (95% CI 83.8 to 97.5); RSDr was 5.4% (95% CI 3.8 to 9.2); and RSDR was 7.9%. In the liver, values were 94.5% (95% CI 91.1 to 98.0), 5.8% (95% CI 4.1 to 9.9), and 6.8%, respectively. In the kidney, values were 91.5% (95% CI 85.3 to 98.1), 5.2% (95% CI 3.7 to 8.8), and 9.0%, respectively. In skin with adhering fat, values were 94.5% (95% CI 89.2 to 100.1), 8.9% (95% CI 6.3 to 15.1), and 8.9%, respectively. In all cases, repeatability and reproducibility were within acceptable limits.ConclusionsThe data and positive feedback support the transition of AOAC Method 2013.07 from First Action to Final Action.HighlightsFinal action status is supported by data collected during routine use of the method rather than a traditional multi-laboratory collaborative study. Data were subjected to statistical analysis using the pC-metamer, and then transformed back to the traditional C-metamer.

Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 μg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 μg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 μg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

Evaluation of the 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate for the Enumeration of E. coli and Coliforms: Collaborative Study, First Action: 2018.13.

The 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate is a selective and differential sample-ready-culture medium designed for the rapid enumeration of Escherichia coli (E. coli) and coliforms in the food and beverage industries. The 3M Petrifilm Rapid E. coli/Coliform Count Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria, the International Organization of Standards (ISO) 4832:2006 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coliforms-Colony-count technique, and ISO 16649-2:2017 Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli-Part 2 Colony-count technique at 44 degrees C using bromo-4-chloro-3- indolyl beta-D-glucuronide methods for the enumeration of E. coli and coliforms in dry dog kibble. The candidate method was evaluated using two diluents, Butterfield's phosphate buffered diluent and peptone salt solution, in a paired study design with each reference method in a multi-laboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels and an uninoculated control level were evaluated. The candidate and reference methods were not statistically different at each contamination level. Reproducibility values obtained during the collaborative study were similar between the candidate and reference methods. These results demonstrate that the candidate method is equivalent to the reference methods. 3M Petrifilm Rapid E. coli/Coliform Count Plate was recommended for Official First Action status for enumeration of E. coli and coliforms in a broad range of foods and environmental surfaces.

Evaluation of the MC-Media PadTM Yeast and Mold Device for the Enumeration of Yeast and Mold: Collaborative Study, First Action 2018.02.

Background: The MC-Media Pad™ Yeast and Mold (YM) is a ready-to use culture device that combines a test pad coated with medium and water-absorption polymers that is designed for the rapid quantification of yeast and mold in food products. Objective: The MC-Media Pad YM was compared with the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the enumeration of yeast and mold in frozen orange juice concentrate. Methods: The candidate method was evaluated using a paired study design in a multilaboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels (low, 10-100 CFU/g; medium, 100-1000 CFU/g; and high 1000-10 000 CFU/g) and an uninoculated control level (0 CFU/g) were evaluated. MC-Media Pad YM devices were enumerated after 48 and 72 h of incubation. Results: Plate count obtained by both methods were log-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and SD were determined for each contamination level. Conclusions: No statistical difference was observed between the MC-Media Pad YM (for both 48 and 72 h) and the FDA BAM for each contamination level. Highlights: The new method offers a convenient alternative to the reference method (FDA BAM) for detection of yeast and mold contamination in food products, yielding reliable and comparable results in 48 h compared to 5 days for the reference method.

Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin

USP’s peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.

