Common Methodologies in the Evaluation of Food Allergy: Pitfalls and Prospects of Food Allergy Prevalence Studies

Common Methodologies in the Evaluation of Food Allergy:Pitfalls and Prospects of Food Allergy Prevalence Studies

Shang-an Shu & Christopher Chang &

Patrick S. C. Leung

# Springer Science+Business Media New York 2012

Abstract Global and regional studies on the prevalence offood allergies are plagued by inconsistent methodologies,variations in interpretation of results, and non-standardizedstudy design. Hence, it becomes difficult to compare theprevalence of food allergies in different communities. Thisinformation would be useful in providing critical data that willenhance research to elucidate the nature of food allergies, andthe role of gene–environment interactions in the sensitizationof children and adults to foods. Testing methodologies rangefrom questionnaires to objective in vitro and in vivo testing, tothe gold standard, double-blind placebo-controlled food chal-lenge (DBPCFC). Although considered the most accurate andreliable method in detecting the prevalence of food allergy,DBPCFC is not always practical in epidemiological studies offood allergy. On the other hand, multiple logistic regressionstudies have been done to determine predictability of theoutcome of food challenges, and it appears that skin pricktesting and in vitro-specific serum IgE are the best predictors.Future studies directed towards confirming the validity ofthese methods as well as developing algorithms to predictthe food challenge outcomes are required, as they may some-day become accessory tools to complement DBPCFC.

Keywords Food allergy . Skin prick testing . Epicutaneoustesting . Specific IgE antibody testing . Double-blindplacebo-controlled food challenge

AbbreviationsDBPCFC Double-blind placebo-controlled food

challengeCAPRAST/CAP-FEIA

ImmunoCAP radioallergosorbent test

OFC Open food challengeRAST Radioallergosorbent testSBPCFC Single-blind placebo-controlled food

challengeSPT Skin prick test

Introduction

Food provides us with the energy and nutrients needed forgrowth and health maintenance. Across different geographicalregions, food ingredients and spices are used to create disheswith distinctive textures, flavors, colors, or smells, whichprovide enjoyment and pleasure. Normally, a food constituentis innocuous and well-tolerated by most individuals.However, if it triggers an allergic response in sensitive indi-viduals, adverse health effects may arise that lower one’squality of life and in rare cases may even bring about death.According to the most current “Guidelines for the Diagnosisand Management of Food Allergy in the United States,” afood allergy is defined as an adverse health effect arising froma specific immune response that occurs reproducibly on ex-posure to a given food [1]. So far, the exact mechanism of foodallergy remains elusive. Hypotheses including the nature ofthe food allergens, the genetics and environmental interaction

S.-a. Shu :C. Chang (*) : P. S. C. Leung (*)Division of Rheumatology, Allergy and Clinical Immunology,University of California at Davis, 451 E Health Sciences DriveSuite 6510,Davis, CA 95616, USAe-mail: [email protected]: [email protected]

C. ChangNemours/A.I. duPont Hospital for Children, Thomas JeffersonUniversity, 1600 Rockland Road,Wilmington, DE 19803, USAe-mail: [email protected]

Clinic Rev Allerg ImmunolDOI 10.1007/s12016-012-8337-8

have been proposed and studied [2]. The most well-knownpathophysiology involves IgE cross-linking on the surface ofmast cells by the allergen, which seems to be an obligatorystep in triggering an allergenic response in a sensitive individ-ual. Not all types of immunologic food allergy follow a type IIgE-mediated hypersensitivity. Because possible adverse out-comes such as anaphylaxis can be life-threatening, food aller-gy has always been a great concern for parents, caretakers,health care practitioners, the food industry, and governmentregulators.

In developed countries, the prevalence of food allergy hasincreased over the last two or three decades, with no uni-versal theory to explain this phenomenon, and no interven-tions appear to curtail a similar increase in developingcountries. The prevalence of food allergy seems to varygreatly with age. Some children may “outgrow” their symp-toms of allergy, but, for others, the food allergies may belifelong. Cross-reactivity between different species of foodsin allergic patients is also common. Dietary avoidance iscurrently the most essential component in the manage-ment of food allergy. However, it may not be beneficialfor children during periods of active growth, as it canlimit their intake of nutrients from different foods.Conflicting information regarding cross-reactivity oftenleads to an inconsistent understanding of food allergiesamong physicians and other health care givers whichlead to disparate recommendations and confusion amongpatients and parents.

Communities all over the world have devoted extensiveresources, including time, energy, and money to determine theprevalence of food allergies within their society. In Westerncountries, studies have been primarily focused on commondietary food groups such as egg, cow’s milk, fish, and peanut[3]. In the Eastern hemisphere, food allergy seems to havebeen on the rise for similar dietary groups, according tolimited available and validated data. It is important to charac-terize food protein sensitization patterns, as these data wouldbe useful in quantifying the prevalence of food allergy andalso help generate information on prevention. The determina-tion of the prevalence of food allergy is often hampered by thelack of reliable methods or tests. Moreover, different tests andmethodologies, standards, and calibrations may vary betweenstudies, rendering comparison and meta-analysis even moredifficult. This review focuses on the various methodologiesand discusses the standardization of the methodology usedwhen conducting studies.

