Background: In the absence of a clear clinical history of reaction, diagnosis of cashew allergy using skin prick tests (SPT) or cashew-specific IgE requires a high number of oral food challenges (OFC). We recently showed that Ana o 3 sIgE alone, or a two-step diagnostic algorithm using cashew sIgE followed by Ana o 3 sIgE can reduce need for OFC. We aimed to determine if either of these approaches can provide a cost reduction to the health system compared to cashew SPT alone. Methods: Pooled individual level data from 6 studies was used to determine diagnostic accuracy and OFC rate. Two studies used cashew SPT (n=567, 198 allergic), with 95% positive and negative predictive values of ≥12mm and <3mm. Four studies were included in the pathways for Ana o 3 sIgE alone or a 2-step algorithm incorporating cashew and Ana o 3 sIgE (n=271, 156 allergic). Cut-offs used were ≥8.5kUA/L and ≤0.1kUA/L for cashew sIgE and ≥0.35kUA/L and ≤0.1kUA/L for Ana o 3 sIgE. Costs were constructed based on unit prices from hospital inpatient admissions, expenses incurred by families, individual patient data on allergic reaction types and rates and adrenaline autoinjector carriage, applying a health system perspective. Results: Modelled data through the Ana o 3 pathway resulted in a 46.43% cost reduction (\euro307,406/1000 patients) compared to using cashew SPT alone (\euro573,854/1000 patients). The 2-step algorithm resulted in a 44.94% cost reduction compared to SPT alone (\euro315,952.82/1000 patients). Both the Ana o 3 pathway and 2-step algorithm resulted in a 79-80% reduction in OFCs compared to SPT. Conclusions: Using Ana o 3 as a standalone test for cashew allergy diagnosis or a 2-step algorithm incorporating cashew sIgE and Ana o 3 sIgE is accurate and results in a large reduction in both OFCs and health system costs compared to cashew SPT alone.

Background: Accidental allergic reactions (AAR) in children are under-studied, especially with precise pediatrician-based exact diagnoses and follow-ups. This study aimed to assess the prevalence and risk factors for AAR in Japanese children with immediate-type food allergies. Methods: This single-center study included children with immediate-type hen’s egg (HE), cow’s milk (CM), wheat, or peanut allergy who had been followed-up regularly at a national center specialized for allergy in Japan. Low-dose reactivity was defined as allergic reactions to a low dose of ≤250, ≤102, ≤53, or ≤133 mg HE, CM, wheat, or peanut protein, respectively. From January to December 2020, pediatricians followed the AAR experience every 2–4 months. Risk factors for AAR were analyzed using multiple logistic regression. Results: Of the 1096 participants, 609, 457, 138, and 90 had HE, CM, wheat, and peanut allergies, respectively. In this cohort, the median age was 5.0 years, 39% had completely eliminated allergenic food, and 24% had low-dose reactivity. The annual AAR rate was 0.130 in all sub-cohorts. Moderate and severe symptoms occurred in 50% and 0.7%, respectively, of children who experienced AAR. Multiple logistic regression revealed that low-dose reactivity was a significant risk factor for AAR in the overall, HE, and CM cohorts, respectively ( p <0.001, p = 0.029 and 0.036). Conclusion: In Japanese children with immediate-type food allergies, the annualized rate of AAR was relatively low; however, half of the participants with AAR had moderate to severe symptoms. Children, especially those with low-dose reactivity, would require careful risk management of AAR.