Total Folate in Infant Formula and Adult Nutritionals by Trienzyme Extraction and LC-MS/MS Quantitation: A Multilaboratory Testing Study, Final Action 2011.06

Background: The need for an updated reference method for folate was identified as a priority by the AOAC's Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) in 2011. An Expert Review Panel (ERP) found AOAC Official MethodSM 2011.06 suitable for the purpose and approved it as a First Action Official Method. Objective: To determine the repeatability and reproducibility of Method 2011.06: Total Folate in Infant Formula and Adult Nutritionals by Trienzyme Extraction and LC-MS/MS Quantitation. Methods: A multilaboratory collaborative study was conducted. Eleven laboratories located in five countries participated and completed analysis of all multilaboratory testing (MLT) samples. The study was divided into two parts. In the first part, the laboratories analyzed two practice samples (blindly coded) using the updated folate Method 2011.06. The laboratories providing results within the expected range qualified for part two, in which they analyzed 11 MLT samples in blind duplicates. Results: The results were compared with the Standard Method Performance Requirements (SMPR 2011.006) established for folate. The precision results met the requirements stated in the SMPR for all of the samples. Repeatability and reproducibility relative standard deviations ranged from 3.5 to 6.6 and from 9.0 to 15.7%, respectively. Horwitz ratio values for all of the samples were well below 2 (0.61-1.06). Conclusions: The ERP determined that the method performance met the SMPR requirements in September 2017 after reviewing the presented MLT data. Highlights: The ERP recommended the method for Final Action status.

Sterols and Stanols in Foods and Dietary Supplements Containing Added Phytosterols: A Collaborative Study

AbstractAn international, multilaboratory collaborative study was carried out to evaluate the performance of Official Method Ce 12‐16 of the American Oil Chemists’ Society (AOCS) for the determination of plant sterols and stanols, collectively referred to as phytosterols, in foods and dietary supplements containing added phytosterols and in the phytosterol food additive concentrates used to prepare such products. AOCS Official Method Ce 12‐16 involves the extraction of free sterols/stanols and saponified steryl/stanol esters followed by the gas chromatographic separation and flame ionization detection of phytosterol trimethylsilyl ether derivatives. A total of 14 laboratories from six countries successfully completed the analysis of collaborative samples of foods (e.g., baked goods, beverages, margarines; n = 9), dietary supplements (n = 5), and phytosterol concentrates (n = 4). Study results for the contents of total phytosterols (weight/weight) were 0.19–8.4% for foods, 8.7–49% for dietary supplements, and 57–97% for concentrates. AOCS Official Method Ce 12‐16 showed acceptable performance for total and individual phytosterols, indicating that this method was suitable for the determination of added phytosterols in a wide variety of market products and concentrates. AOCS Official Method Ce 12‐16 is appropriate for the determination of the five major phytosterols (i.e., campesterol, stigmasterol, β‐sitosterol, campestanol, and sitostanol) that are the subject of the United States Food and Drug Administration's health claim for phytosterols and the reduced risk of coronary heart disease.

Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 - Salmonella for the Detection of Salmonella spp. in Select Foods and Environmental Surfaces: Collaborative Study, First Action 2016.01.

The 3M™ Molecular Detection Assay (MDA) 2 - Salmonella uses real-time isothermal technology for the rapid and accurate detection of Salmonella spp. from enriched select food, feed, and food-process environmental samples. The 3M MDA 2 - Salmonella was evaluated in a multilaboratory collaborative study using an unpaired study design. The 3M MDA 2 - Salmonella was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference method for the detection of Salmonella in creamy peanut butter, and to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 4.08 reference method "Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products and Carcass and Environmental Samples" for the detection of Salmonella in raw ground beef (73% lean). Technicians from 16 laboratories located within the continental United States participated. Each matrix was evaluated at three levels of contamination: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low inoculum level test portions produced difference in collaborator POD values of 0.03 (95% confidence interval, -0.10 to 0.16) for raw ground beef and 0.06 (95% confidence interval, -0.06 to 0.18) for creamy peanut butter, indicating no statistically significant difference between the candidate and reference methods.

Evaluation of the 3M™ Petrifilm™ Rapid Aerobic Count Plate for the Enumeration of Aerobic Bacteria: Collaborative Study, First Action 2015.13.