Methods of Food Testing

The most common methods used in studies of the prevalenceof food allergies include (1) collecting participants’ allergic orrelative information by questionnaire, telephone survey, or

clinical charts; (2) skin prick test (SPT); (3) intradermal skintesting; (4) allergen-specific IgE antibody determination; and(5) food challenges. In general, questionnaires or clinicalhistories are used to screen for potential subjects but may alsobe used as variables in the study itself. SPT and IgE levels arecommonly used for medical diagnosis and for confirmation.Specific IgE levels is measured using a number of differenttechniques, usually enzyme-linked immunosorbent assays(ELISA), ranging from the original and no longer used radio-allergosorbent test (RAST) to the ImmunoCAP blood test.Diagnosis is complicated because detection of a food-specific IgE (sensitization) does not necessarily indicate aclinical reaction. Therefore, diagnosis requires a combinationof a detailed medical history, laboratory data, and in manycases, an oral food challenge, which is confirmatory of eithertolerance or an adverse food reaction (allergy or non-allergic).Double-blind, placebo-controlled food challenge (DBPCFC)is considered to be the gold standard in assessing the preva-lence of food allergies. However, there are relatively fewepidemiological or population-based reports using DBPCFC.A detailed discussion of each methodology follows, and abrief outline including the pros and cons of each method arealso illustrated in Table 1.

Questionnaire/Telephone Survey/Clinical Charts

Questionnaires are often handed out or computerized forstudy subjects to answer. The information collected mayinclude reactions to food, pulmonary symptoms, history ofallergic illnesses, or a family history of atopy. Other relevantinformation such as birth history, age, gender, ethnicity,occupation, smoking history, living environment, breast-feeding history, initial exposure time, diagnosis made by aphysician, previous, or current treatment, and parent educa-tional level as a surrogate of socioeconomic status, might begathered, based on the particular study populations or eth-nicity. Other useful data include whether the participant hada reaction due to food ingestion, whether this reaction al-ways occurred after eating the same food, what type of foodwas ingested, the type and severity of reaction, and thetreatment regimen.

The information collected would typically be used instudies to determine prevalence. However, there are somecaveats in using the questionnaire to predict prevalence.Using questionnaires is the first step in any study to selectout the appropriate participants for further assays or even forprevalence analysis if the sample size is large enough.However, questions can be misleading, depending on thechoice of words or how they were phrased. For infants oryoung children who cannot express or communicate wellenough, the parents or guardians would have to recall theallergic incident and help them answer the questionnaire.The answers for clinical symptoms or the initial exposure

Clinic Rev Allerg Immunol

time can also be subjective or uncertain. This methodologymay also introduce a selection bias as people who are moreaware of their food allergy might be more willing to partic-ipate in the study. Telephone surveys may over-representthose with a higher socioeconomic status because homeswithout telephones are excluded, and homes with multiplevoice lines are more likely to be selected. Participation rateand selection bias may also be caused due to the exclusionof cell phone users, ethnic minorities, immigrants, and lowersocioeconomic groups. These factors may be amplifiedwhen doing studies in developing countries due to therelative lack of landline telephone services.

Designing a standardized questionnaire for a prospective,long-term, population-based study that includes a large sam-ple size is the basis for excluding biases when determining

food hypersensitivity prevalence. A birth cohort design mayprovide better longitudinal data as well as a proper controlgroup. On the other hand, those with higher education or ahigher socioeconomic status tend to be more knowledgeableand more willing to participate in this kind of study. Astrategy to encourage people from all different socialclasses to participate is not only good for revealingthe true prevalence of food allergy in society but wouldalso serve as a good opportunity to provide educationon food-borne allergies.

Skin Prick Test (SPT)

The SPT, which utilizes commercial extracts, is usuallyperformed using a skin prick test device on the back or the

Table 1 Common methods used for food allergy study

Method Procedures Parameters Advantages Disadvantages

Surveys and retrospective chart reviews

Screening Questionnaire Self-report, symptomdiary

Easy to recruit manyparticipants, relative lowcost, no risk

Large sample size needed, time-consuming to go over all the surveys/charts, subjective answersTelephone survey Diagnosis is determined

by different doctors

Clinical charts Symptom diary

Skin test

Skin prick test Skin response is measuredafter allergen extractsare given

Wheal diameter ismeasured(millimeters)

Relative easy to perform, resultis available in minutes,relative low cost, relative lowrisk of side effect, patientcompliance is high