Long-term prognosis after low-dose peanut challenge for patients with history of anaphylaxisNobuko Akamatsu, MD, PhD1,2, Ken-ichi Nagakura, MD, PhD1, Sakura Sato, MD3, Noriyuki Yanagida, MD, PhD1,3,*, Motohiro Ebisawa, MD, PhD3 1Department of Pediatrics, National Hospital Organization, Sagamihara National Hospital, Kanagawa, Japan2Department of Pediatrics and Adolescent Medicine, Tokyo Medical University Hospital, Tokyo, Japan3Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, JapanRunning title: Prognosis after low-dose peanut challenge*Corresponding author: Noriyuki Yanagida, MD, PhDDepartment of Pediatrics, National Hospital Organization, Sagamihara National Hospital,18-1, Sakuradai, Minami-ku, Sagamihara, Kanagawa 252-0392, JapanE-mail: [email protected] count: 1190Number of figures: 2 Number of tables: 0Conflict of interest: Motohiro Ebisawa and Sakura Sato have received speaker honoraria from Viatris. All other authors declare that they have no conflicts of interest.Financial support: This paper has not received any financial support.Keywords : low-dose oral food challenge, peanut, food allergy, low-dose-tolerant, tolerant, pediatric, anaphylaxisTo the Editor,Peanuts cause severe allergic reactions, and only 20% of peanut-allergic patients acquire tolerance.1 Peanut oral food challenge (OFC) has a high risk of severe symptoms such as anaphylaxis, and OFC is avoided in children with an immediate history, especially with a history of anaphylaxis.1Peanut-allergic patients and their guardians have a lower quality of life because of worry that anaphylaxis may occur at any time.2 Low-dose OFC may be useful to manage children with a history of anaphylaxis,3 but it has not been used to evaluate prognosis. This study investigated long-term prognosis after low-dose peanut OFC for patients with a history of immediate reactions, including anaphylaxis.We retrospectively analyzed participants with a history of immediate symptoms due to peanut ingestion, who received baseline low-dose OFC with 133 mg of peanut protein from August 2013–August 2017 at Sagamihara National Hospital (Figure 1), and evaluated two-year tolerance acquisition.We defined tolerance as passing an OFC with 795 mg protein (medium-dose OFC) and ingesting more than 795 mg protein without symptoms at home; consuming this dose enables cessation of strict avoidance in daily life. We defined baseline low-dose OFC negative patients as the low-dose-tolerant group and positive patients as the low-dose-reactive group. The low-dose-tolerant group was instructed to consume 133 mg at home twice a week. Then, based on guardians’ preference, patients received a medium-dose OFC every 6 months or gradually increased peanut ingestion to 795 mg at home under a physician’s direction. The low-dose-reactive group completely avoided peanuts and received low-dose OFC every 6 months. When the low-dose-reactive group passed low-dose OFC, they received medium-dose OFC (Supplementary Figure 1).Anaphylaxis was defined according to the World Allergy Organization Guidelines.4 OFC protocol is described in the supplementary information.5 The percentage of patients who acquired tolerance within two years was estimated using Kaplan-Meier curves. The co-factors for tolerance acquisition were analyzed using Cox regression analysis. Multivariate analysis was performed on the results of low-dose OFC, total IgE, peanut-specific IgE (Pn-sIgE), and history of peanut-related anaphylaxis. SPSS (version 27.0; SPSS Inc., Chicago, IL) was used for all analyses; p <0.05 was considered statistically significant. The Ethics Committee of The Sagamihara National Hospital (2016-015) approved the study according to the Helsinki Declaration. Written informed consent was obtained from all patients’ guardians.Fifty-three patients (median age, 7.1 years) were enrolled; 43% had a history of anaphylaxis. The median Pn-sIgE level was 20.7 (interquartile range 7.0–57.5) kUA/L. The median Ara h 2-specific IgE level was 10.4 (4.98–28.3) kUA/L. The median thresholds of past immediate symptoms were 26.6 mg (13.3–133) (Supplementary Table 1).Twenty-one patients (40%) passed the low-dose OFC and were defined as the low-dose-tolerant group, and 32 (60%) failed and were defined as the low-dose-reactive group (Supplementary Table 1); 35% of patients with a history of anaphylaxis passed the low-dose OFC. During low-dose OFC, oral mucosal symptoms were most common (72%), then gastrointestinal (63%) and respiratory symptoms (63%). Three patients required intramuscular adrenaline (Supplementary Table 2). When the low-dose-tolerant patients ingested low-dose peanuts at home, six (29%) had mild reactions like oral and throat discomfort; most reactions resolved naturally and did not require medical attention.In the low-dose-tolerant group, 13 patients (62%) acquired tolerance within 