The 3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate is a sample-ready culture medium system containing dual-sensor indicator technology for the rapid quantification of aerobic bacteria in food products. The 3M Petrifilm RAC Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA BAM) Chapter 3 (Aerobic Plate Count) for the enumeration of aerobic bacteria in raw easy-peel shrimp and the Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6 (Standard Plate Count Method) for the enumeration of aerobic bacteria in pasteurized skim milk and instant nonfat dry milk (instant NFDM). The 3M Petrifilm RAC Plate was evaluated using a paired study design in a multilaboratory collaborative study following current AOAC validation guidelines. Three target contamination levels (low, 10-100 CFU/g; medium, 100-1000 CFU/g; and high 1000-10 000 CFU/g) were evaluated for naturally occurring aerobic microflora for each matrix. For raw easy-peel shrimp, duplicate 3M Petrifilm RAC Plates were enumerated after 24 ± 2 h incubation at both 32 and 35°C. Pasteurized skim milk 3M Petrifilm RAC Plates were enumerated after 24 ± 2 h incubation at 32°C, and instant NFDM 3M Petrifilm RAC Plates were enumerated after 48 ± 3 h incubation at 32°C. No statistical difference was observed between 3M Petrifilm RAC Plate and FDA BAM or SMEDP reference methods for each contamination level.

High Throughput Analytical Techniques for the Determination and Confirmation of Residues of 653 Multiclass Pesticides and Chemical Pollutants in Tea by GC/MS, GC/MS/MS, and LC/MS/MS: Collaborative Study, First Action 2014.09.

Thirty laboratories from fom North and South America, Europe, and Asia participated in this AOAC collaborative study (15 from China; five from Germany; two each from Italy and the United States; and one each from the Republic of Korea, Canada, Spain, Japan, Belgium, and India). Participants represented government regulatory, commercial testing, university, research institute, and private laboratories. The single-laboratory validated (SLV) tea method was evaluated in the collaborative study to determine the recovery and reproducibility of the method under multilaboratory conditions. Since there were no restrictions regarding the type of analytical instrumentation to use for the analyses, laboratories used a combination of equipment that included GC/MS, GC/MS/MS, and LC/MS/MS instruments from 22 different manufacturers, 21 brands of GC and LC columns, 13 different GC temperature programming profiles, 11 LC gradient elution programs, and six different vendor manufactured SPE cartridges. Even though all the analytical performance parameters for all the 653 compounds had been determined in the SLV study, guidance was obtained from an expert review panel of the AOAC Method-Centric Committee on Pesticide Residues to conduct the multilaboratory collaborative study based on 20 selected compounds that can be analyzed by GC/MS and 20 compounds that can be analyzed by LC/MS/MS. Altogether, 560 samples covering the 40 selected pesticides were analyzed in the study. These samples included green tea and oolong tea samples fortified typically at the European Union maximum residue limit for regulatory guidance and compliance, aged tea samples incurred with 20 pesticides, and green tea and oolong tea samples incurred with five pesticides. The analysis of the 560 samples generated a total of 82 459 test results by the 30 participating laboratories. One laboratory failed to meet the proficiency requirements in the precollaborative study. Therefore, its data submitted for the collaborative study were excluded from further analysis and interpretation. The results presented are therefore the 6638 analytical results obtained from the 29 remaining laboratories, which included 1977 results generated by GC/MS, 1704 results by GC/MS/MS, and 2957 results by LC/MS/MS. It was determined after application of the Grubbs and Dixon tests for outliers to the data sets that there were 65 outlier results from the 1977 GC/MS results (3.3%), 65 outlier results from the 1704 GC/MS/MS results (3.8%), and 57 outlier results out of 2957 LC/MS/MS results (1.9%), representing 0.98, 0.98, and 0.86%, respectively, of the 6638 results generated in the study. Analysis with the AOAC statistical software package also confirmed that the method is rugged, and average recovery, average concentration, RSDr, RSDR, and HorRat values all meet recovery and reproducibility criteria for use in multiple laboratories. The Study Director is recommending this method for adoption as an AOAC First Action Official MethodSM.

Evaluation of 3M™ Molecular Detection Assay (MDA) Listeria for the Detection of Listeria species in Selected Foods and Environmental Surfaces: Collaborative Study, First Action 2014.06.

