Study design and study patients
A prospective study was carried out at the Center of Excellence for Allergy, Thammasat Hospital, Pathum Thani, Thailand between January 2017 and December 2018. Children were enrolled in the study if they met all of the following criteria: 1) a history of at least one symptom such as dermatitis or mucous bloody diarrhea; 2) presented with delayed onset of CMA (i.e., symptom appeared > 2 hours after ingestion); and 3) the symptoms improved when cow’s milk was eliminated.
Children with a history of suspected IgE-mediated CMA (e.g., anaphylaxis, urticaria, angioedema), or severe non-IgE-mediated CMA (e.g., food protein induced enterocolitis syndrome), or subjective symptoms (e.g., pruritus) were excluded. We also excluded children who did not complete the allergy testing.
The study was approved by The Institutional Review Board and the Ethics Committee of the Faculty of Medicine, Thammasat University. Informed consent was obtained from the parents of all children.
All children underwent the skin prick test (SPT) and APT at the first visit. Reading of the APT results was performed at the second and third visits. At the fourth visit, the children underwent the open oral food challenge (OFC). If a reaction did not occur, the intake of cow’s milk continued at home.
Skin prick test
All children were advised to discontinue oral antihistamine for 7 days before testing.
The SPT was carried out using 7 reagents: 1) fresh cow’s milk; 2) 1 gram of powdered skim cow’s milk with 10 mL of isotonic saline solution as a vehicle; 3) commercial solution of cow’s milk; 4) commercial solution of casein; 5) casein reagent solution; 6) a-lactalbumin reagent solution; and 7) β-lactoglobulin reagent solution. Items 3 and 4 were obtained from ALK-Abelló (Port Washington, NY, USA) and items 5, 6, and 7 were provided by Lofarma S.p.A (Milan, Italy). Histamine 1 mg/mL and 50% glycerin were used as positive and negative controls, respectively.
One drop of each reagent was applied to the volar surface of the forearm using a 1-mm single peak lancet by experienced nurses and read after 15‒20 minutes. Each of the 7 reagents were given a code number to maintain blinding for interpretation of the results. The results were read by a nurse who was not involved with the APT or OFC test. Wheal size was measured by the longest and orthogonal diameters, reported as millimeters (mm). SPT positive was defined as a wheal diameter ≥ 3 mm than the negative control.
Atopy patch test
All children were instructed not to apply topical corticosteroids or topical calcineurin to the test site for 5‒7 days before the test. They were also instructed not to put any creams or oils on their backs the morning of the testing .
The APT was carried out using one drop each of the 7 reagents. The solution was put on filter paper using 8-mm aluminum test cups (Finn Chambers on Scanpore; Epitest Ltd Oy, Tuusula, Finland). Vaseline was the negative control. The Finn Chambers were applied to the upper back and covered with non-allergenic plasters. All reagents were assigned a code number to maintain blinding. Evaluation took place 48 hours after application, and the results were read 20 minutes after removing the Finn Chambers by dermatologists who were blinded to the SPT results and independent of the OFC testing. The second reading of the APT was performed 72 hours post-application by the same dermatologist.
Reactions were classified as follows: negative, no reaction; +/- doubtful reaction, faint erythema only; + weak positive reaction, erythema, infiltration, possibly papules; ++ strong positive reaction, erythema, infiltration, papules, vesicles; and +++ extreme positive reaction, intense erythema, intense infiltration, coalescing vesicles .
Open oral food challenge
The open OFC was performed one week after the APT was read. Before the OFC, each child underwent a physical examination. The OFC was performed only when the child was asymptomatic and had a completely normal physical examination. Emergency medications, such as epinephrine, antihistamine, and steroids, and equipment were prepared.
On day 1, the OFC was performed at the hospital. The protocol was applied based on a previous consensus report . Since our cases were at low risk of developing a severe acute reaction, we omitted 3, 10, and 30 mg. The initial dose started with 100 mg of cow’s milk protein. The OFC was performed by administering increasing doses (100, 300, 1000, 3000, 4000 mg) at 20-minute intervals with the total amount of cow’s milk protein being 8400 mg .
During the OFC the children were monitored for vital signs, abnormal signs, and symptoms (e.g., skin rash, vomiting) before escalating each dose. Challenges were discontinued if they developed clinical symptoms. Patients were observed for a minimum of 4 hours after the last dose and if no reaction occurred, they were discharged. The OFC was performed and assessed by an experienced nurse and pediatric allergist who were blinded both of the SPT and APT results.
On days 2‒7, the children continued to receive cow’s milk formula with full servings of 6‒8 ounces at home. The parents were advised to observe for any symptomatic reaction to the milk . If any clinical symptoms occurred during this time, the parents were told to contact and visit our clinic as soon as possible. On the seventh day and 1 month after the OFC the children were followed up and observed for any delayed reaction.
OFC was defined as positive if a child had at least one of the following clinical symptoms: vomiting; diarrhea; hematochezia; and dermatitis. Immediate reactions were defined as those occurring within 2 hours after last dose of OFC and delayed reactions were defined as reactions observed after 2 hours of the last dose.
The statistical analysis was performed using STATA version 14.0. Demographic characteristics were described as mean ± standard deviation for continuous variables as appropriate. Frequency was used for categorical variables. APT accuracy was calculated in terms of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).