Food protein-induced enterocolitis syndrome oral food challenge: Time for a change?

OBJECTIVE: Food protein-induced enterocolitis syndrome (FPIES) is typically diagnosed based on a characteristic clinical history; however, an oral food challenge (OFC) may be necessary to confirm the diagnosis or evaluate for the development of tolerance. FPIES OFC methods vary globally, and there is no universally agreed upon protocol. The objective of this review is to summarize reported FPIES OFC approaches and consider unmet needs in diagnosing and managing FPIES. DATA SOURCES: PubMed database was searched using the keywords food protein-induced enterocolitis syndrome, oral food challenge, cow milk allergy, food allergy, non-immunoglobulin E-mediated food allergy and FPIES. STUDY SELECTIONS: Primary and review articles were selected based on relevance to the diagnosis of FPIES and the FPIES OFC. RESULTS: We reviewed the history of FPIES and the evolution and variations in the FPIES OFC. A summary of current literature suggests that most patients with FPIES will react with 25% to 33% of a standard serving of the challenged food, there is little benefit to offering a divided dose challenge unless there is suspicion of specific immunoglobulin E to the food being challenged, reactions typically appear within 1 to 4 hours of ingestion, and reactions during OFC rarely result in emergency department or intensive care unit admission. CONCLUSION: International standardization in the FPIES OFC approach is necessary with particular attention to specific dose administration across challenged foods, timing between the patient's reaction and offered OFC to verify tolerance, patient safety considerations before the OFC, and identification of characteristics that would indicate home reintroduction is appropriate.

Many Patients With Irritable Bowel Syndrome Have Atypical Food Allergies Not Associated With Immunoglobulin E.

BACKGROUND & AIMS: Confocal laser endomicroscopy (CLE) is a technique that permits real-time detection and quantification of changes in intestinal tissues and cells, including increases in intraepithelial lymphocytes and fluid extravasation through epithelial leaks. Using CLE analysis of patients with irritable bowel syndrome (IBS), we found that more than half have responses to specific food components. Exclusion of the defined food led to long-term symptom relief. We used the results of CLE to detect reactions to food in a larger patient population and analyzed duodenal biopsy samples and fluid from patients to investigate mechanisms of these reactions. METHODS: In a prospective study, 155 patients with IBS received 4 challenges with each of 4 common food components via the endoscope, followed by CLE, at a tertiary medical center. Classical food allergies were excluded by negative results from immunoglobulin E serology analysis and skin tests for common food antigens. Duodenal biopsy samples and fluid were collected 2 weeks before and immediately after CLE and were analyzed by histology, immunohistochemistry, reverse transcription polymerase chain reaction, and immunoblots. Results from patients who had a response to food during CLE (CLE+) were compared with results from patients who did not have a reaction during CLE (CLE-) or healthy individuals (controls). RESULTS: Of the 108 patients who completed the study, 76 were CLE+ (70%), and 46 of these (61%) reacted to wheat. CLE+ patients had a 4-fold increase in prevalence of atopic disorders compared with controls (P = .001). Numbers of intraepithelial lymphocytes were significantly higher in duodenal biopsy samples from CLE+ vs CLE- patients or controls (P = .001). Expression of claudin-2 increased from crypt to villus tip (P < .001) and was up-regulated in CLE+ patients compared with CLE- patients or controls (P = .023). Levels of occludin were lower in duodenal biopsy samples from CLE+ patients vs controls (P = .022) and were lowest in villus tips (P < .001). Levels of messenger RNAs encoding inflammatory cytokines were unchanged in duodenal tissues after CLE challenge, but eosinophil degranulation increased, and levels of eosinophilic cationic protein were higher in duodenal fluid from CLE+ patients than controls (P = .03). CONCLUSIONS: In a CLE analysis of patients with IBS, we found that more than 50% of patients could have nonclassical food allergy, with immediate disruption of the intestinal barrier upon exposure to food antigens. Duodenal tissues from patients with responses to food components during CLE had immediate increases in expression of claudin-2 and decreases in occludin. CLE+ patients also had increased eosinophil degranulation, indicating an atypical food allergy characterized by eosinophil activation.

Cow's milk allergy prevention and treatment by heat-treated whey-A study in Brown Norway rats.

