Differences in egg and milk food challenge outcomes based on tolerance to the baked form

Peter Capucilli 1, Antonella Cianferoni 2, Joel Fiedler 2, Laura Gober 2, Nicholas Pawlowski 2, Gita Ram 2, Rushani Saltzman 2, Jonathan M Spergel 2, Jennifer Heimall 2

Abstract

Background: Previous studies suggest inclusion of baked egg and milk in the diet of children with egg or cow’s milk (CM) allergy might positively affect native tolerance. However, differences in native food reactivity based on historical baked tolerance are not fully understood.
Objective: To assess differences in native egg and CM oral food challenge (OFC) outcomes based on presenting history of tolerance and exposure to these foods in the baked form.
Methods: This study is a retrospective review of all egg and CM OFCs at the Children’s Hospital of Philadelphia (Philadelphia, Pennsylvania) over 4 years (N = 580). History of baked ingestion was compared with OFC pass rate, eliciting dose, epinephrine use, reaction classification, and recent skin test reaction or specific immunoglobulin E level.
Results: There were 115 egg- and 70 CM-positive challenge reactions, with most eliciting anaphylaxis. Children tolerating baked egg passed OFC more frequently (75%) compared with children who avoided baked egg (58%; P = .01) or never ingested egg (45%; P < .0001). For positive reactions, children tolerant of baked egg reacted at higher eliciting doses of native egg (median 3.0 g, range 0.125-15.75 g) compared with those avoiding baked egg (median 0.69 g, range 0.13-10.0 g; P = .03) and those with no egg exposure (median 0.88 g, range 0.13-13.88 g; P = .01). Further, epinephrine use was lower in children tolerating baked egg (10%) compared with children avoiding baked egg (22%; P = .02) and compared with children who never ingested egg (32%; P = .0001). These differences were not observed for CM challenges.
Conclusion: Children who historically tolerated baked egg were less sensitive to native egg during OFC compared with children whose baked reactivity was largely unknown.

INTRODUCTION

Cow's milk (CM) and egg allergy remain the leading causes of food allergy in children, with estimated prevalence of roughly 2% for both foods1-3. There exists a significant economic burden associated with food allergy, both for healthcare systems and for families affected, including costs of routine office visits and oral food challenges (OFC)4. Likewise, the considerable impact on patient and family quality of life contributes to challenges in food allergy management5. The prognosis of childhood CM or egg allergy is generally favorable with roughly 80% of children eventually developing tolerance6. Nevertheless, allergic sensitivity and risk for allergic reaction may persist through childhood, with some patients only developing tolerance in adolescence or later7,8.
It is well described that some children with native CM or egg allergy are able to tolerate these foods in the baked form9. Reports have further suggested that inclusion of baked milk (BM) or baked egg (BE) in the diet may even accelerate native tolerance,9-11 though a recent meta-analysis by Lambert et al. found little evidence to support this hypothesis12. While reports suggest that up to 70-80% of children fall into the baked tolerant category13-17, determining which children will prove to be intolerant remains difficult without performing a direct oral challenge. Several studies have attempted to establish predictive cut off values for baked tolerance using skin prick testing (SPT) and specific IgE levels14,16, but results have shown variability and clear risk for severe reactions even at low cut off values, and thus OFC remains a necessary obstacle to baked introduction9,12. Still, with the potential to positively impact children’s allergenicity, further studies to understand differences in tolerance and reactivity to native egg and CM based on baked tolerance are needed.
In the clinical setting, children with egg or CM allergy often present with history of tolerance, intolerance or avoidance to the baked form, with others who have had no direct exposure, deferring introduction following positive allergy testing. To assess potential differences in native egg and CM reactivity based on children’s presenting history of baked tolerance and exposure, we retrospectively reviewed OFC outcomes to egg and CM in the native form. Specifically, we sought to determine if in children presenting to allergy clinics for egg or CM food challenges, does the historical feature of tolerating BE/BM versus avoiding BE/BM (with largely unknown tolerance) versus never eating any egg/CM predict the rate of passing the challenge, or impact other challenge outcomes. We hypothesized that children who had incorporated BE or BM into their diet would demonstrate more favorable OFC outcomes and increased native tolerance compared to those with baked avoidance or no previous exposure.

