Real-time PCR Detection Methods for Food Allergens (Wheat, Buckwheat, and Peanuts) Using Reference Plasmids.
Specific and sensitive real-time qualitative polymerase chain reaction (PCR) methods for the detection of food allergens including wheat, buckwheat, and peanuts were developed that could cancel between instrument effects and avoid risks of false-positives and false-negatives. In these real-time PCR analysis, the cutoff for determination of positive samples was set in every PCR run by using reference plasmids containing known copies of the target sequences. The copy numbers of the plasmids were used to detect the allergenic ingredients corresponding to 10 ppm (w/w) protein in highly processed foods (cooked for more than 30 min at 122 °C). Reference plasmid analysis for each real-time PCR run helped to minimize variability between runs and instruments (7900HT Real-Time PCR systems and Light Cycler Nano). It also helped to avoid false positives due to trace levels of contaminants from the laboratory environment or agricultural products. The specificity of the real-time PCR method was verified using 79 commonly used food materials and some of their relatives. The method was sensitive enough to detect those allergenic ingredients corresponding to 10 ppm (w/w) in seven types of incurred samples. The current official Japanese method was not able to detect the allergens in some of the incurred samples. The developed method can avoid false negatives due to lack of sensitivity and is useful to confirm positive ELISA screening tests.
Oral immunotherapy for peanut allergy (PACE): a systematic review and meta-analysis of efficacy and safety.
BACKGROUND: Oral immunotherapy is an emerging experimental treatment for peanut allergy, but its benefits and harms are unclear. We systematically reviewed the efficacy and safety of oral immunotherapy versus allergen avoidance or placebo (no oral immunotherapy) for peanut allergy. METHODS: In the Peanut Allergen immunotherapy, Clarifying the Evidence (PACE) systematic review and meta-analysis, we searched MEDLINE, EMBASE, Cochrane Controlled Register of Trials, Latin American & Caribbean Health Sciences Literature, China National Knowledge Infrastructure, WHO's Clinical Trials Registry Platform, US Food and Drug Administration, and European Medicines Agency databases from inception to Dec 6, 2018, for randomised controlled trials comparing oral immunotherapy versus no oral immunotherapy for peanut allergy, without language restrictions. We screened studies, extracted data, and assessed risk of bias independently in duplicate. Main outcomes included anaphylaxis, allergic or adverse reactions, epinephrine use, and quality of life, meta-analysed by random effects. We assessed certainty (quality) of evidence by the GRADE approach. This study is registered with PROSPERO, number CRD42019117930. RESULTS: 12 trials (n=1041; median age across trials 8·7 years [IQR 5·9-11·2]) showed that oral immunotherapy versus no oral immunotherapy increased anaphylaxis risk (risk ratio [RR] 3·12 [95% CI 1·76-5·55], I2=0%, risk difference [RD] 15·1%, high-certainty), anaphylaxis frequency (incidence rate ratio [IRR] 2·72 [1·57-4·72], I2=0%, RD 12·2%, high-certainty), and epinephrine use (RR 2·21 [1·27-3·83], I2=0%, RD 4·5%, high-certainty) similarly during build-up and maintenance (pinteraction=0·92). Oral immunotherapy increased serious adverse events (RR 1·92 [1·00-3·66], I2=0%, RD 5·7%, moderate-certainty), and non-anaphylactic reactions (vomiting: RR 1·79 [95%CI 1·35-2·38], I2=0%, high-certainty; angioedema: 2·25 [1·13-4·47], I2=0%, high-certainty; upper tract respiratory reactions: 1·36 [1·02-1·81], I2=0%, moderate-certainty; lower tract respiratory reactions: 1·55 [0·96-2·50], I2=28%, moderate-certainty). Passing a supervised challenge, a surrogate for preventing out-of-clinic reactions, was more likely with oral immunotherapy (RR 12·42 [95% CI 6·82-22·61], I2=0%, RD 36·5%, high-certainty). Quality of life was not different between groups (combined parents and self report RR 1·21 [0·87-1·69], I2=0%, RD 0·03%, low-certainty). Findings were robust to IRR, trial sequential, subgroup, and sensitivity analyses. INTERPRETATION: In patients with peanut allergy, high-certainty evidence shows that available peanut oral immunotherapy regimens considerably increase allergic and anaphylactic reactions over avoidance or placebo, despite effectively inducing desensitisation. Safer peanut allergy treatment approaches and rigorous randomised controlled trials that evaluate patient-important outcomes are needed.