BACKGROUND: Food processing, including heat-treatment, can affect protein structure and stability, and consequently affect protein immunogenicity and allergenicity. A few studies have shown that structural changes induced by heat-treatment impact the intestinal protein uptake and suggest this as a contributing factor for altered allergenicity. OBJECTIVE: To investigate the impact of heat-treatment of a whey-based protein product on allergenicity and tolerogenicity as well as on intestinal uptake in various animal models. METHODS: Immunogenicity and sensitizing capacity of the heat-treated whey product were compared to that of the unmodified product by intraperitoneal and oral exposure studies, while tolerogenic properties were assessed by oral primary prevention and desensitization studies in high-IgE responder Brown Norway rats. RESULTS: Heat-treatment of whey induced partial protein denaturation and aggregation, which reduced the intraperitoneal sensitizing capacity but not immunogenicity. In contrast, heat-treatment did not influence the oral sensitizing capacity, but the heat-treated whey showed a significantly reduced eliciting capacity compared to unmodified whey upon oral challenge. Heat-treatment did not reduce the tolerogenic properties of whey, as both products were equally good at preventing sensitization in naïve rats as well as desensitizing already sensitized rats. Results from inhibitory ELISA and immunoblots with sera from sensitized rats demonstrated that heat-treatment caused an altered protein and epitope reactivity. Protein uptake studies showed that heat-treatment changed the route of uptake with less whey being absorbed through the epithelium but more into the Peyer's patches. CONCLUSION AND CLINICAL RELEVANCE: These results support the notion that the physicochemical features of proteins affect their route of uptake and that the route of uptake may affect the protein allergenicity. Furthermore, the study highlights the potential for heat-treatment in the production of efficient and safe cow's milk protein-based products for prevention and treatment of cow's milk allergy.

Transforming growth factor beta in human milk and allergic outcomes in children: A systematic review.

BACKGROUND: Human milk (HM) transforming growth factor beta (TGF-ß) is critical for inflammation regulation and oral tolerance promotion. Previous reports suggested that variations in HM TGF-ß levels are associated with allergic outcomes. OBJECTIVE: We undertook a systematic review (PROSPERO 2017 CRD42017069920) to reassess the evidence on the relationships between HM TGF-ß and allergic outcomes in children. METHODS: Electronic bibliographic databases (MEDLINE, EMBASE and Cochrane Library) were systematically searched. Two independent reviewers screened reference lists, extracted the data and assessed risk of bias using the National Institute for Clinical Excellence methodological checklist. RESULTS: A total of 21 studies were identified. Sixteen studies assessed relationships between HM TGF-ß and risk of eczema; 14, allergic sensitization; nine, wheezing/asthma; six, food allergy; three, allergic rhinitis/conjunctivitis. Five cohorts (5/18, 28%) reported a protective effect of TGF-ß1, while 3 (3/10, 30%) suggested increased risk of allergic outcomes development and 1 (1/10, 10%), a protective effect of TGF-ß2 on eczema. Meta-analysis was not possible due to significant heterogeneity in methodology, age of outcome assessment and differing statistical approaches. 71% (15/21) of studies carried a high risk of bias. CONCLUSION AND CLINICAL RELEVANCE: In contrast with previous findings, we did not find strong evidence of associations between HM TGF-ß and allergic outcomes. Differences in studies' methodology and outcomes do not allow unconditional rejection or acceptance of the hypothesis that HM TGF-ß influences the risk of allergy development. Future studies on diverse populations employing standardized methods, accurate phenotyping of outcomes and evaluation of the effect of TGF-ß in combination with other HM immune markers, microbiome and oligosaccharides are required.

Milk processing increases the allergenicity of cow's milk-Preclinical evidence supported by a human proof-of-concept provocation pilot.

