Reproducibility of food challenge to cow's milk: Systematic review with individual participant data meta-analysis.

BACKGROUND: Cow's milk (CM) is an increasingly common cause of severe allergic reactions, but there is uncertainty with respect to severity of reactions at low-level CM exposure, as well as the reproducibility of reaction thresholds. OBJECTIVE: We undertook an individual participant data (IPD) meta-analysis of studies reporting double-blind, placebo-controlled food challenges in CM to determine the rate of anaphylaxis to low-level exposures and the reproducibility of reaction thresholds. METHODS: We performed a systematic review and IPD meta-analysis of studies reporting relevant data. Authors were contacted to provide additional data and/or clarification as needed. Risk of bias was assessed using the National Institute for Clinical Excellence methodologic checklists. RESULTS: Thirty-four studies were included, representing data from over 1000 participants. The cumulative ED01 and ED05 (cumulative doses causing objective symptoms in 1% and 5% of the at-risk allergic population) were 0.3 (95% confidence interval [CI], 0.2-0.5) and 2.9 (95% CI, 1.6-5.4) mg, respectively. At meta-analysis, 4.8% (95% CI, 2.0-10.9) and 4.8% (95% CI, 0.7-27.1) of individuals reacting to ≤5 mg and ≤0.5 mg of CM protein had anaphylaxis (minimal heterogeneity, I2 = 0%). Then 110 individuals underwent repeat double-blind, placebo-controlled food challenges; the intraindividual variation in reaction threshold was limited to a ½-log change in 80% (95% CI, 65-89) of participants. Two individuals initially tolerated 5 mg CM protein but then reacted to this dose at a subsequent challenge, although neither had anaphylaxis. CONCLUSIONS: About 5% of CM-allergic individuals reacting to ED01 or ED05 exposure might have anaphylaxis to that dose. This equates to 5 and 24 anaphylaxis events per 10,000 patients exposed to an ED01 or ED05 dose, respectively, in the broader CM-allergic population. Most of these anaphylactic reactions would be mild and respond to a single dose of epinephrine.

Peanut cross-contamination in randomly selected baked goods.

BACKGROUND: The current standard of care for managing peanut allergy includes avoidance of peanut and use of injectable epinephrine; however, strict avoidance is difficult and accidental ingestion is common with potentially serious consequences. Despite vigilance and efforts to minimize the risk of accidental exposure, peanut protein cross-contamination continues to occur in a variety of foods, including baked goods. OBJECTIVE: To assess and quantify the presence of peanut protein contamination in certain baked goods. METHODS: Randomly selected baked goods were collected from bakeries in the New York and Miami metropolitan areas that sold a variety of ethnic cuisines. A second set of samples from the same bakeries was collected at least 1 week after to evaluate between-batch variability. Samples were sent to the Food Allergy Research and Resource Program to analyze peanut contamination by enzyme-linked immunosorbent assay. Consumption estimates were based on 2003 to 2010 National Health and Nutrition Examination Survey survey data. RESULTS: Of 154 samples from 18 bakeries, 4 (2.6%) had detectable peanut contamination with peanut protein levels ranging from 0.1 mg/100 g to 650 mg/100 g. Consumption estimates for single occasion ingestion of a contaminated item ranged from 0.07 mg to 832 mg of peanut protein. CONCLUSION: In this study, unintended peanut protein was present in a small, but not insignificant, proportion of baked goods, with the potential to trigger a reaction in individuals with peanut allergy. Some products contained high levels of unintended peanut protein. The current data support the potential for accidental exposure to peanut protein with its associated risk.

Using data from food challenges to inform management of consumers with food allergy: A systematic review with individual participant data meta-analysis.

