4

Assessments, Diagnostic Testing, Disease Monitoring, and Prognosis

OVERVIEW

A diagnosis of food allergy carries numerous health, emotional, social, and nutritional consequences. Therefore, a proper diagnosis is imperative. Unfortunately, studies suggest that many individuals needlessly avoid foods on the presumption of a food allergy without seeking medical confirmation, a practice that can lead to unnecessary risk and burden (Boyce et al., 2010; Fleischer et al., 2011; Rona et al., 2007). For example, in one meta-analysis, the rate of self-reported food allergy was 12 percent and 13 percent for children and adults compared to 3 percent when confirmation with testing was applied (Boyce et al., 2010; Rona et al., 2007). One of the major issues in food allergy is the common misconception that having a “positive test,” by a blood test or allergy skin prick test (SPT, otherwise known as sensitization, or a condition in which an individual produces detectable food-specific immunoglobulin E [IgE] antibody), is equivalent to having a clinical food allergy. For example, Fleischer et al. performed 111 supervised feeding tests with 44 children avoiding foods because of positive skin or serum allergy tests and, overall, 93 percent of the children were tolerant of the avoided food (Fleischer et al., 2011). Although this was a subpopulation of children with high rate of atopic dermatitis, on a population level, many more persons are also sensitized to foods than are clinically reactive upon ingestion. For example, 2005-2006 National Health and Nutrition Examination Survey (NHANES) data showed a 7.6 percent rate of positive serum IgE tests to peanut (10.7 percent in children ages 6 to 19 years), clearly higher than the prevalence of clinical peanut allergy (Liu et al., 2010). Compounding

the problem, many physicians lack an understanding of how to apply common diagnostic tests and interpret the results. In a survey of 407 primary care physicians, less than 30 percent of the participants reported that they were comfortable interpreting laboratory tests to diagnose food allergy, and 38 percent indicated incorrectly that skin or blood tests were sufficient for a diagnosis (Gupta et al., 2010). Clearly, the lack of understanding among physicians is compounded among the lay public.

Although overdiagnosis is a concern, conversely, assuming that an allergen has been identified as a trigger of a serious allergic response, a lack of confirmation could lead to re-exposure to the true culprit, with serious consequences. It is therefore imperative that individuals with suspected food allergy seek a medical diagnosis to identify whether the cause of symptoms is a food allergy and to identify culprit foods.

Considering the various symptoms (e.g., rashes, respiratory symptoms, gastrointestinal [GI] symptoms) and medical illnesses (e.g., atopic dermatitis, anaphylaxis) attributable to food allergy, many of which have alternate diagnoses (i.e., intolerance, pharmacologic reactions), or nonfood triggers (i.e., pollen allergy, irritants), food allergy diagnosis is complicated. Additionally, no simple tests exist that, in isolation, diagnose a specific food allergy (Boyce et al., 2010; Sampson et al., 2014). The primary tools currently available for diagnosis include the medical history, elimination diets, SPT, food-specific IgE (sIgE) (serum tests for food-specific IgE against specific proteins in foods), component resolved diagnostics (CRD), and medically supervised oral food challenges (OFCs).

This chapter includes relevant aspects of mechanisms of food allergy in relation to the current accepted methods for diagnostic testing and prognosis, including misconceptions about the methods, limitations, and factors that might affect diagnosis. The chapter also describes some promising methods that need further research, validation, or standardization before being used routinely, and methods that are not recommended for use routinely. The chapter ends with overall conclusions, recommendations, and research needs.

APPROACH TO LITERATURE REVIEW

In preparing this chapter, new individual systematic reviews or meta-analyses were not conducted. The primary resources for discussion, findings, conclusions, and recommendations were derived from the National Institute of Allergy and Infectious Diseases/National Institutes of Health (NIAID/NIH)–supported Guidelines (Boyce et al., 2010), the European Academy of Allergy & Clinical Immunology (EAACI) Guidelines (Muraro et al., 2014), and associated systematic reviews (Soares-Weiser et al., 2014) as well as the American Academy of Allergy, Asthma & Immunology

(AAAAI) Guidelines (Sampson et al., 2014; see Chapter 1, Table 1-1). Additional PubMed searches were selectively performed to identify studies and reports in the literature, especially focusing on papers published after the aforementioned reports. Meta-analyses, systematic reviews, expert reports, and practice guidelines were selected when available and supplemented with more recent publications.

REASONS TO INITIATE ASSESSMENTS FOR FOOD ALLERGY

The NIAID/NIH-supported Guidelines (Boyce et al., 2010) suggest that food allergy should be considered in a number of specific circumstances. Having allergic symptoms within minutes to hours after ingestion, especially from a specific food on more than one occasion, is suggestive of a food allergy and warrants investigation. Symptoms can include skin symptoms of itchy rashes, hives, or swelling; eye symptoms of itching, tearing, redness, or swelling; oral symptoms of itching or swelling of the lips, tongue, or palate; upper airway symptoms of congestion, itching, sneezing, nasal discharge, or hoarseness; lower airway symptoms of cough, chest tightness, wheezing, or trouble breathing; gastrointestinal symptoms of nausea, pain, vomiting, or diarrhea; cardiovascular symptoms of fast or slow heart rate, dizziness, low blood pressure, confusion, loss of consciousness; uterine contractions; and a sense of “impending doom.”