Estimation of Health and Economic Benefits of Commercial Peanut Immunotherapy Products: A Cost-effectiveness Analysis.
IMPORTANCE: Commercial epicutaneous peanut immunotherapy (EPIT) and peanut oral immunotherapy (POIT) may offer significant quality-of-life improvements for patients with peanut allergy, but the cost-effectiveness of commercial peanut immunotherapies is uncharacterized. OBJECTIVE: To evaluate critical inputs associated with the cost-effectiveness of EPIT and POIT from a societal perspective. DESIGN, SETTING, AND PARTICIPANTS: Economic evaluation in which microsimulations with Markov modeling were performed evaluating virtual children aged 4 years over an 80-year time horizon. The base-case costs included a caregiver-reported willingness to pay of $3839 annually for safe and effective food allergy treatment. Estimates of predictive biomarkers or oral challenges were incorporated after the first year of therapy with additional analyses of immunotherapy risk reduction of anaphylaxis and probability of sustained unresponsiveness (SU) to peanut after 4 years. EXPOSURES: Children received EPIT, POIT, or no immunotherapy treatment (n = 10 000 per treatment strategy). MAIN OUTCOMES AND MEASURES: Rates of therapy-associated adverse reactions and quality-of-life improvements associated with changes in eliciting or tolerated peanut doses were modeled along with quality-adjusted life-years (QALYs), anaphylaxis, therapy-associated anaphylaxis, and fatalities. RESULTS: In the base-case analysis without SU to peanut, the EPIT strategy cost less than POIT (mean [SD] cost, $154 662 [$46 716] vs $163 524 [$56 800]) and had fewer total episodes of anaphylaxis (mean [SD], 1.33 [1.55] vs 3.83 [5.02] episodes) and fewer episodes of therapy-associated anaphylaxis (mean [SD], 0.62 [1.30] vs 3.10 [4.94] episodes) but had lower QALY accumulation (mean [SD], 26.932 [2.241] vs 26.945 [2.320] QALYs). The incremental cost-effectiveness ratio was $216 061 for EPIT and $255 431 for POIT. Models were sensitive to therapy cost, SU rates, health state utility, and risk reduction of anaphylaxis. With health state utility sensitivity analyses, the ceiling value-based cost (willingness-to-pay threshold $100 000/QALY) was between $1568 and $6568 for EPIT and between $1235 and $5235 for POIT. If high rates of SU to peanut can be achieved in longer-term models, EPIT and POIT could produce savings in terms of both cost and QALY.CONCLUSIONS AND RELEVANCE: In this simulated analysis, findings showed that EPIT and POIT may be cost-effective under some assumptions. Further research is needed to understand the degree of health state utility improvement associated with each therapy, degree of protection against anaphylaxis, and rates of SU.
Analysis of Oral Food Challenge Outcomes in IgE-mediated Food Allergies to Almond in a Large Cohort.
BACKGROUND: While almond-specific IgE-mediated food allergies have traditionally been equated with other tree nut allergies, outcomes of oral food challenges to almond and the utility of clinical testing to predict IgE-mediated almond hypersensitivity is not well known. OBJECTIVE: To describe almond oral challenge outcomes and assess the predictive value of clinical testing. METHODS: 603 almond challenges performed for 590 patients, aged 1 to 66 years, were analyzed from Massachusetts General Hospital allergy practices. Reactions were graded using the Niggemann and Beyer allergic reaction grading system and the Sampson 2006 NIAID anaphylaxis definition. RESULTS: Almond challenges included 545 passes (92%), 15 (3%) indeterminates, and 30 (5%) failures, in contrast with 31% challenge failures for other foods. Most reactions were mild; 21 (4%) had Grade 2/3 allergic symptoms, and 3 (0.5%) had anaphylaxis. Median almond-specific IgE was 0.89 kU/L (range: <0.35, >100), median skin prick test (SPT) was 4.0 mm (0, 28), and 475 subjects (81%) were sensitized to almond. Failure was associated with higher almond-specific IgE (p<0.001), larger almond SPT (p=0.001), higher peanut IgE (p=0.003), and a history of almond reaction (p<0.029). Almond-specific IgE, almond SPT, and age at challenge combined demonstrated good predictive value for Grade 2/3 allergic reactions by ROC analysis (AUC 0.83). CONCLUSIONS: The proportion of failed almond challenges (5%) was low in contrast with other allergens, suggesting that some almond challenges may be safely conducted with higher patient-to-staff ratios or potentially introduced at home. Though reactions are usually uncommon and mild, anaphylaxis is possible with high almond sensitization.