BACKGROUND: Eliciting doses (EDs) (eg, ED01 or ED05 values, which are the amounts of allergen expected to cause objective symptoms in 1% and 5% of the population with an allergy, respectively) are increasingly being used to inform allergen labeling and clinical management. These values are generated from food challenge, but the frequency of anaphylaxis in response to these low levels of allergen exposure and their reproducibility are unknown. OBJECTIVE: Our aim was to determine (1) the rate of anaphylaxis in response to low-level peanut exposure and (2) the reproducibility of reaction thresholds (and anaphylaxis) at food challenge. METHODS: We conducted a systematic review and individual participant data meta-analysis of studies that reported at least 50 individuals with peanut allergy reacting to peanut at double-blind, placebo-controlled food challenge (DBPCFC) and were published between January 2010 and September 2020. Risk of bias was assessed by using National Institute for Clinical Excellence methodologic checklists. RESULTS: A total of 19 studies were included (covering a total of 3151 participants, 534 of whom subsequently underwent further peanut challenge). At individual participant data meta-analysis, 4.5% (95% CI, 1.9% to 10.1%) of individuals reacted to 5 mg or less of peanut protein with anaphylaxis (moderate heterogeneity [I2 = 57%]). Intraindividual thresholds varied by up to 3 logs, although this variation was limited to a half-log change in 71.2% (95% CI, 56.2% to 82.6%) of individuals. In all, 2.4% (95% CI, 1.1% to 5.0%) of patients initially tolerated 5 mg of peanut protein but then reacted to this dose at subsequent challenge (low heterogeneity [I2 = 16%]); none developed anaphylaxis. CONCLUSION: Around 5% of individuals reacting to an ED01 or ED05 level of exposure to peanut might develop anaphylaxis in response to that dose. This equates to 1 and 6 anaphylaxis events per 2500 patients exposed to an ED01 or ED05 dose, respectively, in the broader population of individuals with peanut allergy.

Comparison of physicochemical and emulsifying properties of commercial pea protein powders.

BACKGROUND: There is an increasing trend in the food industry to utilize plant-based proteins. Pea protein (PP) is one such protein that is a promising alternative emulsifier. However, there is a significant functionality gap between laboratory and commercially produced PP that limits its usage. The physicochemical and emulsification properties of five commercial PP powders were characterized to better understand the functionality gap. RESULTS: Four of the five commercial powders displayed low solubility, high surface hydrophobicity, and an abundance of large insoluble aggregates. High-pressure homogenization was able to break up the aggregates, reduce surface hydrophobicity, and increase solubility. There was a significant correlation between the homogenized solubility and the emulsification properties of the commercial PPs. There was not a significant correlation between the emulsification properties and the other physicochemical properties (unhomogenized solubility, zeta potential, surface hydrophobicity, and interfacial tension). CONCLUSIONS: The conformational changes caused by the commercial isolation process may disrupt the correlations between the physicochemical and emulsification properties of PP. Solubility is a key physicochemical property to enable good emulsification properties for PP. Homogenization is an effective step to improve the solubility of commercial PP and therefore promote its functional properties before industrial usage. © 2021 Society of Chemical Industry.

Tracking Gluten throughout Brewing Using N-Terminal Labeling Mass Spectrometry.

Gluten-containing grains cause adverse health effects in individuals with celiac disease. Fermentation of these grains results in gluten-derived polypeptides with largely uncharacterized sizes and sequences, which may still trigger an immune response. This research used N-terminal labeling mass spectrometry to characterize protein hydrolysates during each stage of bench-scale brewing, including malting, mashing, boiling, fermentation, and aging. Gluten hydrolysates from each brewing step were tracked, and the immunotoxic potential was evaluated by sequence comparison with peptides known to stimulate celiac immune responses. The results indicate that proteolysis and precipitation of gliadins occurring during brewing differ by protein region and brewing stage. The termini of gliadins were hydrolyzed throughout the entire brewing process, but the central regions remained relatively stable. Most hydrolysis occurred during malting, and most precipitation occurred during boiling. The addition of yeast yielded new cleavage sites but did not result in complete hydrolysis. Consistent detection of peptides within the clinically important regions of gliadin corroborated the hydrolytic resistance of this region. N-terminal labeling mass spectrometry served as a novel approach to track the fate of gliadin/gluten throughout bench-scale brewing. Consistently identified fragments could serve as improved targets for the detection of hydrolyzed gluten in fermented products.

Single-dose oral challenges to validate eliciting doses in children with cow's milk allergy.

