Case Report: Allergic Reactivity to Mahaleb (Prunus mahaleb) Spice in a Subject With Almond and Other Tree Nut Allergies.

BACKGROUND: Mahaleb is an aromatic spice prepared from the fruit stone of the St. Lucie Cherry that is used as a flavoring agent in traditional Turkish and Middle Eastern baking. Immunodiagnostic kits for almond, which are based on polyclonal almond-specific IgG antibodies, have been shown to demonstrate considerable cross-reactivity with mahaleb as was incidentally discovered during a cluster of allergen-related food recalls in 2015. OBJECTIVE: Though acute allergy to almond is somewhat common, allergies to mahaleb have not been previously documented. However, based on antigenic similarity observed with almond-specific IgG, it is predicted that mahaleb nut proteins would exhibit some level of cross-reactivity with almond-specific IgE and may therefore potentiate acute allergic symptoms in individuals with food allergy to almond.Case Presentation: Herein, we report on a 40-year old Caucasian female with longitudinal history of multiple tree nut allergies including allergy to almond, presenting with moderate pruritus and oropharyngeal swelling shortly following ingestion of mahaleb seed kernels. METHODS AND RESULTS: Skin-prick testing using extracts compounded from pistachio, almond, and mahaleb revealed positive wheals measuring 8, 3, and 7 mm respectfully. Indirect enzyme-linked immunosorbent assay (ELISA) using plate-bound antigens prepared from pistachio, almond, and mahaleb revealed IgG positive responses to all three targets. ELISA and Western blot analysis performed using goat anti-almond polyclonal IgG demonstrated significant cross-reactivity between almond and mahaleb, but not to pistachio. CONCLUSION: This is the first documented case of acute allergy to mahaleb, co-occurring in the context of plural tree nut allergies, providing novel evidence that mahaleb may pose a risk to nut-allergic individuals and indicating a need for awareness of spice contamination with nut and mahaleb residues.

Opportunities and Challenges Using Non-targeted Methods for Food Fraud Detection.

In recent years, non-targeted methods have been a popular "buzz" phrase in food fraud detection. Using analytical instrumentation techniques, non-targeted methods have been developed and applied in many food and agricultural situations. However, confusion and misstatements remain regarding how the methods are used. This perspective will discuss the definitions related to non-targeted testing, the procedure of developing and validating methods, the techniques and data analysis, and opportunities and challenges regarding the use of this class of analytical methods. The perspective seeks to provide readers with the latest information regarding recent advances in the use of non-targeted methods.

Assessment of Avenins from Different Oat Varieties Using R5-Based Sandwich ELISA.

Gluten derived from wheat and related triticeae cereals possesses distinct amino acid sequences that provoke the immunopathogenic features of celiac disease (CD) in genetically susceptible individuals. However, the role of oat-derived gluten, or avenins, in CD pathogenesis remains a disputed matter, as evidenced by a lack in harmonized legislation regarding gluten classification in relation to gluten-free labeling. In this study, we have analyzed a panel of pure oat cultivars using a sandwich ELISA based on the R5 monoclonal antibody (mAb), which binds to canonical epitopes occurring within celiagenic peptides present in triticeae-derived gluten but reportedly not present in avenins. We have identified three varieties of oats that reproducibly bind R5 antibodies and levels indicating the presence of gluten at more than the 20 ppm gluten regulatory threshold. Nested assessment using Western blot analysis and alternative gluten detection systems corroborated these results. Collectively, these data suggest that select oat varieties may prove problematic to patients with CD and to food companies and regulatory agencies and will extend our basic understanding of current gluten detection systems.

A retrospective analysis of allergic reaction severities and minimal eliciting doses for peanut, milk, egg, and soy oral food challenges.

Food allergy is a public health concern, affecting up to 6% of children and 2% of adults. The severity of allergic reactions can range from mild to potentially life-threatening. In addition, the minimum amount of protein needed to provoke an allergic reaction in an individual patient (the minimal eliciting dose (MED)) ranges from a few micrograms to several grams. To determine whether a retrospective analysis of published data from oral food challenges could be used to assess the potential relationship between MEDs and reaction severities at the MEDs, a three class (mild, moderate, severe) reaction grading system was developed by integrating previously published reaction grading systems. MEDs and symptoms were collected from food challenge studies and each reaction was graded using the integrated grading system. Peanut allergic patients who experienced severe reactions had significantly higher MEDs and threshold distribution doses than those who experienced mild and moderate reactions. No significant differences in threshold distributions according to the severity grading were found for milk, egg and soy. The relationship between threshold dose distribution and reaction severity based on these grading criteria differed between peanut and other allergens, and severe reactions were found to occur in some patients at low MEDs for all of these food allergens.

Analysis of U.S. Food and Drug Administration food allergen recalls after implementation of the food allergen labeling and consumer protection act.