The 3M™ Molecular Detection Assay (MDA) Listeria is used with the 3M Molecular Detection System for the detection of Listeria species in food, food-related, and environmental samples after enrichment. The assay utilizes loop-mediated isothermal amplification to rapidly amplify Listeria target DNA with high specificity and sensitivity, combined with bioluminescence to detect the amplification. The 3M MDA Listeria method was evaluated using an unpaired study design in a multilaboratory collaborative study and compared to the AOAC Official Method of AnalysisSM (OMA) 993.12 Listeria monocytogenes in Milk and Dairy Products reference method for the detection of Listeria species in full-fat (4% milk fat) cottage cheese (25 g test portions). A total of 15 laboratories located in the continental United States and Canada participated. Each matrix had three inoculation levels: an uninoculated control level (0 CFU/test portion), and two levels artificially contaminated with Listeria monocytogenes, a low inoculum level (0.2-2 CFU/test portion) and a high inoculum level (2-5 CFU/test portion) using nonheat-stressed cells. In total, 792 unpaired replicate portions were analyzed. Statistical analysis was conducted according to the probability of detection (POD) model. Results obtained for the low inoculum level test portions produced a difference in cross-laboratory POD value of -0.07 with a 95% confidence interval of (-0.19, 0.06). No statistically significant differences were observed in the number of positive samples detected by the 3M MDA Listeria method versus the AOAC OMA method.

Evaluation of 3M™ Molecular Detection Assay (MDA) Listeria monocytogenes for the Detection of Listeria monocytogenes in Selected Foods and Environmental Surfaces: Collaborative Study, First Action 2014.07.

The 3M™ Molecular Detection Assay (MDA) Listeria monocytogenes combines isothermal amplification and bioluminescence to detect Listeria monocytogenes with high specificity and efficiency in select foods and environmental samples. The 3M MDA Listeria monocytogenes method was evaluated using an unpaired study design in a multilaboratory collaborative study to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook 8.09 (2011) Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, and Egg Products and Environmental Samples for deli turkey, and the AOAC Official Method of Analysis(SM) 993.12 Listeria monocytogenes in Milk and Dairy Products for full-fat (4% milk fat) cottage cheese following the current AOAC guidelines. A total of 16 laboratories located in the continental United States and Canada participated in this collaborative study. For deli turkey, 125 g test portions were evaluated using heat-stressed cells by each method. For full-fat cottage cheese, 25 g test portions were evaluated using nonheat-stressed cells. Each matrix had three inoculation levels: an uninoculated control level (0 CFU/test portion), and two levels artificially contaminated with L. monocytogenes, a low inoculum level (0.2-2 CFU/test portion) and a high inoculum level (2-5 CFU/test portion). In total, 1584 unpaired replicate samples were analyzed. Statistical analysis was conducted according to the probability of detection (POD) model. Results obtained for the low inoculum level full-fat cottage cheese test portions produced a difference in cross-laboratory POD (dLPOD) value of -0.08 with a 95% confidence interval (CI) of (-0.20, 0.05). For the low-level deli turkey test portions, a dLPOD value of -0.02 with a 95% CI of (-0.14, 0.11) was obtained.

Evaluation of the 3M™ Petrifilm™ Rapid Yeast and Mold Count Plate for the Enumeration of Yeast and Mold in Food: Collaborative Study, First Action 2014.05.