BACKGROUND: There is increasing interest in the use of eliciting doses (EDs) to inform allergen risk management. The ED can be estimated from the distribution of threshold doses for allergic subjects undergoing food challenges within a specified population. Estimated ED05 values for cow's milk (the dose expected to cause objective allergic symptoms in 5% of the milk-allergic population) range from 0.5 mg to 13.9 mg cow's milk protein. We undertook a single-dose challenge study to validate a predicted ED05 for cow's milk of 0.5 mg protein. METHODS: Participants were recruited from 4 clinical centres. Predetermined criteria were used to identify patients reacting to 0.5 mg cow's milk protein (approximately 0.015 mL of fresh cow's milk). Children over 1 year underwent formal challenge to cow's milk to confirm clinical reactivity. RESULTS: 172 children (median age 6.0 (IQR 0.7-11) years, 57% male) were included in this analysis. Twelve (7.0%, 95% CI 3.7%-11.9%) children experienced objective symptoms that met the predetermined criteria. One participant had mild anaphylaxis that responded to a single dose of adrenaline, the remainder experienced only mild symptoms with no treatment required. We did not identify any baseline predictors of sensitization that were associated with objective reactivity to the single-dose challenge using 0.5 mg cow's milk protein. CONCLUSIONS: These data support an estimated ED05 for cow's milk of 0.5 mg protein. Values for ED05 above 0.5 mg for cow's milk protein proposed for allergen risk management need to be reviewed.

Can we define a level of protection for allergic consumers that everyone can accept?

Substantial progress has been made in characterising the risk associated with exposure to allergens in food. However, absence of agreement on what risk is tolerable has made it difficult to set quantitative limits to manage that risk and protect allergic consumers effectively. This paper reviews scientific progress in the area and the diverse status of allergen management approaches and lack of common standards across different jurisdictions, including within the EU. This lack of regulation largely explains why allergic consumers find Precautionary Allergen Labelling confusing and cannot rely on it. We reviewed approaches to setting quantitative limits for a broad range of food safety hazards to identify the reasoning leading to their adoption. This revealed a diversity of approaches from pragmatic to risk-based, but we could not find clear evidence of the process leading to the decision on risk acceptability. We propose a framework built around the criteria suggested by Murphy and Gardoni (2008) for approaches to defining tolerable risks. Applying these criteria to food allergy, we concluded that sufficient knowledge exists to implement the framework, including sufficient expertise across the whole range of stakeholders to allow opinions to be heard and respected, and a consensus to be achieved.

Deriving individual threshold doses from clinical food challenge data for population risk assessment of food allergens.

BACKGROUND: Food allergies are a significant public health issue, and the only effective management option currently available is strict avoidance of all foods containing the allergen. In view of the practical impossibility of limiting risks to zero, quantitative allergen risk assessment and management strategies are needed. OBJECTIVE: We sought to develop appropriate methods for informing population-based risk assessments and risk management programs to benefit all stakeholders but particularly patients with food allergy. METHODS: Individual thresholds for food allergens (maximum tolerable doses and minimum eliciting doses) can ideally be established through double-blind, placebo-controlled food challenges. If double-blind, placebo-controlled food challenge data are not available, data from widely used open food challenges using predefined objective criteria can also provide useful data regarding minimum eliciting doses. For more than 20 years, the Netherlands Organisation for Applied Scientific Research and the Food Allergy Research and Resource Program at the University of Nebraska-Lincoln have been collecting individual maximum tolerable doses and minimum eliciting doses that produce objective symptoms from published and unpublished clinical data to better refine knowledge regarding the sensitivity of the population to food allergens. RESULTS: In this article we provide in-depth insights into the methodology applied by the Netherlands Organisation for Applied Scientific Research and Food Allergy Research and Resource Program to derive individual maximum tolerable doses and minimum eliciting doses for objective symptoms from clinical food challenge data. More than 90 examples for determining individual allergic thresholds are presented. CONCLUSION: With the methodology presented in this article, we aim to stimulate harmonization and transparency in quantitative food allergen risk assessment and risk management programs, encouraging their wider adoption.

Evidence-based approaches to the application of precautionary allergen labelling: Report from two iFAAM workshops.

Food allergy is a major public health concern with avoidance of the trigger food(s) being central to management by the patient. Food information legislation mandates the declaration of allergenic ingredients; however, the labelling of the unintentional presence of allergens is less defined. Precautionary allergen labelling (PAL) was introduced by the food industry to help manage and communicate the risk of reaction from the unintended presence of allergens in foods. In its current form, PAL is counterproductive for consumers with food allergies as there is no standardized approach to applying PAL. Foods with a PAL often do not contain the identified food allergen while some products without a PAL contain quantities of common food allergens that are capable of inducing an allergic reaction. Integrated Approaches to Food Allergen and Allergy Risk Management (iFAAM) was an EU-funded project that aimed to improve the management of food allergens by the food industry for the benefit of people with food allergies. Within iFAAM, a clinically validated tiered risk assessment approach for food allergens was developed. Two cross-stakeholder iFAAM workshops were held on 13-14 December 2016 and 19-20 April 2018. One of the objectives of these workshops was to develop a proposal to make PAL effective for consumers. This paper describes the outcomes from these workshops. This provides the basis for the development of more informative and transparent labelling that will ultimately improve management and well-being in consumers with food allergy.