2 years, including five patients with a history of anaphylaxis, whereas in the low-dose-reactive group, one patient (3%), with no history of anaphylaxis, acquired tolerance (p <0.001) (Figure 2). In the low-dose-reactive group, 6% of patients passed low-dose OFC within two years.The predictive factors of failure to acquire tolerance have been positive reactions to low-dose OFC (crude hazard ratios of total IgE, Pn-sIgE: 0.37 [95% confidence interval 0.15–0.94, p =0.04), and log (Pn-sIgE) 2.23 [1.01–4.92], p =0.048) (Supplementary Table 3). In 23 patients with a history of anaphylaxis, five (22%) acquired tolerance. In 30 patients with no history of anaphylaxis, nine (30%) acquired tolerance. History of anaphylaxis did not significantly affect tolerance acquisition (Supplementary Figure 2).This is the first report showing that low-dose OFC can be undergone relatively safely with tolerance acquisition in some peanut-allergic patients, including patients with a history of anaphylaxis. Patients with a history of anaphylaxis have a lower quality of life because of worry that anaphylaxis may occur at any time.6 Therefore, these results are significant because if patients realize that low-dose peanuts can be ingested, complete avoidance becomes unnecessary, and tolerance acquisition could be assessed. The low-dose-reactive group was less likely to develop tolerance and required careful follow-up to prevent accidental ingestion.Previous studies of long-term prognosis after peanut OFC excluded patients with a history of anaphylaxis,1 therefore, their tolerance acquisition based on the results of OFC was unknown. In our current study, more than half of patients in low-dose-tolerant groups acquired tolerance within two years, even those with a history of anaphylaxis. Furthermore, one-fifth of patients with a history of anaphylaxis tolerated peanuts, and there was no significant difference between patients with and without a history of anaphylaxis in acquiring tolerance. Therefore, passing low-dose OFC could be considered to assess tolerance acquisition, regardless of the history of anaphylaxis.It has been reported that peanut OFC is high risk because it often causes anaphylaxis and other serious symptoms.1 In the previous studies of peanut OFC, subjects have no history of anaphylaxis or as few as 10%, while this study had 40%, but the occurrence of anaphylaxis in OFC was comparable.1,7 Furthermore, there is a report of a group of subjects, 83% of whom had a history of anaphylaxis, and all patients reacted with anaphylaxis in OFC.8 It has been reported that the incidence of anaphylaxis with OFC was higher with progression up to the total OFC ingested.7 Therefore this study suggests that low-dose OFC is relatively safe in patients with a history of anaphylaxis.Recently, some trials of low-dose oral immunotherapy showed that ingesting low-dose peanuts (133-300 mg) could induce immunological changes and allow the intake of larger amounts.9Similarly, daily ingestion of low-dose wheat is effective in increasing consumption dose and preventing accidental symptoms, even in patients with a history of anaphylaxis.10 Therefore, twice weekly ingestion of 133 mg in the low-dose-tolerant group may yield oral tolerance. In addition, few serious reactions were observed in the low-dose-tolerant group during the subsequent at-home dose escalation in this study.This study has several limitations. First, 33 subjects were excluded because their two-year course could not be tracked. Although the excluded and included patients’ backgrounds were similar (Supplementary Table 4), predictors of tolerance acquisition, such as anaphylaxis may be different with more subjects. Second, there was a lack of information on several points. Although at-home intake methods were unified, the frequency of home peanut intake and adherence was unknown. Additionally, we couldn’t confirm thresholds of past immediate symptoms in 25% of subjects. However, the median thresholds were 26.6 mg in 75% of subjects who were able to assess the threshold. Therefore, we assume that thresholds in the remaining children were similar.For peanut-allergic patients with a history of anaphylaxis, low-dose OFC is relatively safe and effective in the assessment of tolerance acquisition. Low-dose OFC results may effectively stratify peanut-allergic patients with anaphylaxis history with good and poor tolerance acquisition to select optimal management plans.Nobuko Akamatsu, MD, PhD1,2, Ken-ichi Nagakura, MD, PhD1, Sakura Sato, MD3, Noriyuki Yanagida, MD, PhD1,3, Motohiro Ebisawa, MD, PhD3,1Department of Pediatrics, National Hospital Organization, Sagamihara National Hospital, Kanagawa, Japan2Department of Pediatrics and Adolescent Medicine, Tokyo Medical University Hospital, Tokyo, Japan3Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japan