BACKGROUND: Several studies demonstrated the adverse effect of milk processing on the allergy-protective capacity of raw cow's milk. Whether milk processing also affects the allergenicity of raw milk is hardly investigated. OBJECTIVE: To assess the allergenicity of raw (unprocessed) and processed cow's milk in a murine model for food allergy as well as in cow's milk allergic children. METHODS: C3H/HeOuJ mice were either sensitized to whole milk (raw cow's milk, heated raw cow's milk or shop milk [store-bought milk]) and challenged with cow's milk protein or they were sensitized and challenged to whey proteins (native or heated). Acute allergic symptoms, mast cell degranulation, allergen-specific IgE levels and cytokine concentrations were determined upon challenge. Cow's milk allergic children were tested in an oral provocation pilot with organic raw and conventional shop milk. RESULTS: Mice sensitized to raw milk showed fewer acute allergic symptoms upon intradermal challenge than mice sensitized to processed milk. The acute allergic skin response was low (103 ± 8.5 µm vs 195 ± 17.7 µm for heated raw milk, P < 0.0001 and vs 149 ± 13.6 µm for shop milk, P = 0.0316), and there were no anaphylactic shock symptoms and no anaphylactic shock-induced drop in body temperature. Moreover, allergen-specific IgE levels and Th2 cytokines were significantly lower in raw milk sensitized mice. Interestingly, the reduced sensitizing capacity was preserved in the isolated native whey protein fraction of raw milk. Besides, native whey protein challenge diminished allergic symptoms in mice sensitized to heated whey proteins. In an oral provocation pilot, cow's milk allergic children tolerated raw milk up to 50 mL, whereas they only tolerated 8.6 ± 5.3 mL shop milk (P = 0.0078). CONCLUSION AND CLINICAL RELEVANCE: This study demonstrates that raw (unprocessed) cow's milk and native whey proteins have a lower allergenicity than their processed counterparts. The preclinical evidence in combination with the human proof-of-concept provocation pilot provides evidence that milk processing negatively influences the allergenicity of milk.

The Basophil Activation Test reduces the need for a food challenge test in children suspected of IgE-mediated cow's milk allergy.

BACKGROUND: The gold standard for the diagnosis of cow's milk allergy is the Double-Blind Placebo-Controlled Food Challenge (DBPCFC) test. However, disadvantages of the DBPCFC are the potential risk of anaphylactic reactions, the time-consuming procedure and high costs. OBJECTIVE: The aim of this study was to determine the reliability of the Basophil Activation Test (BAT) both for the initial diagnosis of cow's milk allergy in children and for the determination of tolerance in children with cow's milk allergy. METHODS: Ninety-seven BATs and cow's milk-specific IgE (sIgE) tests were performed in 86 infants/young children, suspected of (persistent) cow's milk allergy, who were qualified for an in-hospital DBPCFC. The BAT was performed with cow's milk extract and the purified major allergens casein, α-lactalbumin, ß-lactoglubulin. Basophil activation was determined by CD63 upregulation measured by flow cytometry. The BAT results were compared to the DBPCFC outcomes. RESULTS: Based on unequivocal DBPCFC and BAT result combinations (80%), the BAT had a sensitivity and specificity of 100% (CI: 86%-100% and 68%-100%, respectively) in IgE-sensitized children (41% of the tested children). All non-IgE-sensitized children (59%) had a negative DBPCFC and BAT, except for five patients. These latter showed delayed and relatively mild symptoms in the DBPCFC with a negative BAT, supporting a non-IgE-mediated allergy in these children. CONCLUSIONS AND CLINICAL RELEVANCE: The BAT seems reliable and cost-effective to diagnose patients with an IgE-mediated cow's milk allergy. In IgE-sensitized patients, a BAT might replace a DBPCFC. For non-IgE-sensitized patients presenting with mild symptoms, we propose to consider a (double-blind) extended (time) challenge test at home.

Outcomes of Baked Milk and Egg Challenge in Cow's Milk and Hen's Egg Allergy: Can Tolerance Be Predicted with Allergen-Specific IgE and Prick-to-Prick Test?