The 3M™ Petrifilm™ Rapid Yeast and Mold (RYM) Count Plate is a simple, ready-to-use chromogenic culture method for the rapid detection and enumeration of yeast and mold in food products. The 3M Petrifilm RYM Count Plate method was compared to the U. S. Food and Drug Administration Bacteriological Analytical Manual (FDA BAM) Chapter 18, Yeasts, Molds and Mycotoxins and the ISO 21527:2008 Microbiology of Food and Animal Feeding Stuffs-Horizontal Method for the Enumeration for Yeast and Molds - Part 1: Colony Count Technique in Products with Water Activity Greater Than 0.95 and Part 2: Colony Count Technique in Products with Water Activity Less Than or Equal to 0.95 reference methods for raw almonds and raw frozen ground beef patties (77% lean). The 3M Petrifilm RYM Count Plate method was evaluated using a paired study design in a multi-laboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels (low, 10-100 CFU/g; medium, 100-1000 CFU/g; high 1000-10 000 CFU/g) as well as an uninoculated control level (0 CFU/g) were evaluated for each matrix. Samples evaluated by the 3M Petrifilm RYM Count Plate method were prepared in duplicate and incubated at both 25°C and 28°C. Plates at both temperatures were enumerated after 48 and 60 h of incubation. No significant difference was observed between the 3M Petrifilm RYM Count Plate method and the FDA BAM or ISO 21527 reference methods for each contamination level. No statistical differences were observed between samples analyzed by the 3M Petrifilm RYM Count Plate method (at either 25°C or 28°C) and the reference methods. No statistical significant differences were observed between enumeration of colonies at 48 and 60 h on the 3M Petrifilm RYM Count Plate method and the reference methods.

Evaluation of the 3M™ Petrifilm™ Salmonella express system for the detection of Salmonella species in selected foods: collaborative study.

The 3M™ Petriflm™ Salmonella Express (SALX) System is a simple, ready-to-use chromogenic culture medium system for the rapid qualitative detection and biochemical confirmation of Salmonella spp. in food and food process environmental samples. The 3M Petrifilm SALX System was compared using an unpaired study design in a multilaboratory collaborative study to the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) Microbiology Laboratory Guidebook (MLG) 4.07 (2013) Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products and Carcass and Environmental Sponges for raw ground beef and the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) Chapter 5, Salmonella (2011) reference method for dry dog food following the current AOAC validation guidelines. For this study, a total of 17 laboratories located throughout the continental United States evaluated 1872 test portions. For the 3M Petrifilm SALX System, raw ground beef was analyzed using 25 g test portions, and dry dog food was analyzed using 375 g test portions. For the reference methods, 25 g test portions of each inatrix were analyzed. The two matrices were artificially contaminated with Salmonella at three inoculation levels: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). Each inoculation level was statistically analyzed using the probability of detection statistical model. For the raw ground beef and dry dog food test portions, no significant differences at the 95% confidence interval were observed in the number of positive samples detected by the 3M Petrifilm SALX System versus either the USDA/FSIS-MLG or FDA/BAM methods.

Evaluation of VIDAS Listeria monocytogenes Xpress (LMX) for the detection of Listeria monocytogenes in a variety of foods: First Action 2013.11.

The VIDAS Listeria monocytogenes Xpress (LMX) is an automated rapid screening enzyme immunoassay for the detection of Listeria monocytogenes in food products. The VIDAS LMX method was compared in a multi-laboratory collaborative study to AOAC Official Method 993.12 Listeria monocytogenes in Milk and Dairy Products reference method following current AOAC guidelines. A total of 14 laboratories participated, representing government and industry, throughout the United States. One matrix, queso fresco (soft Mexican cheese), was analyzed using two different test portion sizes, 25 and 125 g. Samples representing each portion size were artificially contaminated with L. monocytogenes at three levels: an uninoculated control level [0 colony forming units (CFU)/test portion], a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). For this evaluation, 1800 unpaired replicate test portions were analyzed by either the VIDAS LMX or AOAC 993.12. Each level was analyzed using the Probability of Detection (POD) statistical model. For the low-level inoculated test portions, difference in collaborator POD (dLPOD) values of 0.04, (-0.08, 0.15) and 0.01, (-0.10, 0.13), with 95% confidence intervals, were obtained, respectively, for 25 and 125 g test portions. The range of the confidence intervals for dLPOD values for both the 25 and 125 g test portions contain the point 0.0 indicating no statistically significant difference in the number of positive samples detected between the VIDAS LMX and the AOAC method. In addition to Oxford Agar (OXA), VIDAS LMX test portions were confirmed using Agar Listeria Ottavani and Agosti (ALOA), a proprietary chromogenic agar for the identification and differentiation of L. monocytogenes and Listeria species. No differences were observed between the two selective agars. The VIDAS LMX method, with the optional ALOA agar confirmation method, was adopted as Official First Action status for the detection of L. monocytogenes in a variety of foods.