Background: Approximately 50–90% of children with immediate-type cow’s milk allergy (CMA) acquire tolerance by pre-school age. We aimed to investigate the tolerance acquisition rate of CMA in children aged 6–12 years. Methods: Children with CMA that persisted until 6 years of age were included. Tolerance acquisition was defined as either passing an oral food challenge with 200 mL of unheated cow’s milk (CM) or consuming CM of any quantity without symptoms. Persistent CMA was defined as fulfilling neither of these criteria by 12 years of age. Children undergoing oral immunotherapy were defined as having persistent CMA. Risk factors associated with persistent CMA were assessed using Cox regression analysis. Results: Of the 123 children analyzed, 60 (49%) had previous CM anaphylaxis, 82 (67%) eliminated CM from their diet, and the median CM-specific immunoglobulin E (sIgE) level was 23.3 kU A/L at 6 years of age. Twenty-five children (20%) acquired tolerance by 9 years of age, and 46 (37%) by age 12. At baseline, higher CM-sIgE levels (hazard ratio: 2.58 [95% confidence interval: 1.62–4.12], optimal cutoff level: 34.4 kU A/L), previous CM anaphylaxis (2.42 [1.24–4.69]), and complete CM elimination (5.18 [2.45–10.99]) were associated with persistent CMA. None of the children with CMA who had all three risk factors (n = 26) acquired tolerance. Conclusion: At least one-third of the children with CMA at 6 years of age acquired tolerance by 12 years of age. Children with CMA who have the risk factors are less likely to acquire tolerance.