BACKGROUND: Adding baked food into the diets of patients with cow's milk allergy (MA) and hen's egg allergy (EA) has several benefits. OBJECTIVE: We aimed to determine baked and unbaked food tolerance and evaluate the effectiveness of laboratory findings on the prediction of baked and unbaked food tolerance in patients with MA and EA. METHODS: Clinical outcomes of the patients with MA and EA who had been exposed to oral food challenge with baked food were retrospectively analyzed. RESULTS: Ninety-one patients were evaluated. The median age of the study group was 22 months. Forty-nine and 42 patients had IgE-mediated MA and EA, respectively. While all patients with EA tolerated baked egg, 24.5% patients with MA could not tolerate baked cow's milk (BM). In patients with MA, BM tolerance showed negative association with milk-specific IgE, skin prick test (SPT), and prick-to-prick test (PTP), and the PTP was the most significant parameter (sensitivity 83.8%, specificity 91.7% for PTP ≤7 mm). Negative association was seen between milk-specific IgE, SPT, PTP, and unbaked milk (UBM) tolerance, and PTP was the most significant parameter (sensitivity 100%, specificity 55% for PTP ≤4 mm). In patients with EA, at the end of 6 months of baked hen's egg (BE) consumption, scrambled egg tolerance showed negative association with egg white-specific IgE level, egg white SPT and PTP. Egg white PTP was the most significant parameter (sensitivity 82.4%, specificity 96.0% for PTP ≤5 mm). CONCLUSION: Specific-IgE, SPT, and PTP should be kept in mind as parameters that can be used to predict tolerance to BM and BE for patients with MA and EA.

Early Onset Allergic Proctitis in a Preterm Neonate-A Case Report and Review of the Literature.

Cow's milk protein allergy/intolerance (CMPA/CMPI) is a common entity in the pediatric population with a nonspecific presentation ranging from gastrointestinal symptoms to systemic manifestations. Most infants with CMPI are term, and symptoms often appear in the week following the introduction of cow's milk-based formula. There is typically a significant delay in the onset of milk allergy in premature infants compared to full term. We report a rare case of a premature neonate who presented with symptoms of CMPA within the first 2 days of life.

Preventive effects of royal jelly against anaphylactic response in a murine model of cow's milk allergy.

CONTEXT: Royal jelly (RJ) has long been used to promote human health. OBJECTIVE: The current study investigated the preventive effects of RJ against the development of a systemic and intestinal immune response in mice allergic to cow's milk proteins. MATERIALS AND METHODS: Balb/c mice treated orally for seven days with RJ at doses of 0.5, 1 and 1.5 g/kg were sensitized intraperitoneally with ß-lactoglobulin (ß-Lg). Serum IgG and IgE anti-ß-Lg were determined by an enzyme-linked immunosorbent assay (ELISA). Plasma histamine levels, symptom scores and body temperature were determined after in vivo challenge to ß-Lg. Jejunums were used for assessment of local anaphylactic responses by an ex vivo study in Ussing chambers and morphologic changes by histological analysis. RESULTS: RJ significantly decreased serum IgG (31.15-43.78%) and IgE (64.28-66.6%) anti-ß-Lg and effectively reduced plasma histamine level (66.62-67.36%) (p < 0.001) at all the doses tested. Additionally, no clinical symptoms or body temperature drops were observed in RJ-pretreated mice. Interestingly, RJ significantly reduced (p < 0.001) intestinal dysfunction by abolishing the secretory response (70.73-72.23%) induced by sensitization and prevented length aberrations of jejunal villi by 44.32-59.01% (p < 0.001). DISCUSSION AND CONCLUSIONS: We speculate that using RJ may help prevent systemic and anaphylactic response in allergic mice. These effects may be related to its inhibitory effects on the degranulation of mast cells.

Functional improvements in ß-lactoglobulin by conjugating with soybean soluble polysaccharide.

Soybean soluble polysaccharide (SSPS) was hydrolyzed by autoclaving for 6 and 12 h to obtain SSPS (6 h) and SSPS (12 h). Bovine ß-lactoglobulin (BLG) was conjugated with each SSPS by the Maillard reaction to improve its function. Conjugation between BLG and each SSPS was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. BLG-SSPS (6 h) and BLG-SSPS (12 h), respectively, retained approximately 56 and 43% of the retinol-binding activity of BLG. Structural analyses by intrinsic fluorescence and enzyme-linked immunosorbent assay with monoclonal antibodies indicated that the native structure of BLG had almost been maintained in each conjugate and that the surface structure was covered by conjugated SSPS. The emulsifying properties of BLG were improved in each conjugate at pH 3, 5, and 7 in the presence of 0.2 M NaCl. The antibody responses to BLG-SSPS (6 h) and BLG-SSPS (12 h) were considerably reduced in BALB/c mice. We conclude that conjugation with SSPS was very effective for improving the function of BLG and this study would contribute to greater utilization of SSPS.