Interpretation maybe difficult inpatients with eczema ordermatographism, systemic andtopical antihistamines maysuppress the weal and flarereaction

Intradermal skintest

Serum IgE level

RAST,ImmunoCAP,Hycor, Immulite

A blood test is measuredaccording to the allergen-specific IgE level

Classes 0 to 6 differentcut-off lines (kU/L)

Relative easy to perform, candetect multiple food allergensat once, relative low cost,relative low risk; notnecessary to remove thepatient from medication

Less sensitive than skin prick test,need to be confirmed with foodchallenge

Food challenge

OFC All the participants, medicalteam, and the investigatorsknow the food forchallenge

Reaction or allergicsymptom is reported

If the patient has an allergicreaction only to the suspectedfoods and not to the otherfoods tested, the diagnosis offood allergy is confirmed.

It requires more effort and time ofthe participants, the medical team,and the investigator; its usefulnessrequires strict adherence to thetechniques; relative high risk ofside effect; and as with allmethods of food allergy testing, afalse-negative reaction can occurto foods if they have not beeneaten for a long time

SBPCFC Only the medical team andthe investigators know thefood for challenge

DBPCFC None of the participants,medical team, or theinvestigators knows thefood for challenge.Allergen is prepared by thethird party.

Gold standard for food allergystudy; raw, de-shelledAmerican market types

RAST radioallergosorbent test, OFC open food challenge, SBPCFC single-blinded placebo-controlled food challenge, DBPCFC double-blindedplacebo-controlled food challenge


volar surface of the forearm. There is a wide variety of skintesting devices available worldwide [4]. Devices that facil-itate simultaneous application of multiple tests are alsoavailable. Standardized allergen extracts are used whenavailable commercially, but most extracts are not standard-ized. Skin responses are measured in 15 or 20 min andcompared with positive or negative controls consisting of10 mg/ml of histamine or saline, respectively. Based on thewheal diameter, a measurement greater that is 3 or 5 mmgreater than the wheal for the negative control is considereda positive skin reaction (Table 2). The limitation of skintesting to foods is that the results may not correlate with aclinically significant allergy. SPT is also prone to examinervariability in the measurement and interpretation of the sizeof the reaction, and to device variability.

Allergen-Specific IgE Antibody Determination

One of the major problems with using in vitro testing to studyepidemiology of food allergies is that, while the methodologyis the same in principle, there are several test standards for thisprocedure. These include the ImmunoCAP, the Hycor system,and the Immunlite System from Siemens.

In vitro tests to measure specific IgE were developed begin-ning in 1974 with the well-known radioallergosorbent orRAST test. This was a proprietary test done developed byPharmacia, and although many allergists and lay people recog-nize and use the termRASTor CAPRAST, this test is no longerused because of the development of newer and better assays.

ImmunoCAP

Currently, the most widely used test is the ImmunoCAPdeveloped by Pharmacia, which was subsequently acquiredby Pfizer. There are currently several different ImmunoCAPsystems, designated as Phadia 100 to Phadia 2500. Thenewest version of the ImmunoCAP is ImmunoCAP specificIgE 0–100, which in 2004 received Food and DrugAdministration (FDA) approval to report results down to asensitivity of 0.1 kIU/l. Phadia has also recently developedan ImmunoCAP Rapid system, which is meant for point-of-care testing and will generate an “allergenic profile” tocommon pre-assigned allergens. The ImmunoCAP ISAC isanother one of Phadia products and which utilizes the prin-ciples of component-resolved diagnostics and biochip tech-nology to test multiple allergenic determinants in amicroarray system. This test is reserved for the diagnosisof complex cases.

Immulite

Immulite 2000 by Siemens utilizes an ELISA immunoassayto detect antibodies to different food allergens. It is based on

a solid-phase assay using specific antibody-coated polysty-rene wells. Sensitivity and specificity of the Immulite 2000are similar to that of the ImmunoCAP system [5], but thespecific result values may be different [6]. The allergensources used in the testing to detect specific IgE differsignificantly among the three available assay systems.

Hycor

The third group of commonly used ELISAs for the evalua-tion of IgE to food allergies is developed by Agilent. In2010, Agilent sold its Hycor Biomedical Business to LindenLLC. The Hycor system is an ultra-sensitive enzyme immu-noassay system that is FDA-cleared for the measurement ofallergen-specific IgE. The detection limit of this system is0.043KU/l. This system is compliant with ISO9001 andCLSI guidelines and is capable of generating over 1,500test results per run. An alternative system developed byHycor is known as the HYTEC 288 Plus system. This isan activated cellulose-based solid-phase assay with demon-strated equivalence to fluorescence enzyme immunoassay[7]. While the Hycor system is an accurate and standardizedtest, it is less popular in the United States than theImmunoCAP. The different reference laboratories in theUnited States utilize different specific methodologies.When comparing studies, it is important to realize that theresults may differ based on the test that is used.