Non-digestible oligosaccharides scFOS/lcFOS facilitate safe subcutaneous immunotherapy for peanut allergy.
BACKGROUND: Improving the safety of subcutaneous immunotherapy (SCIT) for food allergy is necessary to reduce side effects and achieve long-term tolerance. We determined the effect of dietary supplementation with 1% non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) on safety and efficacy of SCIT using a peanut allergy mouse model. METHODS: After sensitization, mice received a scFOS/lcFOS or control diet for the rest of the study. To study safety of SCIT, mice were dosed with a single subcutaneous injection of peanut extract (PE) or PBS. To study efficacy, mice were dosed subcutaneously (SCIT, 3 times/week) with PE or PBS for 3 weeks. Hereafter, acute allergic skin responses, anaphylactic shock symptoms and body temperature were assessed. To study the mechanism in vitro, the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) line was sensitized with an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation. RESULTS: scFOS/lcFOS reduced anaphylaxis caused by a single PE SCIT dose. scFOS/lcFOS alone also reduced the acute allergic skin response. Moreover, scFOS/lcFOS supplementation resulted in lower MMCP-1 levels in serum after PE SCIT dose compared to control diet, while antibody levels were not affected by the diet. In vitro incubation with scFOS/lcFOS at 0.5% suppressed the degranulation of IgE-sensitized RBL cells. However, dietary supplementation with scFOS/lcFOS did not improve the efficacy of SCIT. CONCLUSIONS: We show that scFOS/lcFOS diet improves the safety of SCIT, as evidenced by lower anaphylactic responses without compromising the efficacy in a mouse model for peanut allergy. This effect is likely to result from the suppression of mast cell effector function.
First real-world safety analysis of preschool peanut oral immunotherapy.
BACKGROUND: In 2017, a clinical trial of 37 subjects demonstrated that preschool peanut oral immunotherapy (P-OIT) was safe, with predominantly mild symptoms reported and only 1 moderate reaction requiring epinephrine. OBJECTIVES: We sought to examine whether these findings would be applicable in a real-world setting. METHODS: As part of a Canada-wide quality improvement project, academic and community allergists administered P-OIT to preschool-age children who had (1) skin prick test wheal diameter greater than or equal to 3 mm or specific IgE level greater than or equal to 0.35 kU/L and history of reaction and/or positive baseline oral food challenge, or (2) no ingestion history and specific IgE level greater than or equal to 5 kU/L. Over 16 to 22 weeks, patients had biweekly clinic visits for updosing, and consumed the dose daily at home between visits. Target maintenance dose was 300 mg peanut protein. Symptoms were classified using a modified World Allergy Organization Subcutaneous Immunotherapy Reaction Grading System (1 mildest, 5 fatal). RESULTS: Of 270 patients who started P-OIT in the period 2017 to 2018, 243 reached maintenance, and 27 dropped out (10.0%); 67.8% of patients experienced reactions during buildup: 36.3% grade 1, 31.1% grade 2, and 0.40% grade 4. Eleven patients (4.10%) received epinephrine (10 patients received 1 dose, 1 patient received epinephrine on 2 separate days), representing 2.23% of reactions (12 of 538) and 0.029% of doses (12 of 41,020). CONCLUSIONS: We are the first group to describe preschool P-OIT in a real-world multicenter setting. The treatment appears to be safe for the vast majority of patients because symptoms were generally mild and very few reactions received epinephrine; however, life-threatening reactions in a minority of patients (0.4%) can still occur.
Food Allergen Sensitisation Patterns in Omani Patients with Allergic Manifestations.
OBJECTIVES: This study aimed to evaluate the relationship between food allergen sensitisation patterns and allergic manifestations in Omani patients and highlight the importance of specific immunoglobulin E (IgE) testing. METHODS: This retrospective study included all patients referred due to allergic manifestations to the Sultan Qaboos University Hospital (SQUH), Muscat, Oman, from November 2012 to November 2016. Specific IgE blood testing was performed to determine sensitisation to common foods known to cause allergic reactions. RESULTS: A total of 164 patients were referred to SQUH over the study period, with 35.4% presenting with one allergic manifestation, 48.8% with 2-3 and 15.9% presenting with more than three manifestations. There was a family history of allergies in 70.7% of patients. Eosinophil counts and total and specific IgE levels were elevated in 18.9%, 54.9% and 73.2% of patients, respectively. Patients demonstrated sensitisation to cow milk (47.6%), wheat (41.5%), chicken eggs (34.8%), mixed tree nuts (34.1%), lentils (33.5%), peanuts (32.9%), soy (32.3%), shrimp (23.2%) and fish (15.2%). Overall, 19.5% were sensitised to a single allergen, 14% were sensitised to 2-3 and 39.6% were sensitised to more than three allergens. Almost one-third (29.3%) of patients suffered from food-induced anaphylaxis, of which 85.4% were prescribed self-injectable adrenaline. CONCLUSION: To the best of the authors' knowledge, this study is the first to describe food allergen sensitisation patterns among Omani patients with allergic manifestations. In conjunction with clinical symptoms, the correct interpretation of specific IgE levels is important to diagnose food allergies and make safe decisions about reintroducing foods.