METHODS

We conducted a retrospective chart review of all patients who underwent native egg and CM OFCs at the Children’s Hospital of Philadelphia (CHOP), Philadelphia, PA, between January 2012 and December 2015. We elicited patient clinical data including previous histories of asthma, atopic dermatitis, allergic rhinoconjunctivitis, and history of other food allergies as well as skin prick test (SPT) and CM or egg-specific IgE antibody levels. Food challenge outcomes, reaction type, eliciting dose and treatments were also obtained. In addition, we elicited patients’ previous history of known ingestion or exposure to CM or egg in the baked form prior to challenge date. As depicted in Figure 1, subjects were then defined into three subgroups for primary analysis: (a) those with history of allergic reaction to native egg or CM, with subsequent tolerance (Tol) of the respective food in the baked form (BE/BM Tol), (b) those with prior reaction to native egg or CM, who maintained strict avoidance (Avoid) of the baked form (BE/BM Avoid), and (c) those who never ingested (NI) egg or CM in the native or baked form, maintaining strict avoidance following positive food allergy testing (Egg/Milk NI). All children had positive SPT and/or specific IgE antibody level prior to food challenge to confirm allergic sensitization to the given food. We then compared differences in OFC outcomes, eliciting doses, and epinephrine use based on these subgroup differentiations. Additionally, we evaluated differences in reaction classification for positive (failed) challenges. Finally, we sought to determine if differences in subgroup OFC outcomes were reflected in SPT or IgE levels prior to challenge date. The study was reviewed and approved by the CHOP Institutional Review Board, IRB number 07-005420.

Food Challenge Protocol

We performed open OFCs based on previously described challenge protocols18,19. Challenge doses administered were 125mg, 250mg, 500mg,1g, 2g, 4g, 8g, and ad lib. In certain clinical scenarios, a lower starting dose (40-60mg) was used with concern for high risk of reaction. Challenges were administered using egg or CM powdered protein (Barry Farm Enterprises, Wapakoneta, OH, USA) that was masked in foods such as applesauce, pudding or juice20,21. Doses were administered in 20-minute observation intervals until the patient reached the final dose or experienced an objective reaction. Following completion of the challenge, patients were monitored for 2.5 hours prior to discharge. Challenges were stopped for respiratory, gastrointestinal, cardiovascular, and neurologic symptoms as well as non-contact cutaneous reactions or multi-system reactions. The protocol was standardized among providers with the usual anaphylaxis rescue medications available during all challenges. All children withheld antihistamines for at least 3 days prior to OFC.

Classification of Reactions

Reactions were categorized based on previously established classifications19 as either isolated cutaneous, respiratory, gastrointestinal, or multi-organ system anaphylaxis. Cutaneous reactions included hives, atopic dermatitis flare, flushing, or angioedema, but did not include mild contact reaction around the mouth. Respiratory symptoms included rhinitis, sneezing, throat closing, voice change as well as lower respiratory symptoms consisting of cough, wheeze, shortness of breath, increased work of breathing, tachypnea. Gastrointestinal reactions included abdominal pain or discomfort, emesis, and diarrhea. Anaphylaxis was characterized by symptoms involving multiple organ systems as per established guidelines22. Patients were monitored for 2.5 hours following any allergic reaction.

Skin Prick and Serum IgE Testing

Skin testing was performed by the prick method using bifurcated needles and commercial extracts (Bayer Laboratories, Spokane, WA; Greer Laboratories, Lenoir, NC, USA). All skin tests were performed within 1 year of challenge date. Maximum wheal and flare diameter were measured at 15 minutes with a wheal of 3mm greater than the negative control, accompanied by a flare, considered positive. A positive control of 10mg/mL of histamine dihydrochloride was also placed. Serum samples included whole egg or CM-specific IgE antibody concentrations by the Pharmacia CAP system FEIA (Pharmacia and Upjohn Diagnostics, Uppsala, Sweden). IgE levels were drawn at variable intervals prior to OFC date (Median: 6 months, Range: 0.25-81 months), with the most recent level used for analysis. A value of 0.35 kU/L was considered the lower limit of detection, as previously detailed18.