An illustration of the different values obtained by thesetests can be illustrated in Table 3, which shows that not onlyare the numerical values vastly different, but so are theranges corresponding to artificially designated levels ofclinical significance (i.e., class). The level of specific IgErepresents a likelihood of being allergic to that particularallergen but has no predictive value regarding the severity ofa potential reaction.

Even when the same methods are used to measure theserum-specific IgE, the positive predictive values of reactingto a particular food in a population is quite different(Table 2). For example, the range for peanut protein IgE tobe considered positive is from 0.1 to 15 kU/L. This is morethan a tenfold difference [8–10]. Similar variations are ob-served for other food allergens, milk (0.5–32 kU/L) [11, 12],and egg (0.35–6 kU/L) [13–15].

Food Challenge

Three types of challenges may be performed in a foodallergy study: open, single-blind, placebo-controlled, orDBPCFC. The open challenge is an unblinded feeding witha food in its natural form. It is used if the concern forparticipant bias is low and clinical symptoms are notexpected to occur. It is usually performed in the officesetting with a 1–2-h observation period, and it is acceptable


Table 2 Variability in food allergy testing

Methods Variations References

Skin prick test Wheal size for positive

≥2 mm [22, 40, 41]

≥3 mm [8, 10, 11, 14, 15, 19, 21, 23,26, 30, 31, 34, 37–39, 42–55]

Positive/negative control

10 mg/ml histamine hydrochloride/saline [49]

10 mg/ml histamine/50 % glycerosaline [8]

A wheal≥3 mm of the negative control was taken as a positive [50]

10 mg/ml histamine/saline [56]

10 mg/ml histamine dihydrochloride/diluent [48]

10 mg/ml histamine/not specified [31, 39]

1 mg/ml histamine phosphate/saline [23]

RAST Class for positive

≥2+ [11, 36]

3+ [24]

ImmunoCAP IgE level for positive (kU/L)

0.1 (peanut) [8]

0.35 (peanut) [9, 10, 15, 49]

0.7 (peanut) [14, 22, 57]

14 KIUA/L (peanut) [58]

15 (peanut) [12, 15, 21, 43]

0.5 (milk) [11]

15 KIUA/L (milk) [58]

32 (milk) [12]

0.35 (egg) [13, 15]

0.7 (egg) [14, 57]

6 (egg) [12]

7 KIUA/L (egg) [58]

20 (fish) [12, 43, 58]

Low 1.43–4 SU/Lml, moderate 4–20, high 20–100, very high 100–300,extremely high >300

[46]

Not mentioned how to determine “positive” [55]

Class 0 (0.35 kUa/l), class 1 (0.35–0.7), class 2 (0.7–3.5), class3 (3.5–17.5), class 4 (17.5–<50), class 5 (>50), class 6 (>100)

[59]

Food challenge Mask

OFC Cow’s-milk-based formula, allomin 15 g protein/L; after 12 months ofage, pasteurized and homogenized whole cow’s milk (35 g protein/L)

[11]

Pancake [24]

Low-fat milk or contained in pancakes [44]

Peanut butter spread on bread or a flapjack biscuit that contained peanut [19]

Baked egg in a cake or uncooked pasteurized whole egg [15]

Peanuts were hidden in apple compote [8]

Baked egg was offered, not cow’s milk or shellfish [56]

A drop of ordinary formula/milk on the lip, boiled egg were used [23]

SBPCFC Stewed apples or mashed potatoes [25]

Dried biscuit powder to simulate crushed peanut [25]

DBPCFC Codfish and peanut were disguised in chocolate bars; milk and egg weremasked in colored cup with basic ingredients of sugar, cocoa, andvanilla sugar.

[10]

Peanut flour-based biscuits [20]

[22]


for use in challenging young children in most cases.Because it is not blinded to the investigators, physicians,and participants, the interpretation of the challenge can besubjective. However, it is still useful in eliminating potentialfood culprits when the questionnaire or laboratory testingindicates that the food is unlikely to be causative.

Another approach is blinding or masking the challengefood with a vehicle by placing the food in an opaquetitanium-dioxide-coated gelatin capsule or by mixing inanother food to reduce bias. For example, nonfat milkpowder can be mixed in applesauce or egg in pancake; drybiscuit powder may be included to simulate crushed peanutor peanut meal mixed in oatmeal flakes or mashed potatoes(Table 2). The placebo is usually a food that is similar to thechallenge food in terms of taste, consistency, texture, ap-pearance, smell, and sensation within the mouth. In theSBPCFC, the patient is unaware of the challenge content,but the physician is. As long as the investigators remaincareful not to inadvertently reveal to the patient what he orshe will be receiving and be consistent when they hand outthe food, this approach is fairly reliable.