Prognosis of infantile food protein-induced enterocolitis syndrome to wheat: A case series Makoto Nishino1,2 | Noriyuki Yanagida1,3,* | Sakura Sato1 | Ken-ichi Nagakura3,4 | Kyohei Takahashi3 |Kiyotake Ogura3| Motohiro Ebisawa11 Clinical Research Center for Allergy and Rheumatology, National Hospital Organization Sagamihara National Hospital, Kanagawa, Japan2 Department of Pediatrics, Ushiku Aiwa General Hospital, Ibaraki, Japan3 Department of Pediatrics, National Hospital Organization Sagamihara National Hospital, Kanagawa, Japan4 Department of Pediatrics, Jikei University School of Medicine, Tokyo, Japan*Corresponding author: Noriyuki YanagidaDepartment of Pediatrics, National Hospital Organization Sagamihara National Hospital,18-1, Sakuradai, Minami-ku, Sagamihara, Kanagawa 252-0392, JapanE-mail:[email protected] count: 1,191Number of figures: 2 Number of tables: 1

Background: Food allergy is a disease with a diverse and variable natural history, and some patients may react to two or more food antigens. This study aimed to classify and characterize the long-term prognosis of infantile-onset, immediate-type food allergies in children, focusing on three major antigens in Japan: egg, milk, and wheat. Methods: All children visited to our hospital with food allergies, including suspected cases, were prospectively registered in our medical database. From this database, infants who had immediate-type symptoms or were sensitized to above three antigens were included. Cox regression analysis and repeated-measures latent class analysis were performed to reveal risk factors and tolerance patterns for food allergies. Results: Of 2,830 patients registered in the database, we included 915 patients with immediate-type food allergy symptoms and 276 sensitized asymptomatic patients in this study. The number of patients with immediate-type symptoms to egg, milk, and wheat was 609, 443, and 235, respectively. The number of patients with multiple food allergies was 302. Ratios of acquiring tolerance to egg, milk, and wheat at the age of 6 years were 74%, 69%, and 75%, respectively. Latent class analysis revealed 10 classes of prognosis for food allergies, including five with multiple food allergies. The largest class was transient egg allergy alone (21.4%), and there were severe cases of persistent allergy to three major allergens (3.2%). Conclusions: This study demonstrated the prognosis of food-allergy classes in Japan, including multiple food allergies, with 10 classes with its own characteristics.

Background The current diagnostics of fish allergy lack sufficient accuracy such that more reliable tests such as component-resolved diagnosis (CRD) are urgently needed. This study aimed at identifying fish allergens of salmon and grass carp and evaluating the sensitization pattern towards the identified allergens in fish allergic subjects from two distinct populations in Asia. Methods One hundred and three fish allergic subjects were recruited from Hong Kong (67 subjects) and Japan (46 subjects). Western blot and mass spectrometry were used to identify allergens from salmon and grass carp. Fish allergens were purified and tested against 96 sera on ELISA to analyze patients’ sensitization pattern. The protein profiles of salmon meat prepared under different cooking methods until core temperature reached 80°C were evaluated by SDS-PAGE and mass spectrometry. Results Three common allergens between salmon and grass carp, namely enolase, glycerldehyde-3-phosphate dehydrogenase (GAPDH) and parvalbumin, and two salmon-specific allergens collagen and aldolase were identified. Parvalbumin was the major allergen for both fishes showing an overall sensitization rate of 74.7%, followed by collagen (38.9%), aldolase (38.5%) and enolase (17.8%). Japanese subjects showed more diverse allergen sensitization pattern and more frequent IgE-binding to heat-labile salmon allergens. Compared with steaming and boiling, cooking by baking and frying retained more fish proteins inclusive of heat-labile allergens. Conclusions Fish allergic patients from different Asian populations show varying fish allergen sensitization profiles. The relevant extracts and components for diagnosis are population-dependent but parvalbumin and collagen are important biomarkers. Cooking methods modify allergen composition of salmon and appear to influence patients’ allergic manifestations.

Background: In the absence of a clear clinical history of reaction, diagnosis of cashew allergy using skin prick tests (SPT) or cashew-specific IgE requires a high number of oral food challenges (OFC). By using Ana o 3 sIgE alone, or a two-step diagnostic algorithm using cashew sIgE followed by Ana o 3 sIgE, there is a reduced need for OFC. We aimed to perform a cost comparison for both of these approaches compared to cashew SPT alone. Methods: Pooled individual level data from 6 studies was used to determine diagnostic accuracy and OFC rate. Two studies used cashew SPT (n=567, 198 allergic), with 95% positive and negative predictive values of ≥12mm and <3mm. Four studies were included in the pathways for Ana o 3 sIgE alone or a 2-step algorithm incorporating cashew and Ana o 3 sIgE (n=271, 156 allergic). Cut-offs used were ≥8.5kUA/L and ≤0.1kUA/L for cashew sIgE and ≥0.35kUA/L and ≤0.1kUA/L for Ana o 3 sIgE. Costs were constructed based on unit prices from hospital inpatient admissions, expenses incurred by families, individual patient data on allergic reaction types and rates and adrenaline autoinjector carriage, applying a health system perspective. Results: Modelled data through the Ana o 3 pathway resulted in a 46.43% cost reduction (\euro307,406/1000 patients) compared to using cashew SPT alone (\euro573,854/1000 patients). The 2-step algorithm resulted in a 44.94% cost reduction compared to SPT alone (\euro315,952.82/1000 patients). Both the Ana o 3 pathway and 2-step algorithm resulted in a 79-80% reduction in OFCs compared to SPT. Conclusions: Using Ana o 3 as a standalone test for cashew allergy diagnosis or a 2-step algorithm incorporating cashew sIgE and Ana o 3 sIgE is accurate and results in a large reduction in both OFCs and health system costs compared to cashew SPT alone.