Evaluation of VIDAS UP Listeria assay (LPT) for the detection of Listeria in a variety of foods and environmental surfaces: First Action 2013.10.

The VIDAS UP Listeria (LPT) is an automated rapid screening enzyme phage-ligand based assay for the detection of Listeria species in human food products and environmental samples. The VIDAS LPT method was compared in a multi-laboratory collaborative study to AOAC Official Method 993.12 Listeria monocytogenes in Milk and Dairy Products reference method following current AOAC guidelines. A total of 14 laboratories participated, representing government and industry, throughout the United States. One matrix, queso fresco (soft Mexican cheese), was analyzed using two different test portion sizes, 25 and 125 g. Samples representing each test portion size were artificially contaminated with Listeria species at three levels, an uninoculated control level [0 colony-forming units (CFU)/test portion], a low-inoculum level (0.2-2 CFU/test portion), and a high-inoculum level (2-5 CFU/test portion). For this evaluation, 1800 unpaired replicate test portions were analyzed by either the VIDAS LPT or AOAC 993.12. Each inoculation level was analyzed using the Probability of Detection (POD) statistical model. For the low-level inoculated test portions, difference in collaborator POD (dLPOD) values of 0.01, (-0.10, 0.13), with 95% confidence intervals, were obtained for both 25 and 125 g test portions. The range of the confidence intervals for dLPOD values for both the 25 and 125 g test portions contains the point 0.0 indicating no statistically significant difference in the number of positive samples detected between the VIDAS LPT and the AOAC methods. In addition to Oxford agar, VIDAS LPT test portions were confirmed using Agar Listeria Ottavani and Agosti (ALOA), a proprietary chromogenic agar for the identification and differentiation of L. monocytogenes and Listeria species. No differences were observed between the two selective agars. The VIDAS LPT method, with the optional ALOA agar confirmation method, was adopted as Official First Action status for the detection of Listeria species in a variety of foods and environmental samples.

Evaluation of 3M™ Molecular Detection Assay (MDA) Salmonella for the Detection of Salmonella in Selected Foods: Collaborative Study

The 3M Molecular Detection Assay (MDA) Salmonella is used with the 3M Molecular Detection System for the detection of Salmonella spp. in food, food-related, and environmental samples after enrichment. The assay utilizes loop-mediated isothermal amplification to rapidly amplify Salmonella target DNA with high specificity and sensitivity, combined with bioluminescence to detect the amplification. The 3M MDA Salmonella method was compared using an unpaired study design in a multilaboratory collaborative study to the U.S. Department of Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook (USDA/FSIS-MLG 4.05), Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products for raw ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 5 Salmonella reference method for wet dog food following the current AOAC guidelines. A total of 20 laboratories participated. For the 3M MDA Salmonella method, raw ground beef was analyzed using 25 g test portions, and wet dog food was analyzed using 375 g test portions. For the reference methods, 25 g test portions of each matrix were analyzed. Each matrix was artificially contaminated with Salmonella at three inoculation levels: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). In this study, 1512 unpaired replicate samples were analyzed. Statistical analysis was conducted according to the probability of detection (POD). For the low-level raw ground beef test portions, the following dLPOD (difference between the POD of the reference and candidate method) values with 95% confidence intervals were obtained: -0.01 (-0.14, +0.12). For the low-level wet dog food test portions, the following dLPOD with 95% confidence intervals were obtained: -0.04 (-0.16, +0.09). No significant differences were observed in the number of positive samples detected by the 3M MDA Salmonella method versus either the USDA/FSIS-MLG or FDA/BAM methods.