The last method is the double-blind, placebo-controlledfood challenge, which is the gold standard for diagnosing

food allergies for both clinical and research purposes. Thepatients, investigators, and physicians should not be awareof the challenge food content. The tested foods and placebosare prepared and coded by a third party not involved in theevaluation or analysis of results to minimize any possiblebias. Once the study is completed, the code is revealed foranalysis. This design requires more time and work but is thebest way to evaluate for food allergy. Attending physiciansare usually required to take medical precautions before,during, and after the challenges to ensure the safety of theparticipants.

Ideally, because DBPCFC provides valuable and accuratedata, it should be performed whenever possible.Unfortunately, DBPCFC may not be a practical way toevaluate food allergies in large-scale epidemiologic studies.In reality, parents are reluctant to consent to their childrenundergoing this kind of food challenge, especially if theyhave a history of bad allergic symptoms or even worse,anaphylaxis. Food challenges are time-consuming, and ifhundreds or thousands of subjects are needed for analysisin prevalence studies, it may become a logistical nightmare.

Diagnostic testing using recombinant allergens and inhi-bition assays can be used but is limited by the number ofallergens commercially available. In addition, results fromthese tests do not always correlate with clinical reactivity.Although many of the major allergens are commerciallyavailable, food allergies are limited. A common feature ofthese food allergens is that they are water-soluble glycopro-teins and are relatively stable to heat, acid, and proteases.Therefore, the purification process and the selection of theappropriate food species for extraction can be critical. Morerecently, a more precise in vitro method of evaluating foodallergies, referred to as component resolved diagnostics ormolecular allergology, involves evaluating for individualproteins or peptides rather than by using crude mix as theallergen source for the ELISA. This may provide a much

Table 2 (continued)

Methods Variations References

Peanut meal were disguised in mashed potatoes or oatmeal flakes forhigher doses.

Defatted peanut flour [9]

Allergen ingredients were disguised in pancakes or muffins. [24]

Milk substitute [44]

Peanut flakes were disguised in applesauce or grape jelly. [21]

Did not mention what the allergens (egg, fish, peanut) were disguised in [30, 52, 53]

Disguised in Sinlac, orange flavor, carotene, cereal flakes, and/or purecacao powder

[55]

Disguised in opaque, titanium-dioxide-coated gelatin capsules [40]

Disguised in stewed apple or mashed potatoes [60]

Did not mention what the allergens (egg, fish, milk) were disguised in [50]

Table 3 Interpretation of specific IgE ELISA tests

Class ImmunoCAP (kU/l) Hycor Immulite

0 <0.35 <0.08 <0.35

0/1 N/A N/A N/A

1 0.35–0.70 0.08–0.15 0.35–0.70

2 0.71–3.50 0.16–0.50 0.71–3.50

3 3.51–17.5 0.51–2.50 3.51–17.5

4 17.5–50 2.51–12.50 17.51–52.50

5 51–100 12.51–62.50 52.50–100

6 >100 62.51–>100 >100


greater level of understanding of the patient’s allergies butmay be impractical for large-scale epidemiological studies atthis time.

Most laboratories that study peanut food allergies wouldspecify the particular kind of peanut they used and how theyprepared the allergen in their food challenge (Table 4). InDean’s group, partially defatted Runner roasted peanut mealwas used, whereas raw, de-shelled American market typeswere prepared in Brugman’s experiment [16, 17]. On the otherhand, this is often not the case for other food allergens. Forexample, milk whey and casein concentrations may vary withdifferent preparation methods. Heating and other methods offood processing may also have different effects on food aller-gens, including those contained in the same food [18].

Even though food challenge, especially the DBPCFC, isthe best way to confirm food allergy, this method relies onan estimation of average daily intake of a food to determinethe amount used in a challenge. During a prevalence study,an underestimation or overestimation of the prevalence mayoccur if the study design is not standardized, as in somecases less allergen may be given than in other studies. Thechallenge may also be influenced by participant wishes,which may even lead to a discordant amount of allergenadministered within a study. According to Morisset et al.,they observed that the lowest reactive threshold is set at lessthan 2 mg for egg, 5 mg for peanut, and 0.1 ml for milk.However, without a standardized protocol, the thresholddoses for eliciting allergic reaction can vary from study tostudy. For example, the starting dose for peanut ranges from0.01 to 25 mg, and the total peanut allergen given to theparticipants varies from 2 to 25 g [8–10, 19–22]. A similarissue is observed in other studies with regard to milk, egg,seafood, or other food allergens [11, 23–25].

Interpretation of Food Allergy Study Data

In general, eggs, fish, milk, and peanuts are the most com-mon foods that cause hypersensitivity in most Western andAsian countries. However, there is remarkable heterogeneityin the prevalence or ranking of these foods due to differentdesigns, methods, tests, or populations recruited in variousstudies. Even if similar methods or tests are used for food

allergy studies, estimation of allergy prevalence is predom-inantly based on how the individual investigator deciphersthe data collected from participants.

