Study 1: patient recruitment and data collection
100 participants were recruited from the allergy clinic at McMaster University and community allergy outpatient clinics in the greater Hamilton area.
All participants were at least 6 years of age and of either sex. Exclusion criteria for the study were uncontrolled or severe asthma, receipt of allergy injections in the past, and use of injectable epinephrine 1 month prior to the start of the study. Individuals taking daily antihistamines, leukotriene receptor antagonists, or nasal, inhaled, or oral corticosteroids were also excluded. These interventions may have interfered with our study measurements, particularly cytokine secretion.
We collected the following data on each participant: age, sex, peanut SPT wheal size, clinical peanut allergy status, peanut ImmunoCAP, total IgE, supernatants from peripheral blood mononuclear cells (PBMC) under unstimulated and peanut-stimulated conditions, immediate family history of peanut allergy, asthma, rhinitis, and eczema status.
Participants were divided into 4 groups according to their peanut allergy status based on history and peanut skin prick test.
Group 1 consisted of peanut allergic individuals. These individuals had a prior history of an allergic reaction to peanut on ingestion and a positive SPT to peanut. Allergic symptoms included, but were not limited to, urticaria, angioedema, dyspnea, cough, wheeze, nausea, vomiting, lightheadedness, rash, and/or shock.
Group 2 consisted of individuals who had a positive skin prick test to peanut, but could tolerate peanut ingestion without difficulty. Thus, these individuals were not allergic to peanut and their skin test results were designated as “false positives”.
Group 3 consisted of individuals who had a positive skin prick test, but no known history of peanut ingestion. Many of these individuals may have avoided peanut for specific reasons, such as a family history of peanut allergy. It was therefore uncertain whether they would react to peanut on ingestion and they were considered to be at risk of clinical reactivity based on the presence of sensitization.
Group 4 consisted of individuals who had a negative skin prick test to peanut and had previously ingested peanut without problems. Consequently, they served as a negative control group. This group did not have any other food or environmental allergies.
This study was approved by the Research Ethics Board at McMaster University and all participants, or their guardians, provided written informed consent.
Skin prick test measurements
The forearm was prepped with alcohol and peanut extract (ALK-Pharmaceuticals, Mississauga, ON, Canada) was applied to the skin of the dorsal forearm. A sterile metal lancet (HollisterStier, Spokane, WA, USA) was used to puncture the skin below the allergen droplet. Skin prick test wheal size was measured after 15 min. Tape was placed on the dorsal forearm and an outline of the wheal was traced. The widest diameter of the wheal was measured by two different study nurses.
Peanut and total IgE plasma measurements
Total IgE was measured using the Immage 800 (Beckman Coulter, Mississauga, ON, Canada) and peanut-specific IgE antibodies were measured using the Phadia 250 (Thermo Scientific, Waltham, MA, USA).
Mononuclear cells were isolated from 30 to 40 ml of blood by density gradient centrifugation after red blood cells were lysed with AKC lysis buffer. Cells were re-suspended in RPMI supplemented with 10% FBS, 1% l-glutamine, 1% penicillin/streptomycin, 55 µM 2-mercaptoethanol (Thermo Scientific), 1 mM sodium pyruvate, 10 mM HEPES and 0.1 mM MEM NEAA (Thermo Scientific). Viable cells were counted via Trypan Blue (Thermo Scientific) exclusion and re-suspended at 8 × 106 cells/mL. 125,000 live cells per well were cultured in triplicates in medium alone or with 50 µg/mL/well of crude peanut extract in flat-bottom 96-well plates (BD Biosciences, Mississauga, ON, Canada). After 5 days of culture at 37 °C and 5% CO2, the triplicates were pooled, spun down and cell-free supernatants harvested and stored at −80 °C until further analysis. Cytokines in cell-free supernatants were quantified using Luminex (Millipore Canada Ltd, Etobicoke, ON, Canada) following the manufacture’s instructions.