Statistical Analysis

Median and range values were calculated for patient age at time of challenge, SPT, specific IgE level and eliciting dose for positive reactions. Prevalence rates for baseline characteristics, epinephrine use and overall challenge outcomes were also determined. The Mann-Whitney U test (to compare two subgroups) and the Kruskal Wallis test (to compare three groups) were used to compare differences in age, SPT wheal diameter, specific IgE level, and eliciting dose between subgroups, assuming these variables were not normally distributed. The chi-square analysis of independent variables was used to compare differences in subgroup history of asthma, atopic dermatitis, allergic rhinitis, presence of other food allergy, and gender.
Dichotomous variables were analyzed using the Fisher’s exact test, including rates for challenge outcome and use of epinephrine. We created a Kaplan-Meier survival curve to further assess differences in OFC pass rate by increasing eliciting dose and calculated differences between cohorts using a Log-rank (Mantel-Cox) test for trend. A p value <0.05 was considered statistically significant. A multivariable logistic regression analysis for all primary outcomes (pass rate, eliciting dose, epinephrine use) considering age as a variable was performed using Stata v15 (StataCorp LP, College Station, Tex). All other statistical analysis performed with GraphPad Version 7 (Prism Software, San Diego, CA).

RESULTS

Subjects and Demographics

A total of 580 charts were reviewed with 11 challenges (4 egg and 7 CM) excluded due to insufficient intake or illness at time of challenge, leaving 569 challenges that were included in our final analysis. The total number and percentage of subjects in each cohort is depicted in Figure 1. For the Egg NI and Milk NI cohorts, the documented indication for allergy testing prior to ingestion is also shown. Reported reasoning for food allergy testing included five primary indications with roughly half of patients (52%) tested in the setting of atopic dermatitis thought to be secondary to food allergy. Another 1/3rd were tested following diagnosis of IgE-mediated allergy to another food or with history of sibling with food allergy. Patients ranged from 11 months - 17 years of age with a median overall age of 5 years. Differences in patient demographics were compared by three-way analysis of egg or CM subgroups. For egg, a significant difference was found for overall patient age between the three subgroups (P=0.02). Subjects who never ingested egg (Egg NI) showed higher rates of atopic dermatitis (P=0.01), history of other food allergy (P=0.002) and had higher overall SPT values (P=0.004) and specific-IgE levels (P=0.049) comparatively. A difference in overall patient age between CM cohorts was also appreciated (P=0.0023), with the rate of atopic dermatitis also notably highest in the Milk NI subgroup (P=0.004). Specific IgE levels to CM were highest in BM tolerant subjects (P=0.03). Analysis of other subject demographics found no differences between egg and CM subgroups for gender, history of asthma, or history of allergic rhinoconjunctivitis (Table 1).

Oral Food Challenge Outcomes

Of the 569 challenges analyzed, there were 342 egg challenges, 115 (34%) of which elicited a positive reaction. Of the 227 challenges to CM, 70 (31%) were positive. For egg challenges, a higher proportion of the BE Tol group (n=158) passed OFC compared to the BE Avoid group(n=42)(75% vs. 58%; P=0.01), and compared to the Egg NI group (n=27)(75% vs. 45%; P<0.0001). No significant difference was found in pass rate between BE Avoid and Egg NI subgroups (P=0.16). For CM, no differences were found for OFC pass rate between subgroups (Fig.2A).
For positive reactions, BE Tol subjects (n=53) reacted at higher eliciting doses (Median 3.0g, range 0.125-15.75g) compared to BE Avoid subjects (n= 30) (Median 0.69g, range 0.1310.0g; P=0.03) and Egg NI subjects (n=32) (Median 0.88g, range 0.13-13.88g; P=0.01). Eliciting doses were no different between BE Avoid and Egg NI subgroups (P=0.85). Further, no differences in eliciting dose by subgroup analysis were appreciated in challenges to CM (Fig. 2B). Log-rank survival analysis for percent of negative (passed) OFCs compared to increasing eliciting dose (g) was also significantly different between subgroups for egg challenges (P<0.0001), with no differences appreciated for CM (P=0.98) (Fig. 3 A/B).
Finally, a total of 57 reactions to egg and 34 reactions to CM required epinephrine. This accounted for 50% of all positive reactions to egg and 49% of all positive reactions to CM and 17% and 15% of total challenges to egg and CM, respectively. Compared to BE Tol subjects(n=22), BE Avoid subjects (n=16) required epinephrine more frequently for positive reactions (10% vs. 22%; P=0.02). Egg NI subjects (n=19) similarly required a higher rate of epinephrine administration (32%) compared to BE Tol subjects(P=0.0001), but not compared to BE Avoid subjects (P=0.24). As with pass rate and eliciting dose outcomes, no significant differences in epinephrine use were appreciated for CM challenges between subgroups (Fig. 2C).
In a multivariable logistic regression analysis model for all primary outcomes (pass rate, eliciting dose, epinephrine use), age was not found to be a significant contributing factor for differences between egg or milk cohorts.