Questionnaire/Survey

Many food studies utilize only questionnaires or telephonesurvey to determine prevalence regardless the sample size[16, 26–30]. However, data from this approach are based onthe perceptions of the participants and can be subjective.There could also be a significant selection bias as investi-gators are dependent upon the response rates of differentsubpopulations. Inevitably, the prevalence tends to be higherin these studies compared with those that involve more tests,such as SPT, specific IgE level, or food challenge. Anotherissue for the questionnaire design is whether the participantsremember all the incidences correctly. For example, theinitial exposure time has been hypothesized to sensitize theinfant even during pregnancy or through breast milk whenfeeding. Studies focused on young children often includequestions regarding first exposure time. Recall may be aproblem. Not all the children would have obvious symptomsimmediately which the parents will note and record forresearchers. As a result, parents can only estimate the timeof initial exposure, which may not always be accurate.

Ethnicity/Diet

Some countries have extremely heterogeneous populationswith diverse cultural backgrounds which can subsequentlyexert influence on diets and lifestyles. The differences in thedietary content of various groups may partly explain whyallergy prevalence varies throughout the world (Table 5).Indeed, with easy access to exotic ingredients these days,people are exposed to food proteins that their ancestors mightnot have ingested. The reactions to these new allergens as wellas allergy reactions to the foods specifically consumed bydifferent ethnic groups are increasingly being reported inliterature [23, 31, 32]. Another example can be seen in howfoods are prepared. The amount of peanuts consumed is moreor less the same in Western countries and Asia. However,when preparing this item for consumption, roasting is thepreferred method in Western countries, while people preferboiling in Asia. It has been shown that high heat will increasethe allergenicity of peanuts when they are roasted as comparedwith boiled or fried peanuts [18].

Although early life exposure, including prenatal expo-sure, to certain foods can be associated with the subsequentdevelopment of food allergies, there are limited reports onthe optimal timing of when potential food allergens shouldbe introduced. In addition, there is scant evidence support-ing the necessity for altering or restricting maternal dietduring pregnancy or lactation to prevent food allergies [33].

Table 4 Source of food allergens used for studies

Sources Reference

Peanut flour-based biscuits [20]

Partially defatted Runner roasted peanut meal [22]

Raw, de-shelled American market types Runner,Valencia, Spanish, and Virginia

[45]

Defatted Runner cultivar peanut [9]


Cut-Off Line

Many of the known patterns and documented natural historyfor food hypersensitivities in children seem to be age-related. Studies have shown that many patients with cow’smilk or egg allergy tend to outgrow their allergy withinchildhood, whereas peanut or seafood allergy tends to bemore persistent throughout life. It has also become recentlyevident that the natural history depends on prognostic fac-tors such as the initial titer of specific IgE. In a large-scaleseafood allergy study done in the United States by a randomtelephone survey, the prevalence is shown to be more com-mon in adults compared with children [34]. However, based

on their age group breakdown, which included from 0–5, 6–17, 18–40, 41–60, and 61 years old and up, the cut-off linesfor children and adults were not defined in the study.Another example is from Green’s group which showed apredominance of male patients being affected at a youngerage while female patients were affected at an older age.However, the subjects’ ages were not specified [35].Arbitrary age cut-off lines have been set for different re-search groups, which makes it difficult to compare studieseven though they are for the same food allergens.

Cow’s milk and egg allergies are the two most commonfood allergies in children, but many will outgrow them bytheir mid-teens. Therefore, the prevalence will be age-

Table 5 Common FA (withranking) in different geographi-cal regions

Europe Food allergens References

Denmark Egg, milk, seafood, wheat, peanut, soy [10, 11]

Vegetables>milk, fruit>nuts>legumes>seafood>wheat [61]

Estonia Egg, milk [46]

Finland Milk>fruits>vegetables>seafood>eggs>nuts>wheat [61]

Egg, fish, milk, nuts, fruits, wheat, [62]

France Egg, peanut, milk, mustard, codfish, hazelnut, kiwi, sesame [19, 25, 38]

Germany Nuts>fruits>milk>seafood>peanut>egg [41]

Fruit>milk>nuts, wheat>vegetables>egg>seafood [61]

Hazelnut>fruits>milk>flour>cacao [55]

Italy Fish>milk>nuts>egg>fruit>cereals [38]

Milk>fruits>egg, wheat>nuts, vegetables>seafood>nuts [61]

The Netherlands Peanut, fruits [22, 49]

Norway Hazelnut>egg>seafood [24, 30]

Spain Egg, fish, milk, peach, nuts, lentil, peanut, chick pea [36, 38, 42]

Sweden Nuts, peanut, milk, egg, fish, fruits, vegetables [59]

UK Peanut, milk, egg, fish, wheat, sesame, soy, cheese, chocolate [19, 20, 27, 37, 63]