To avoid potentially life-threatening reactions, food allergic consumers rely on information on food labels to help them avoid exposure to a food or ingredient that could trigger a reaction. To help consumers in the United States obtain the information that they need, the Food Allergen Labeling and Consumer Protection Act of 2004 defined a major food allergen as being one of eight foods or food groups and any ingredient that contains protein from one of these foods or food groups. A food that contains an undeclared major food allergen is misbranded under the U.S. Food, Drug, and Cosmetic Act and is subject to recall. Food allergen labeling problems are the most common cause of recalls for U.S. Food and Drug Administration (FDA)-regulated food products. To help understand why food allergen recalls continue to occur at a high rate, information on each food allergen recall that occurred in fiscal years 2007 through 2012 was obtained from the FDA recall database. This information was analyzed to identify the food, allergen, root cause, and mode of discovery for each food allergen recall. Bakery products were the most frequently recalled food type, and milk was the most frequently undeclared major food allergen. Use of the wrong package or label was the most frequent problem leading to food allergen recalls. These data are the first reported that indicate the importance of label and package controls as public health measures.

A survey of food allergen control practices in the U.S. food industry.

Despite awareness of the importance of food allergy as a public health issue, recalls and adverse reactions linked to undeclared allergens in foods continue to occur with high frequency. To reduce the overall incidence of such problems and to ensure that food-allergic consumers have the information they need to prevent adverse reactions, it is important to understand which allergen control practices are currently used by the food industry. Therefore, the U.S. Food and Drug Administration carried out directed inspections of registered food facilities in 2010 to obtain a broader understanding of industry allergen control practices in the United States. The results of these inspections show that allergen awareness and the use of allergen controls have increased greatly in the last decade, but that small facilities lag in implementing allergen controls.

Workshop proceedings: challenges and opportunities in evaluating protein allergenicity across biotechnology industries.

A workshop entitled "Challenges and Opportunities in Evaluating Protein Allergenicity across Biotechnology Industries" was held at the 51st Annual Meeting of the Society of Toxicology (SOT) in San Francisco, California. The workshop was sponsored by the Biotechnology Specialty Section of SOT and was designed to present the science-based approaches used in biotechnology industries to evaluate and regulate protein allergenicity. A panel of experts from industry and government highlighted the allergenicity testing requirements and research in the agricultural, pharmaceutical/biopharma, and vaccine biotechnology industries and addressed challenges and opportunities for advancing the science of protein allergenicity. The main learning from the workshop was that immunoglobulin E-mediated allergenicity of biotechnology-derived products is difficult to assess without human data. The approaches currently being used to evaluate potential for allergenicity across biotechnology industries are very different and range from bioinformatics, in vitro serology, in vivo animal testing, in vitro and in vivo functional assays, and "biosimilar" assessments (ie, biotherapeutic equivalents to innovator products). The challenge remains with regard to the different or lack of regulatory requirements for allergenicity testing across industries, but the novel approaches being used with bioinformatics and biosimilars may lead to opportunities in the future to collaborate across biotechnology industries.

The regulatory challenge of food allergens.

Food allergy is an important public health issue worldwide. Allergic consumers must avoid eating foods that could provoke potentially life-threatening reactions, and successful avoidance depends on having complete and accurate information on food labels. Regulatory agencies support allergic consumers by working with industry to ensure that all food allergens that are intended to be present in a food are declared on the label and that effective controls are used to prevent the presence of unintended allergens. These regulatory activities take place in a complex legal and policy environment both domestically and internationally. Protecting allergic consumers in this complex environment requires effective use of public health data and risk assessments.

Comparison of international food allergen labeling regulations.

Food allergy is a significant public health issue worldwide. Regulatory risk management strategies for allergic consumers have focused on providing information about the presence of food allergens through label declarations. A number of countries and regulatory bodies have recognized the importance of providing this information by enacting laws, regulations or standards for food allergen labeling of "priority allergens". However, different governments and organizations have taken different approaches to identifying these "priority allergens" and to designing labeling declaration regulatory frameworks. The increasing volume of the international food trade suggests that there would be value in supporting sensitive consumers by harmonizing (to the extent possible) these regulatory frameworks. As a first step toward this goal, an inventory of allergen labeling regulations was assembled and analyzed to identify commonalities, differences, and future needs.

AllerML: markup language for allergens.

Many concerns have been raised about the potential allergenicity of novel, recombinant proteins into food crops. Guidelines, proposed by WHO/FAO and EFSA, include the use of bioinformatics screening to assess the risk of potential allergenicity or cross-reactivities of all proteins introduced, for example, to improve nutritional value or promote crop resistance. However, there are no universally accepted standards that can be used to encode data on the biology of allergens to facilitate using data from multiple databases in this screening. Therefore, we developed AllerML a markup language for allergens to assist in the automated exchange of information between databases and in the integration of the bioinformatics tools that are used to investigate allergenicity and cross-reactivity. As proof of concept, AllerML was implemented using the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/) database. General implementation of AllerML will promote automatic flow of validated data that will aid in allergy research and regulatory analysis.

Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil.

The Key Events Dose-Response Framework: a foundation for examining variability in elicitation thresholds for food allergens.