Understanding how consumers with food allergies make decisions based on precautionary labelling.

BACKGROUND: Understanding consumer perceptions is crucial if effective food safety policy and risk communication are to be developed and implemented. We sought to understand how those living with food allergy assess risk with precautionary allergen labelling (PAL) and their preference in how risks are communicated within a quantitative risk assessment (QRA) framework. METHODS: The Integrated Approaches to Food Allergen and Allergy Risk Management (iFAAM) labelling online survey was developed for adults and parents of children with food allergy and distributed across Germany, Ireland, Netherlands, Spain and UK via patient support groups. RESULTS: There were 1560 complete responses. 'This product is not suitable for' was selected as first choice for PAL by 46% overall and 'May contain' was selected as the first choice by 44%. Seventy-three percent reported that it would improve their trust in a product if a QRA process had been used to make a decision about whether to include 'may contain'. Overall, 66% reported that a 'statement + symbol' on the label indicating a QRA, would help them to understand the risk assessment process that had been used by the food manufacturer. CONCLUSIONS: Consumers want to know what process has actually taken place for the placing of a PAL and/or risk assessment statement on a particular food product. Our findings provide a basis for the development of more informative communication around food allergen risk and safety and support evidence-based policy-making in the context of the legislative requirements of the European Union's Food Information for Consumers Regulation.

Peanut Allergen Threshold Study (PATS): Novel single-dose oral food challenge study to validate eliciting doses in children with peanut allergy.

BACKGROUND: Eliciting doses (EDs) of allergenic foods can be defined by the distribution of threshold doses for subjects within a specific population. The ED05 is the dose that elicits a reaction in 5% of allergic subjects. The predicted ED05 for peanut is 1.5 mg of peanut protein (6 mg of whole peanut). OBJECTIVE: We sought to validate the predicted peanut ED05 (1.5 mg) with a novel single-dose challenge. METHODS: Consecutive eligible children with peanut allergy in 3 centers were prospectively invited to participate, irrespective of previous reaction severity. Predetermined criteria for objective reactions were used to identify ED05 single-dose reactors. RESULTS: Five hundred eighteen children (mean age, 6.8 years) were eligible. No significant demographic or clinical differences were identified between 381 (74%) participants and 137 (26%) nonparticipants or between subjects recruited at each center. Three hundred seventy-eight children (206 male) completed the study. Almost half the group reported ignoring precautionary allergen labeling. Two hundred forty-five (65%) children experienced no reaction to the single dose of peanut. Sixty-seven (18%) children reported a subjective reaction without objective findings. Fifty-eight (15%) children experienced signs of a mild and transient nature that did not meet the predetermined criteria. Only 8 (2.1%; 95% CI, 0.6%-3.4%) subjects met the predetermined criteria for an objective and likely related event. No child experienced more than a mild reaction, 4 of the 8 received oral antihistamines only, and none received epinephrine. Food allergy-related quality of life improved from baseline to 1 month after challenge regardless of outcome (η2 = 0.2, P < .0001). Peanut skin prick test responses and peanut- and Ara h 2-specific IgE levels were not associated with objective reactivity to peanut ED05. CONCLUSION: A single administration of 1.5 mg of peanut protein elicited objective reactions in fewer than the predicted 5% of patients with peanut allergy. The novel single-dose oral food challenge appears clinically safe and patient acceptable, regardless of the outcome. It identifies the most highly dose-sensitive population with food allergy not otherwise identifiable by using routinely available peanut skin prick test responses or specific IgE levels, but this single-dose approach has not yet been validated for risk assessment of individual patients.

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA.

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.

Prospective investigation on the transfer of Ara h 2, the most potent peanut allergen, in human breast milk.