Differences in Reaction Classification

Symptoms were categorized for egg and CM reactions for positive (failed) challenges by subgroup differentiation in Table 2. For all groups, the majority of reactions involved symptoms from multiple organ systems (anaphylaxis), with isolated respiratory reactions being most rare, as similarly reported in previous studies19,23. Within the baked egg subgroups, isolated cutaneous reactions were seen with higher frequency in the BE Tol cohort compared to other groups, a difference that was not appreciated between the CM cohorts. No other significant differences in reaction classification were determined.

Differences in SPT and IgE Values

In the evaluation of passed OFCs, we found a significant difference in specific-IgE levels between BM Tol subjects (Median 1.7 kU/L, Range 0-22 kU/L), BM Avoid subjects (Median 0.4 kU/L, Range 0.05-68.5 kU/L), and those in the Milk NI subgroup (0.4 kU/L, Range 0.2-0.6)(P=0.0009). No other statistically significant differences were appreciated in the comparison of tolerated and failed OFCs between egg and CM subgroups for both SPT and specific IgE levels (Table 3).

DISCUSSION

In this study, we evaluated OFC outcomes to assess for differences in native egg and CM tolerance based on subjects’ previous known history of tolerance, avoidance and exposure to these foods in the baked form. We found that egg allergic children tolerating BE were more likely to pass native egg challenges compared to egg allergic children actively avoiding BE and compared to those with no previous exposure to egg, maintaining avoidance secondary to positive allergy testing. Additionally, BE tolerant subjects were able to consume higher doses of native egg before eliciting a positive reaction compared to those avoiding BE and compared to those with no prior egg ingestion. Finally, subjects tolerant to BE required less epinephrine during positive challenges compared to both the baked avoidant and no exposure cohorts. We found no differences in primary challenge outcomes comparing those avoiding BE and those with no egg exposure. Interestingly, we also did not appreciate any differences in CM challenge outcomes based on differences in BM tolerance or avoidance history. Together these results suggest that patients tolerant of BE are less sensitive to native egg compared to other subgroups. These data are consistent with previous reports suggesting children who tolerate BE show increased tolerance to native egg,9,11-12,24 but are inconsistent with other reports6,9-10 suggesting the same influence for BM in children with CM allergy. The exact mechanism by which BE or BM ingestion might influence native food allergy is not fully known. Children’s tolerance of their allergen in the baked form has been attributed to altered protein epitopes in heated or matrixed goods, with less allergenic IgE recognition25,26.
Related to this, studies have demonstrated immunologic patters of tolerance in those ingesting BE or BM consistent with development of natural tolerance or tolerance via subcutaneous immunotherapy10,11. In contrast, a recent randomized control trial of BE introduction in children with egg allergy found no impact of BE ingestion on native egg tolerance with both the control and intervention group demonstrating a more egg-tolerant immune profile over time27. In our study, we demonstrated a unique discrepancy in the apparent impact of BE compared to BM on native tolerance, which might be attributed to several potential factors. We considered the possibility that patients ingesting BE had more frequent ingestion and subsequent effective desensitization. Peters et al. found that children were 3 times more likely to outgrow their egg allergy by 1 year of age with frequent ingestion of BE, compared to infrequent ingestion or avoidance24. Second, given our patient population is made up of a large referral base, it is possible that our CM allergy patients were those that selectively had more persistent and unremitted allergy and thus showed no significant response to BM exposure. Finally, we considered theoretical differences in epitope bioavailability in BE products compared to BM products. While this might have contributed to outcomes seen in our patient population, future studies would be necessary to explore these hypotheses.
Regarding our primary outcomes, the tolerance patterns observed between our egg cohorts may reflect intrinsic differences in baseline allergy severity between each group. That is, children with BE tolerance were those children more likely to outgrow their native egg allergy. This hypothesis would be consistent with result from the aforementioned meta-analysis12. We might next assess the severity of children’s diagnostic (initial) reaction to egg or CM and prospectively follow baked tolerance and native eliciting dose using our defined cohorts over time to better assess the degree to which BE directly contributed to desensitization.