Food allergies are caused by immunological reactions in individuals sensitized to normal protein components of foods. For any given sensitized individual, the severity of a reaction is generally assumed to be proportional to the dose of allergenic protein. There is substantial clinical evidence that "threshold" doses exist for the elicitation of an allergic reaction; however, the threshold (i.e., lowest dose that elicits a reaction) varies substantially across the sensitized population. Current approaches to protecting sensitized individuals from exposure to food allergens are highly qualitative (i.e., they rely on food avoidance). The Key Events Dose-Response Framework is an analytical approach for refining understanding of the biological basis of the dose-response. Application of this approach to food allergy provides a foundation for a more rigorous quantitative understanding of variability in allergic response. This study reviews the allergic disease process and the current approaches to identifying thresholds for food allergens. The pathway of key biological events occurring between food intake and allergic response is considered, along with factors that may determine the nature and severity of response to food allergens. Data needs, as well as implications for identifying thresholds, and for characterizing variability in thresholds, are also discussed.

Allergen databases and allergen semantics.

The efficacy of any specific bioinformatic analysis of the potential allergenicity of new food proteins depends directly on the nature and content of the databases that are used in the analysis. A number of different allergen-related databases have been developed, each designed to meet a different need. These databases differ in content, organization, and accessibility. These differences create barriers for users and prevent data sharing and integration. The development and application of appropriate semantic web technologies, (for example, a food allergen ontology) could help to overcome these barriers and promote the development of more advanced analytic capabilities.

Cleaning and other control and validation strategies to prevent allergen cross-contact in food-processing operations.

Food allergies affect an estimated 10 to 12 million people in the United States. Some of these individuals can develop life-threatening allergic reactions when exposed to allergenic proteins. At present, the only successful method to manage food allergies is to avoid foods containing allergens. Consumers with food allergies rely on food labels to disclose the presence of allergenic ingredients. However, undeclared allergens can be inadvertently introduced into a food via cross-contact during manufacturing. Although allergen removal through cleaning of shared equipment or processing lines has been identified as one of the critical points for effective allergen control, there is little published information on the effectiveness of cleaning procedures for removing allergenic materials from processing equipment. There also is no consensus on how to validate or verify the efficacy of cleaning procedures. The objectives of this review were (i) to study the incidence and cause of allergen cross-contact, (ii) to assess the science upon which the cleaning of food contact surfaces is based, (iii) to identify best practices for cleaning allergenic foods from food contact surfaces in wet and dry manufacturing environments, and (iv) to present best practices for validating and verifying the efficacy of allergen cleaning protocols.

Allergen sequence databases.

A number of specialized databases have been developed to facilitate studies of human allergens. These include molecular databases focused on protein sequences and structures, informational databases focused on clinical, biochemical and epidemiological data related to protein allergens, a database on allergen nomenclature, and other knowledge bases or informational websites that are peripherally-related to research on allergens. Examples of each type of databases are listed and described briefly in this review. Database construction and maintenance and their impact on database quality and usefulness are also discussed.

Bioinformatics and food allergens.

Bioinformatics can play an important role in developing improved technology for the detection and characterization of food allergens. However, the full realization of this potential will depend on the development of allergen-specific databases as well as improved methods for data mining within these databases. Examples of existing allergen databases and analysis tools are described, as are the most important issues that need to be addressed in the next stage of database development.

Molecular, serological, and virulence characteristics of Vibrio parahaemolyticus isolated from environmental, food, and clinical sources in North America and Asia.

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.

Sequence analysis for assessing potential allergenicity.

Sequence analysis plays an important role in assessing the potential allergenicity of proteins used in transgenic foods, particularly for proteins that have not previously been part of the food supply. Sequence comparisons are used to indicate potential unexpected cross reactivity to existing allergens and to assess the potential for developing new sensitivities. Although the concept of using sequence analysis is straightforward, implementing a bioinformatic analysis that is accurate and complete can be complex. Several factors need to be considered, including the design and content of the sequence database, the analysis strategy, and the criteria for evaluating the results.

Fluorescent Antibodies Applied to Direct Epifluorescent Filter Technique for Microscopic Enumeration of Escherichia coli 0157:H7 in Milk and Juice.

In a modification of the direct epifluorescent filter technique (DEFT), direct fluorescent antibody staining was used for the rapid (<1 h), specific enumeration of foodbome Escherichia coli 0157:H7 by epifluorescence microscopy. Cell counts obtained by this method (Ab-DEFT) correlated well with DEFT counts obtained by acridine orange staining and with viable plate counts ranging from 108 to 101 cells per ml for pure cultures in buffer. Ab-DEFT also was effective for counting E. coli 0157:H7 cells inoculated into milk and juice; the sensitivity limit was about 103 for milk. The highly specific nature of the technique was demonstrated by enumeration of E. coli 0157:H7 cells in the presence of large numbers of indigenous spoilage microorganisms in milk. This is the first known demonstration of the combination of DEFT and antibody probe technology for the specific enumeration of a microbe directly in food without a growth or enrichment step.