America

Canada Peanut, fish, shellfish, sesame [21, 43]

USA Egg, seafood, milk, peanut, nuts, vegetables, fruits, cereal, soy [64] [8, 34, 36]

Eastern

China Egg, milk, peanut, nuts, seafood [23, 32]

Hong Kong Shellfish>egg>peanut>beef>milk>tree nuts [65]

Milk>egg>shellfish>peanut [66]

Japan Soy, egg, milk, seafood, wheat, beans, chicken, vegetables [38, 67]

Korea Egg>milk>nuts [68]

Philippine

Singapore Egg>shellfish>peanut [31]

Milk>egg>shellfish>peanut [69]

Bird's nest>seafood>egg>milk>Chinese herbs [38]

Thailand Milk [60]

Middle East

Israel Egg, milk, sesame, peanut, soy, nuts, strawberry [38]

Turkey Milk [48]

Pacific

Australia Egg, milk, peanut, nuts, sesame, wheat, soy, fish [30, 31, 38, 39, 56]


Table 6 Prevalence estimation for different food allergens at different countries

Dietaryfood

Country Samplesize

Age(years)

Gender Prevalence(%)

Methods used in the study Reference

Peanut Denmark 111 <3 241 F/245 M 0 Q, SPT, IgE, OFC, DBPCFC [10]

Denmark 486 03 0.2 Q, SPT, IgE, OFC, DBPCFC [10]

Denmark 301 3–21 0 Q, SPT, IgE, OFC, DBPCFC [10]

Denmark 936 22–59 0.4 Q, SPT, IgE, OFC, DBPCFC [10]

Spain 355 0–11 111 F/244 M 3.9 IgE, SPT, RAST [36]

Israel 102 0–2 43 F/59 M 0.04 Q, SPT [38]

UK 1,072 4–5 1.8 Q, SPT, DBPCFC [20]

Canada 4,339 5–9 1.5 Q, SPT, IgE, DBPCFC [21]

Germany 4,093 18–79 2.4 Q, SPT, IgE, DBPCFC [55]

UK 2,878 3–4 1.5 Q, SPT, OFC [19]

France 33,110 <61 1 Q [70]

Australia 2,848 1–1.5 8.9 Q, SPT, IgE [56]

Hong Kong 3,827 2–7 1,797 F/1,856 M 8.1 Q [65]

Canada 9,667 1 Q [43]

Cow's milk Denmark 111 <3 241 F/245 M 0 Q, SPT, IgE, OFC, DBPCFC [10]





Israel 102 0–2 43 F/59 M 0.3 Q, SPT [38]

Germany 1,537 25–74 775 F/762 M 2.8 Q, SPT, CAP-RAST [41]

Denmark 8,825 0–17 22.7 Telephone survey [61]

Finland 8,825 0–17 41.7 Telephone survey [61]

Germany 8,825 0–17 23.8 Telephone survey [61]

Italy 8,825 0–17 33.3 Telephone survey [61]

Denmark 1,749 0–1 2.2 Q, SPT, IgE, RAST, OFC, DBPCFC [71]

USA 140 0–16 47 F/93 M 20 Medical charts [35]

Japan 1,336 0–6 31.8 Q [28]

Norway 2,721 0–2 1.1 Q, SPT, OFC, DBPCFC [44]

The Netherlands 4,450 4–15 2,180 F/2,270 M 22 Q [16]


France 33,110 <61 8 Q [70]


Hong Kong 3,827 2–7 1,797 F/1,856 M 5.7 Q [65]

China 497 0–2 220 F/257 M 1.3 Q, SPT, OFC [23]

Egg Denmark 111 <3 241 F/245 M 0 Q, SPT, IgE, OFC, DBPCFC [10]





Israel 102 0–2 43 F/59 M 0.5 Q, SPT [38]

Germany 1,537 25–74 775 F/762 M 0.4 Q, SPT, CAP-RAST [41]

Denmark 8,825 0–17 0 Telephone survey [61]





Norway 2,721 0–2.5 1.2–2.6 [44]


dependent, and comparisons between different studies maybe difficult, due to variations in patient inclusion criteria.Children from different geographic areas may not only beexposed to the same species of a particular food, but themethod of preparation may be different which will affect theresults of a questionnaire or phone survey. Protein extractsused for analysis may vary from region to region which mayaffect the results of objective skin or serum testing.Therefore, determining the cut-off criteria for food allergyis different for each food substance and may also differbased on the study, making comparison difficult.