Background: Cross-reactivity between wheat and other cereals is an essential issue in the management of wheat allergy. Few studies have reported in vitro cross-reactivity in immediate-type wheat allergy. This study aimed to examine cross-reactivity of the three fractions (albumin/globulin, gliadin, and glutenin fractions) among cereals in children with wheat allergy. Methods: Sera from 128 children with immediate-type wheat allergy were collected. We measured specific immunoglobulin E (sIgE) levels against each fraction of wheat, barley, and rye by using an enzyme-linked immunosorbent assay (ELISA). Cross-reactivities of each fraction among wheat, barley, and rye were examined via inhibition ELISA. Results: All subjects were sensitized to all the fractions of wheat, and also those of barley and rye. The wheat sIgE levels were significantly higher than those of barley and rye in all the fractions (p ≤ 0.001) and were significantly correlated with sIgE levels to them in each fraction (r = 0.887–0.969, p < 0.001). On inhibition ELISA, wheat inhibited the IgE binding to most of the solid phases at the lower protein levels compared to barley and rye in all fractions. Conclusions: In children with immediate-type wheat allergy, sensitization to all the three fractions of wheat was observed. In addition, they showed sensitization to barley and rye caused by in vitro cross-reactivity with wheat in each fraction. When managing children with wheat allergy, sensitization to barley and rye caused by the cross-reactivities should be considered.

Threshold and safe ingestion dose among infants sensitized to hen’s eggMasatoshi Mitomori,a Noriyuki Yanagida,aMakoto Nishino,a Kyohei Takahashi,aKen-ichi Nagakura,a Kiyotake Ogura,aMari Takei,b Sakura Sato,b Motohiro EbisawabaDepartment of Pediatrics, National Hospital Organization, Sagamihara National Hospital, Kanagawa, JapanbClinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, JapanRunning title: Threshold and safe ingestion dose of egg

Clinical cross-reactivity to quail’s egg in patients with hen’s egg allergyMasatoshi Mitomoria, Noriyuki Yanagidaa, Mari Takeib, Kinji Tadab, Makoto Nishinoa, c, Sakura Satob, Motohiro EbisawabaDepartment of Pediatrics, National Hospital Organization, Sagamihara National Hospital, Kanagawa, JapanbClinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, JapancCourse of Allergy and Clinical Immunology, Juntendo University Graduate School of Medicine, Tokyo, JapanRunning title: Clinical cross-reactivity of HE & QECorresponding author:Noriyuki Yanagida18-1, Sakuradai, Minamiku, Sagamihara, Kanagawa 252-0392, JapanTel.: +81 042 742 8311 Fax: +81 042 742 5314E-mail: [email protected]

Previous history of immediate reactions changes predictive accuracy for pediatric peanut allergyNaoko Fusayasua, MD, Tomoyuki Asaumia, MD, Kyohei Takahashia,b, MD, Ken-ichi Nagakuraa,c , MD, PhD, Makoto Nishinoa,b, MD, Sakura Satob,d, MD, Noriyuki Yanagidaa,d, MD, Motohiro Ebisawad, MD, PhD,aDepartment of Pediatrics, National Hospital Organization Sagamihara National Hospital, Kanagawa, JapanbCourse of Allergy and Clinical Immunology, Juntendo University Graduate School of Medicine, Tokyo, JapancDepartment of Pediatrics, Jikei University School of Medicine, Tokyo, JapandDepartment of Allergy, Clinical Research Center for Allergy and Rheumatology, National Hospital Organization Sagamihara National Hospital, Kanagawa, Japan