Each predictor was entered into a univariate logistic regression analysis to determine if it was associated with the primary outcome—clinical peanut allergy status. We then generated cumulative models composed of multiple predictors using multivariable logistic regression. All univariate and multivariable analyses included the 69 study participants from Groups 1, 2, and 4.
For all models, parameter estimates were obtained for each predictor and expressed as odds ratios with corresponding 95% confidence intervals and associated p values. p values are reported to 4 decimal places.
Hierarchical models were compared to determine if the model with the greater number of predictors was statistically significantly better at predicting the primary outcome than the model with fewer predictors. This was done by comparing the models’ −2 Log Likelihood statistics. For each model, the area under the Receiver Operating Characteristics (ROC) curve was reported as a measure of discriminability. The best model was used to predict the peanut allergy status of participants in Group 3 and to determine the predicted probability (Pr) of each participant having clinical peanut allergy. Using Pr, we classified each individual as having a peanut allergy or not based on a specific cutpoint. We chose this cutpoint to eliminate false negatives and maximize true positives in the data set.
All analyses were conducted in SAS version 9.4.
All individuals in Group 3 underwent a peanut challenge to determine peanut allergy status. The food challenge took place in the Allergy Clinic at McMaster University Medical Centre under the supervision of a study physician. A research/Critical Care nurse and study physician were present at all times with the appropriate set-up to deal with any and all allergic reactions.
All subjects had baseline vital signs taken, body weight measured, and an intravenous inserted prior to oral food challenge.
Each subject was given either 1 mg of peanut or placebo orally mixed with grape jelly or applesauce. Peanut flakes were the source of peanut and cracker crumbs were used as the placebo. The dose of peanut was increased to 5 mg and increased every 15–30 min to 10, 25, 50, 100, 250, 500 mg, 1, and 2.5 g until the maximum dose of 2.5 g was reached or objective findings of allergic reaction were observed. 2.5 g is the equivalent of 5 peanuts.
Subjects were carefully observed for the following signs of allergic reaction: rash (erythema, morbilliform rash, urticaria, angioedema), ocular (conjunctival swelling, scleral edema, tearing), nasal (congestion, rhinorrhea, sneezing), respiratory (wheezing, cough, drop of PEF or FEV1 by >20%), gastrointestinal (vomiting, diarrhea, abdominal pain), systemic (blood pressure drop by >20%).
Vital signs (oxygen saturation, blood pressure, heart rate, respiratory rate) were assessed before each dose, with every new symptom reported, and when objective findings were observed.
If a subject developed any two mild symptoms (generalized itchiness or flushing, runny nose, watery eyes, or sneezing) or any one severe symptom (persistent cough, significant abdominal pain, nausea, vomiting, diarrhea, swelling of the lips or face, difficulty breathing, wheezing, or fainting) the challenge was immediately stopped and the subject was considered to be peanut allergic .
Subjects who experienced allergic reactions were treated with appropriate medications, namely intramuscular epinephrine, intravenous antihistamines, and corticosteroids (1 mg/kg for 3 days). The subjects were observed for 4–8 h after an allergic reaction to ensure that it had been adequately treated and resolved .
If 2.5 g of peanut was tolerated, 10 g was administered in an open challenge and subjects were monitored for signs of allergic reaction. In the event of a reaction, each subject received appropriate medication and monitoring.
The results of the oral peanut challenges were then compared to patients’ predicted peanut allergy status.
Study 2: patient recruitment and data collection
We conducted a retrospective chart review of a separate cohort of 194 subjects: 97 with confirmed clinical peanut allergy, and 97 sex- and age-matched controls without clinical peanut allergy. Peanut allergy was defined as: the participant had consumed peanuts in the past and displayed peanut allergy-compatible symptoms, as described earlier, and had undergone confirmatory testing. For each participant, we collected date of birth, sex, peanut skin prick test wheal size, allergic rhinitis, asthma, and eczema status. We also recorded food allergy status for milk, egg, wheat, individual nut, and nut mix.
The predictive value of each variable was analyzed using exact conditional logistic regression. All analyses were conducted in SAS version 9.4.