Histopathologic findings in children diagnosed with cow's milk protein allergy.

BACKGROUND: Cow's milk protein allergy is the most common cause of food allergy. The challenge test, either open or doubled-blind with a placebo control, is regarded as the criterion standard. Endoscopy and histologic findings are considered a method that can aid in the diagnosis of this entity. AIMS: The aim of this study was to describe the histopathologic findings in children suspected of cow's milk protein allergy that were seen at our hospital. MATERIAL AND METHODS: A descriptive, observational study was conducted on 116 children clinically suspected of presenting with cow's milk protein allergy that were seen at the Department of Gastroenterology and Nutrition of the Instituto Nacional de Pediatría. Upper endoscopy and rectosigmoidoscopy with biopsies were performed and the findings were described. RESULTS: Of the 116 patients, 64 (55.17%) were girls and 52 (44.83%) were boys. The rectum was the site with the greatest presence of eosinophils per field in both groups, followed by the duodenum. In general, more than 15 eosinophils were found in 46% of the patients. CONCLUSIONS: Between 40 and 45% of the cases had the histologic criterion of more than 15 to 20 eosinophils per field and the rectosigmoid colon was the most affected site. Therefore, panendoscopy and rectosigmoidoscopy with biopsy and eosinophil count are suggested.

Chronic food protein-induced enterocolitis syndrome caused by cow's milk proteins passed through breast milk.

We describe 2 cases of food protein-induced enterocolitis syndrome (FPIES) caused by cow's milk (CM) passed through breast milk. The onset in both cases was characterized by chronic symptoms (regurgitation, colic, diarrhea, failure to thrive); in one patient, two acute episodes due to the direct consumption of CM formula by the infant were also reported. The diagnosis of FPIES through breast milk can be easily overlooked, especially in milder cases. We also discuss some important issues concerning the general management of the disease. In conclusion, (1) the diagnosis of chronic FPIES should be taken into account even in exclusively breast-fed infants who present suggestive symptoms such as persistent regurgitation, small amounts of vomiting, lethargy, failure to thrive, dehydration, diarrhea (sometimes bloody) and abdominal distention. A 2-week maternal elimination diet should be considered even in apparently mild cases. (2) CM seems to be the most frequently reported culprit food. (3) In those cases in which acute FPIES is elicited by the direct consumption of the culprit food in breast-fed infants, maternal diet may be unrestricted.

What affects quality of life among caregivers of food-allergic children?

BACKGROUND: Food allergy is associated with decreased caregiver quality of life (QoL). The influence of accurate reaction perception, allergen, and sociodemographic factors on caregiver QoL is poorly understood. OBJECTIVE: To determine factors influencing caregiver food allergy QoL within an academic center cohort. METHODS: Caregivers of children allergic to milk, egg, peanut, or tree nut evaluated from 2009 to 2011 completed a questionnaire assessing the details of the children's most severe food reaction and caregiver QoL using the Food Allergy Quality of Life-Parental Burden index. Survey responses were verified through chart review. A multiple linear regression model was used to determine factors influencing QoL score. RESULTS: Of 305 caregivers surveyed, 65% had accurate reaction perception. This was higher in caregivers of children with provider-confirmed anaphylaxis (P = .008). Milk or egg allergy was associated with higher total and independent domain-specific QoL scores compared with peanut or tree nut allergy, and an income lower than $50,000 was associated with higher total QoL scores and higher scores for 5 of 17 individual domains. In a linear regression model, age at most severe reaction (-0.02, P = .01), peanut or tree nut allergy (-0.81, P = .004) and milk allergy (-1.12, P = .001) vs egg allergy, income higher than $50,000 (-0.53, P = .04), multiple food allergies (0.45, P = .007), accurate reaction perception (-0.37, P = .04), eczema (+0.49, P = .004), and caregiver report that the child had anaphylaxis (+0.48, P = .02) were significantly associated with QoL score. CONCLUSION: Food allergen, having multiple food allergies, age at reaction, income, eczema, parent-reported anaphylaxis, and reaction perception can significantly affect caregiver food allergy QoL. Milk or egg allergy was associated with worse total and domain-specific caregiver QoL scores vs peanut or tree nut allergy, representing a high-risk subgroup.

Cow's milk protein allergy: immunological response in children with cow's milk protein tolerance.