Evaluation of VIDAS® UP Salmonella (SPT) Assay for the Detection of Salmonella in a Variety of Foods and Environmental Samples: Collaborative Study

The VIDAS UP Salmonella (SPT) uses recombinant phage proteins to detect Salmonella species in human and animal food products and production environmental samples after 18-26 h of enrichment. The VIDAS SPT assay is performed with the automated VIDAS or mini-VIDAS instruments. The VIDAS SPT method was compared in a multilaboratory collaborative study to the U.S. Department of Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook (USDA/FSIS-MLG) 4.05 (2011) Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products reference method following the current AOAC guidelines. A total of 15 laboratories representing government, academia, and industry throughout the United States participated. One matrix, raw ground beef, was analyzed using two different test portion sizes, 25 and 375 g. Each test portion was artificially contaminated with Salmonella at three inoculation levels, an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFUltest portion), and a high inoculum level (2-5 CFU/test portion). In this study, 1656 unpaired replicate samples were analyzed. Of those unpaired replicates, 476 were presumptive positive by the VIDAS method, with 475 confirmed positive by the traditional confirmation procedures and 476 confirmed positive by an alternative confirmation procedure. There were 411 confirmed positive replicates by the USDA/FSIS-MLG reference method. Statistical analysis was conducted according to the probability of detection (POD). For the low-level 375 g test portions, the following dLPOD values, with 95% confidence intervals, were obtained: 0.01 (-0.12, +0.15) for samples confirmed following the traditional confirmation; 0.02 (-0.18, +0.2) for samples confirmed following traditional confirmation on IBISA and ASAP; and 0.03 (-0.18, +0.24) for samples confirmed following the alternative confirmation on IBISA and ASAP. For the low-level 25 g test portions, the following dLPOD values, with 95% confidence intervals, were obtained: 0.41, (0.32, +0.49) for samples confirmed following the traditional confirmation, the traditional confirmation on IBISA and ASAP, and the alternative confirmation on IBISA and ASAP. With 0.0 within the confidence intervals for the 375 g test portions, there was no statistically significant difference in the number of positive samples detected by the VIDAS SPT method and the USDA/FSIS-MLG method at the 0.05 level. For the 25 g test portions, a statistically significant difference was observed between the VIDAS SPT method and the reference method for the low inoculum level, where the VIDAS SPT method recovered a higher number of positive results than the reference method. It is recommended that the VIDAS SPT method with the optional ASAP and IBISA agar confirmation method be adopted for Official First Action status for the detection of Salmonella in a variety of foods and environmental samples.

Multilaboratory Validation of a Luminex Microbead-Based Suspension Array for the Identification of the 11 Most Clinically Relevant Shiga Toxin--Producing Escherichia coli O Serogroups

AbstractRapid and high-throughput identification and serotyping of Shiga toxin--producing Escherichia coli (STEC) O serogroups is important for detecting, investigating, and controlling STEC infection outbreaks and removing hazardous products from commerce. A Luminex microbead-based suspension array has been developed to identify the 11 most clinically relevant STEC serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. Here we present results of a blinded multilaboratory collaborative study involving 10 participants from nine laboratories using 55 unknown strains. From the total 495 analyses, two false-positive and three false-negative results were obtained, indicating the assay to be a rapid, high-throughput, and robust method for identifying clinically relevant STEC serogroups.

Standardized Methods and Quality Control Limits for Agar and Broth Microdilution Susceptibility Testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum

An international multilaboratory collaborative study was conducted to develop standard media and consensus methods for the performance and quality control of antimicrobial susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum using broth microdilution and agar dilution techniques. A reference strain from the American Type Culture Collection was designated for each species, which was to be used for quality control purposes. Repeat testing of replicate samples of each reference strain by participating laboratories utilizing both methods and different lots of media enabled a 3- to 4-dilution MIC range to be established for drugs in several different classes, including tetracyclines, macrolides, ketolides, lincosamides, and fluoroquinolones. This represents the first multilaboratory collaboration to standardize susceptibility testing methods and to designate quality control parameters to ensure accurate and reliable assay results for mycoplasmas and ureaplasmas that infect humans.