We unexpectedly found specific IgE levels to be highest in BM tolerant subjects who then passed food challenge. Differences in SPT and specific IgE levels based on baked tolerance and exposure history were otherwise not appreciated. While the exact etiology of these findings is not immediately apparent, they also do not appear to be consistent in clinical outcomes and should likely be reevaluated in future studies.
We also found no difference in the rate of multi-systemic anaphylaxis between subgroups, though differences in epinephrine use between egg subgroups was notable. At time of OFC, the same rescue medications were prepared individually for each patient, with all medications equally and immediately accessible to providers upon request. While epinephrine would be indicated for any multi-system reaction, consistent with anaphylaxis, we considered that the severity of reactions in the closely observed food challenge setting may be assessed with subjective variability by providers, despite meeting strict clinical criteria for anaphylaxis, and thus may have impacted the decision to administer epinephrine. In the design of a prospective study using identical cohorts, one could ensure well-structured indications for epinephrine administration during OFC, in order to decrease bias in clinical interpretation of reaction severity.
Our study is unique in the comparison of OFC outcomes in subjects with reaction-defined allergy and those never exposed to the food in either native or baked form. In our population, the majority of children in the Milk NI or Egg NI cohorts were those with positive skin prick or specific IgE testing done in the setting of atopic dermatitis. Previous studies have suggested that children with atopic dermatitis are more likely to develop other atopic disease, including food allergy28,29. This is particularly interesting given that the rate of atopic dermatitis was found to be highest in both the Egg and Milk NI cohorts, the subgroups also found to have the poorest OFC outcomes. In this case, allergen testing prior to ingestion may have selected for children with the most severe food allergy. However, we also acknowledge work by Fleischer and colleagues, which found a high false-positive rate for food allergy testing in children with atopic dermatitis30. Thus, we suggest the possibility that avoidance of egg and possibly CM secondary to allergen testing might have increased patient’s risk for iatrogenic development of food allergy of profound severity. Future studies are needed to assess food avoidance patterns and subsequent food allergy development by prospective and longitudinal methods.
We considered limitations in our data outcomes and interpretation, notably in our design of the study as a retrospective chart review. Further, our data comprise outcomes from a single tertiary care center with a large referral base and may be prone to selection bias. We attempted to minimize bias in defining our subgroup populations through systematic chart review for all patients. All progress notes from the day of OFC were reviewed with previous outpatient visit notes reviewed to elicit a complete clinical history. For those with documented tolerance to BE or BM, we assumed tolerance at time of OFC. We notably did not investigate, however, differences in dose and frequency of baked food ingestion in these subjects, which might have impacted our results and would be worthwhile to control for in future studies. Subjects were defined as baked avoidant with documentation of strict avoidance to the respective allergen following initial reaction. This included subjects with native egg or CM reaction who never attempted introduction of BE or BM, as well as those who had directly reacted to the food in the baked form, though the latter group was found to be considerably smaller. In this cohort, as well as the Egg/Milk NI cohorts, we acknowledge inclusion of subjects who may have developed tolerance to egg or CM in the baked form prior to OFC date, thus potentially biasing our data through misclassification.With a larger and more powerful study, specific analysis of patients reactive to both native and baked egg or CM may further expand upon our findings, as this cohort was too small for independent analysis in our population.
Our study is strengthened, however, by the practicality of our subgroup differentiation. In clinical practice, children with egg or CM allergy may present with history of exposure and tolerance to egg or CM in the baked form, reaction, avoidance or no previous exposure. In each case, our data can be considered in the decision to pursue OFC and might provide insight regarding severity associated with positive challenge reactions.
In summary, our study found that children who tolerate BE are less sensitive to native egg. They are more likely to pass egg challenges, tolerate higher overall doses of egg and experience less severe reactions compared to those avoiding BE and compared to those never exposed. These differences were not seen in CM challenge outcomes. Additionally, food allergic

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