BACKGROUND: Peanut allergy is one of the most severe food allergies. Whether breastfeeding induces tolerance to peanuts or on the contrary, pre-disposes at risk-babies to occult allergic sensitization to peanuts is still a matter of discussion. We sought to investigate the transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German breast milk study and to shed light on the time kinetics of Ara h 2 appearance. METHODS: We recruited 32 lactating, non-peanut-allergic women and collected breast milk samples at different time points after consumption of 100 g dry roasted peanuts. Breast milk samples were investigated for Ara h 2 with different immunological methods: by 2D immunoblotting with a patient's serum, by affinity enrichment using a monoclonal antibody against Ara h 2 followed by LC-MS/MS-based detection and by a competitive inhibition ELISA for the detection of Ara h 2 and its digestion-resistant peptides (DRP-Ara h 2). RESULTS: In a qualitative analysis, Ara h 2 could be identified in a breast milk sample by 2D immunoblot by means of a patient's serum and furthermore by immunoaffinity enrichment followed by LC-MS/MS analysis. In a semi-quantitative analysis, Ara h 2 and its digestion-resistant peptides were detected in the breast milk of 9 of 32 subjects. Evidence suggests that Ara h 2 is excreted individually either rapidly (after 1, 2, 3 or 4 h) or delayed (after 8 or 12 h) and in different concentrations. CONCLUSIONS: Time and concentration of secreted Ara h 2 in breast milk appears to be individually regulated. The identification of Ara h 2 in breast milk is the prerequisite for the investigation of its sensitizing or tolerogenic properties.

Allergen reference doses for precautionary labeling (VITAL 2.0): clinical implications.

BACKGROUND: There has been a dramatic proliferation of precautionary labeling by manufacturers to mitigate the perceived risk from low-level contamination from allergens in food. This has resulted in a significant reduction in choice of potentially safe foods for allergic consumers. OBJECTIVES: We aimed to establish reference doses for 11 commonly allergenic foods to guide a rational approach by manufacturers based on all publically available valid oral food challenge data. METHODS: Reference doses were developed from statistical dose-distribution modeling of individual thresholds of patients in a dataset of more than 55 studies of clinical oral food challenges. Sufficient valid data were available for peanut, milk, egg, and hazelnut to allow assessment of the representativeness of the data used. RESULTS: The data were not significantly affected by the heterogeneity of the study methodology, including little effect of age on results for those foods for which sufficient numbers of adult challenge data were available (peanut and hazelnut). Thus by combining data from all studies, the eliciting dose for an allergic reaction in 1% of the population estimated for the following were 0.2 mg of protein for peanut, 0.1 mg for cow's milk, 0.03 mg for egg, and 0.1 mg for hazelnut. CONCLUSIONS: These reference doses will form the basis of the revised Voluntary Incidental Trace Allergen Labeling (VITAL) 2.0 thresholds now recommended in Australia. These new levels will enable manufacturers to apply credible precautionary labeling and provide increased consumer confidence in their validity and reliability, as well as improving consumer safety.

Novel post-translationally cleaved Ara h 2 proteoforms: Purification, characterization and IgE-binding properties.

The 2S albumins Ara h 2 and Ara h 6 have been shown to be the most important source of allergenicity in peanut. Several isoforms of these allergens have been described. Using extraction and liquid chromatography we isolated proteins with homology to Ara h 2 and characterized hitherto unknown Ara h 2 proteoforms with additional post-translational cleavage. High-resolution mass spectrometry located the cleavage site on the non-structured loop of Ara h 2 while far UV CD spectroscopy showed a comparable structure to Ara h 2. The cleaved forms of Ara h 2 were present in genotypes of peanut commonly consumed. Importantly, we revealed that newly identified Ara h 2 cleaved proteoforms showed comparable IgE-binding using sera from 28 peanut-sensitized individuals, possessed almost the same IgE binding potency and are likely similarly allergenic as intact Ara h 2. This makes these newly identified forms relevant proteoforms of peanut allergen Ara h 2.

Validation procedures for quantitative gluten ELISA methods: AOAC allergen community guidance and best practices.

The food allergen analytical community is endeavoring to create harmonized guidelines for the validation of food allergen ELISA methodologies to help protect food-sensitive individuals and promote consumer confidence. This document provides additional guidance to existing method validation publications for quantitative food allergen ELISA methods. The gluten-specific criterion provided in this document is divided into sections for information required by the method developer about the assay and information for the implementation of the multilaboratory validation study. Many of these recommendations and guidance are built upon the widely accepted Codex Alimentarius definitions and recommendations for gluten-free foods. The information in this document can be used as the basis of a harmonized validation protocol for any ELISA method for gluten, whether proprietary or nonproprietary, that will be submitted to AOAC andlor regulatory authorities or other bodies for status recognition. Future work is planned for the implementation of this guidance document for the validation of gluten methods and the creation of gluten reference materials.