Food-induced anaphylaxis: role of hidden allergens and cofactors.
Food anaphylaxis is on the increase, with those who have an allergy to peanuts, tree nuts, milk, and seafood at the highest risk of developing such a reaction. However, the diet in many societies is increasingly varied, much of the food consumed is prepared outside the home, and meals are often composed of many different ingredients. Anaphylaxis may occur to a composite food, and it may be unclear whether the reaction is due to contamination or to a culprit allergen present in an added ingredient. Composite foods can contain many allergic proteins present in small amounts, which do not always have to be labeled, unless they feature in European or US labeling regulations. These "hidden" allergens include mustard, celery, spices, lupine, pea, natural food colourings, and preservatives, but can occasionally include allergenic material from contaminants such as cereal mites. Hidden allergens can provoke severe reactions to seemingly unconnected foods which might then lead to a diagnosis of idiopathic anaphylaxis. The same problem can arise with two well-known types of food allergy; wheat-dependant exercise induced anaphylaxis and allergy to non-specific Lipid Transfer Protein allergens, both of which might only manifest when linked to a cofactor such as exercise. Many of these risk factors for food anaphylaxis have a common link; the public's engagement with popular concepts of health and fitness. This includes the development of a food and exercise culture involving the promotion and marketing of foods for their health-giving properties i.e., meat substitutes, wheat substitutes, supplements and alternative, or "natural" remedies for common ailments. Some of these foods have been reported as the cause of severe allergic reactions, but because they are often viewed as benign unlikely causes of severe allergic reactions, could be considered to be hidden allergens. The best resource to elicit the likelihood of a hidden allergen provoking an allergic reaction is to take a detailed history of the allergic reaction, presence of co-factors, foods suspected, type of food and where it was consumed. A good knowledge of commonly used ingredients, and list of potential hidden allergen suspects are essential tools for the food allergy detective.
Detection and Quantification of Allergens in Foods and Minimum Eliciting Doses in Food-Allergic Individuals (ThRAll).
Risk-based approaches to managing allergens in foods are being developed by the food industry and regulatory authorities to support food-allergic consumers to avoid ingestion of their problem food, especially in relation to the traces of unintended allergens. The application of such approaches requires access to good quality data from clinical studies to support identification of levels of allergens in foods that are generally safe for most food-allergic consumers as well as analytical tools that are able to quantify allergenic food protein. The ThRAll project aims to support the application of risk-based approaches to food-allergen management in two ways. First, a harmonized quantitative MS-based prototype reference method will be developed for the detection of multiple food allergens in standardized incurred food matrices. This will be undertaken for cow's milk, hen's egg, peanut, soybean, hazelnut, and almond incurred into two highly processed food matrices, chocolate and broth powder. This activity is complemented by a second objective to support the development and curation of data on oral food challenges, which are used to define thresholds and minimum eliciting doses. This will be achieved through the development of common protocols for collection and curation of data that will be applied to allergenic foods for which there are currently data gaps.
Simultaneous Detection of 13 Allergens in Thermally Processed Food Using Targeted LC-MS/MS Approach.
Food allergy is a major concern for public health and food industries. Because of the large numbers of food ingredients to be tested, MS is considered an alternative to existing techniques in terms of high selectivity, sensitivity, and capability to analyze multiple allergens simultaneously. In this study, we developed the method for monitoring significant peptides derived from 13 food allergens (milk, eggs, cod, shrimp, lobster, almonds, brazil nuts, cashew nuts, hazelnuts, walnuts, peanuts, wheat, and soybeans) and evaluated it in thermally processed foods (bread, cookie, fried fish, and frozen pasta). To select significant peptides to monitor, we used a bioinformatics-based approach and experimental confirmatory analysis. It was demonstrated that the developed method could detect target food ingredients from thermally processed foods successfully.