OBJECTIVE: To study changes in immunological responses in patients with CMPA during symptomatic and asymptomatic episodes of cow's milk protein tolerance status. METHODS: 27 CMPA patients were enrolled and underwent diagnostic evaluation, including CM challenge test, skin prick test and specific IgE to CM. Blood samples were collected in two periods from those who became tolerant (n = 13) and those with persistent CMA (n = 14), in order to measure in vitro PBMC responses to cow's milk protein (IL-10, IFN-γ, IL-5), IgG4 to ß-lactoglobulin, casein, BLG-IgG4/IgE ratio and the CAS-IgG4/IgE ratio. RESULTS: Seventy percent of CMPA patients in our study were male with a mean age at diagnosis of 8 months and mean age of onset of 3 months. The reaction time to CM ranged from within 7 minutes to within 14 days. Positive IgE-sensitization was defined as either a specific IgE to CM of more than 0.35 kUA/L (N=11) or SPTs positive for CM and/or fresh cow's milk (N=20). Forty-eight percent of the patients (n = 13) could tolerate CM by 13.38 months (8-19 months). Mean specific-IgE levels to CM were 4.1 kUA/L (range 0.35-14.3 kUA/L). Determination of the cytokine (IL-10, IFN-γ, IL-5) response to BLG revealed significantly higher IL-10 levels during the tolerance phase (212.93 vs 142.46 pg/ml, P = .011). There was a significant increase in BLG-IgG4 and the BLG-IgG4/IgE ratio in the tolerance phase when compared to the symptomatic phase. CONCLUSIONS: IL-10, BLG-IgG4 and the BLG-IgG4/IgE ratio were higher in CMPA patients during the tolerance phase compared to the symptomatic phase.

Dietary long chain n-3 polyunsaturated fatty acids prevent allergic sensitization to cow's milk protein in mice.

BACKGROUND: Cow's milk allergy is one of the most common food allergies in children and no treatment is available. Dietary lipid composition may affect the susceptibility to develop allergic disease. OBJECTIVE: Assess whether dietary supplementation with long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) prevents the establishment of food allergy. METHODS: Mice were fed a control or fish oil diet before and during oral sensitization with whey. Acute allergic skin response, serum immunoglobulins as well as dendritic cell (DC) and T cell subsets in mesenteric lymph nodes (MLN), spleen and/or small intestine were assessed. RESULTS: The acute allergic skin response was reduced by more than 50% in sensitized mice fed the fish oil diet compared to the control diet. In addition, anti-whey-IgE and anti-whey-IgG1 levels were decreased in the fish oil group. Serum transfer confirmed that the Th2-type humoral response was suppressed since sera of fish oil fed sensitized mice had a diminished capacity to induce an allergic effector response in naïve recipient mice compared to control sera. Furthermore, the acute skin response was diminished upon passive sensitization in fish oil fed naïve recipient mice. In addition, the percentage of activated Th1 cells was reduced by fish oil in spleen and MLN of sham mice. The percentage of activated Th2 cells was reduced in both sham- and whey-sensitized mice. In contrast, whey-sensitized mice showed an increased percentage of CD11b+CD103+CD8α- DC in MLN in association with enhanced FoxP3+ regulatory T cells (Treg) in spleen and intestine of fish oil fed whey-sensitized mice compared to sham mice. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary n-3 LCPUFA largely prevented allergic sensitization in a murine model for cow's milk allergy by suppressing the humoral response, enhancing local intestinal and systemic Treg and reducing acute allergic symptoms, suggesting future applications for the primary prevention of food allergy.

Anal fissures in infants may be a pathognomonic sign of infants with cow's milk allergy.

OBJECTIVE: To study the association between analfissures and cow's milk allergy (CMA) in infants. METHODS AND METHOD: In a prospective study, 72 confirmed cases of CMA in infants were examined for anal fissure by pediatricians with five years' experience. A positive finding was defined as when an anal fissure was detected by at least two out of three examiners. RESULTS: Of infants with CMA with and without gastrointestinal GI symptoms, 79% and 83% had anal fissures, respectively The prevalence of anal fissure in these infants is significantly higher than in normal infants. CONCLUSION: Anal fissure may be a pathognomonic sign of cow's milk allergy in infants.