Statistics for Determining the Food Allergy Prevalence

Epidemiological and prevalence studies for food allergiesmay include as many as thousands to as few as 10–20participants. Besides determining the prevalence of a partic-ular food allergy in a particular segment of the world, the

aim of these studies is also to characterize the food hyper-sensitivity, so that the researcher can gain a better under-standing of the mechanism or pathophysiology. The resultsof these studies may provide information for the physiciansto make better recommendations to parents regarding avoid-ance and also to better evaluate the food allergy with theappropriate tests. However, the statistics used to estimate theprevalence of a food allergy is often inconsistent from paperto paper. In a study of 355 children from Spain, the preva-lence of different foods was calculated based on percentageof the total allergic reactions in their study [36]. Many othergroups use SPSS for their statistics analysis; however, theweighting of the study data with regard to, for example, age,sex, breastfeeding, education, environmental factor, andother allergic illnesses, are not standardized and often notspecified. Table 6 summarizes the prevalence estimation fordifferent food allergens in different countries. The variationin prevalence for individual food allergies was high, for

Table 6 (continued)

Dietaryfood

Country Samplesize

Age(years)

Gender Prevalence(%)

Methods used in the study Reference

Q, SPT, IgE, RAST, OFC,DBPCFC

Japan 1,336 0–6 52.3 Q [28]

Norway 3,623 0–2 1,811 F/1,812 M 4.4 Q [26]


France 33,110 <61 4 Q [70]


Hong Kong 3,827 2–7 1,797 F/1,856 M 9.1 Q [65]

China 497 0–2 220 F/257 M 2.5 Q, SPT, OFC [23]

Fish Denmark 111 <3 241 F/245 M 0 Q, SPT, IgE, OFC, DBPCFC [10]

Denmark 486 03 0 Q, SPT, IgE, OFC, DBPCFC [10]




Israel 102 0–2 43 F/59 M 0.01 Q, SPT [38]

Denmark 8,825 0–17 0 Telephone survey [61]





Norway 3,623 0–2 1,811 F/1,812 M 3 Q [26]

The Netherlands 4,450 4–15 2,180 F/2,270 M 30 Q [16]

Australia 2,848 1–1.5 0.9a Q, SPT, IgE [56]

Hong Kong 3,827 2–7 1,797 F/1,856 M 15.8 a Q [65]

USA 23,426 4–6 12,552 F/10,635 M 5.9 a Q [34]

Canada 9,667 0.51 Q [43]

F female, M male, Q questionnaire, SPT skin prick test, OFC open food challenge, DBPCFC double-blinded placebo-controlled food challengea For shellfish


example, prevalence varied from 0.04 to 8.9 for peanut, 0.3to 41.7 for cow’s milk, 0.1 to 52.3 for egg, and 0.01 to 30 forfish. In general, in the studies that only used questionnaireor telephone survey, the estimated food allergy prevalencetended to be much higher than studies using other quantifiedassays, such as SPT, serum allergen-specific IgE level, orfood challenge.

Discussion

Data from Venter’s group revealed that peanut allergy prev-alence for young children born in the same geographicalarea has changed over time [37]. In the past few decades, anincreasing prevalence of food allergies in general has alsobeen observed throughout the world. Egg, cow’s milk, pea-nut, and shellfish are common foods that cause allergiesworldwide; however, the order of highest prevalence mayvary in different geographic areas at different time points.For example, sesame is a common food ingredient and isintroduced to children early in life in Israel. In a cross-sectional study, Dalal et al. has shown that sesame is thethird most common food allergy after egg and cow’s milk inyoung children in Israel [38]. Seafood allergy is a commonfood allergy in Australia and the United States [39]. Inreviewing various reports and literature in this paper, wehave noted numerous limitations in determining prevalenceor incidence of food allergy. Moreover, a variety ofpopulation-based studies have generated new hypothesesabout risk factors for the development food allergy.Limitations which include definitions for food allergy, meth-odological design, and the study’s population size, environ-mental/cultural and genetic/racial differences indeed mayhinder researchers from making accurate assessments offood allergy. Therefore, a large-scale and cross-cultural foodallergy study that involves the joint effort of multiple labo-ratories worldwide should be conducted. By following thesame design, protocols, or even the same model of instru-ments, a “standardized” food allergy study will help mini-mize the bias as well as possible technical differences anderrors among the participating laboratories.

Although there remains uncertainty about the exact prev-alence and incidence of food allergies, it is clear that thedisease commonly impacts huge populations around theworld. Similar to other atopic diseases, many current studiessuggest that the prevalence of food allergy is increasing.This paper extensively discussed the advantages and disad-vantages of the current methods used in most food allergyprevalence studies. Well-defined guidelines and protocolsfor food allergy are still required. Large population-basedcohort studies will likely provide additional insights on theprevalence, natural course, and risk factors for disease.Future research to establish a standardized algorithm for

the conduct of all food allergy prevalence studies worldwidewould be invaluable. This knowledge will improve preven-tion and treatment and greatly benefit the general public.

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Documents

Common Methodologies in the Evaluation of Food Allergy: Pitfalls and Prospects of Food Allergy Prevalence Studies