Food allergy diagnostic testing also may be warranted for infants, young children, and selected older individuals with moderate to severe atopic dermatitis because a higher rate of food allergy occurs in these populations, whether or not the food allergy may be contributing to the rash (Boyce et al., 2010; Sidbury et al., 2014). Disorders with subacute or chronic symptoms that indicate food-related disorders, such as food protein–induced enterocolitis (FPIES), enteropathy, and allergic colitis, also warrant investigation for food-allergic triggers. Food allergy also should be considered in children and adults with eosinophilic esophagitis (Boyce et al., 2010; Liacouras et al., 2011; Markowitz et al., 2003). Importantly, food allergy is not a typical trigger of chronic asthma or chronic rhinitis in childhood (Boyce et al., 2010; Sampson et al., 2014), although it can cause occupational asthma in certain groups, such as bakers or shellfish handlers.

The initiation of food allergy diagnostic testing also has some areas of uncertainty. For example, one expert panel (Boyce et al., 2010) concluded that there was insufficient evidence to recommend routine food allergy testing before introducing highly allergenic foods to children at high risk of food allergy, such as those with pre-existing severe allergic disease or family history of food allergy. However, they indicated value in such evaluations for selected patients, such as those having a peanut allergy or evidence of

another underlying food allergy. For example, testing for tree nut allergy in a child with peanut allergy who has not yet been exposed to tree nuts would be appropriate. Similarly, consensus recommendations regarding introduction of peanut to high-risk infants with early-onset atopic disease, such as severe eczema or egg allergy, have suggested that infants might benefit from evaluation to diagnose any food allergy and to evaluate an infant for introduction of peanut (Fleischer et al., 2015).

A common misconception or concern among caregivers is that if one sibling develops a food allergy, other siblings also will become allergic. However, a recent study of a large cohort of families with food allergies found that only a small proportion of siblings are both sensitized (based on SPT and IgE) and clinically reactive to a food (based on history of typical symptoms of an allergic reaction to a food) (Gupta el al., 2016). In support of NIAID/NIH-supported Guidelines (Boyce et al., 2010), the authors concluded that testing for food allergy in siblings without a history of clinical reactivity appears to be unjustified and that screening may lead to negative consequences related to potential misdiagnosis and unnecessary avoidance of a food.

MECHANISMS OF FOOD ALLERGY IN RELATIONSHIP TO DIAGNOSTIC TESTING

Chapter 2 described specific food allergic disorders and pathophysiology. With regard to diagnostic testing, the pathophysiology of the disorder is relevant. For example, tests for food-specific IgE antibodies (i.e., SPT, sIgE, and CRD) are relevant for IgE-mediated disorders. These tests may sometimes be performed in disorders that are non-IgE-mediated to identify a potential for acute allergic reactions if the previously consumed food has been removed from the diet after having been a part of the diet (Liacouras et al., 2011), or to determine whether there has been a change in pathophysiology to an IgE-mediated disorder, as can occur with FPIES (Caubet et al., 2014). In contrast, the medical history, elimination diets, and physician-supervised OFCs are useful in all food allergic disorder evaluations.

CURRENTLY AVAILABLE MODALITIES ROUTINELY USED TO DIAGNOSE FOOD ALLERGY

A number of modalities have been recommended for diagnosing food allergy (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). These are reviewed briefly in the following section with an emphasis on utility and limitations. The diagnostic tests discussed below are generally not used in isolation (see “General Diagnostic Algorithms”).

Medical History and Physical Examination

A thorough medical history and physical examination are imperative in the diagnosis of food allergy (Boyce et al., 2010; Muraro et al., 2014). They can help to identify the likelihood of the diagnosis, and suggest whether the pathophysiology is IgE or non-IgE, which is important for test selection. The history and physical examination also identify potential triggers, which help to hone specific test selection. Importantly, details of the history may disclose alternative reasons for symptoms, other than a food allergy. For example, an acute allergic reaction attributed to a food may actually be triggered by other allergens, such as medications or insect stings. Numerous triggers, such as environmental irritants, change in temperature, and infections, can initiate atopic dermatitis flares. Chronic GI symptoms can be attributed to food but may actually be caused by medical conditions such as reflux or inflammatory bowel disease. In fact, a broad differential diagnosis exists to distinguish food allergy from other allergic disorders or from disorders that are not immunologically mediated and associated with food. Food poisoning or pharmacologic effects from food components may be masqueraders of a food allergy. Many patients confuse food allergy and food intolerance (Sicherer et al., 2012). Food intolerance is not mediated by the immune system, and is characterized by symptoms such as gas, bloating, and diarrhea in the case of lactose intolerance.

No evidence-based, standard series of questions has been developed for use in taking a medical history to evaluate a possible food allergy, although creating this type of question set is under study (Skypala et al., 2015). The clinical history should include possible eliciting allergens, the timing and chronicity of the ingestion and symptoms, symptom severity, reproducibility, risk factors, identification of foods that are tolerated, and coexisting medical and allergic problems. The use of structured questionnaires on symptoms, foods, and other background information may be beneficial. However, based on limited data, the predictive value of the clinical history for immediate symptoms, either alone or in combination with SPT or sIgE, ranges from 50 percent to 100 percent (Muraro et al., 2014). Nonetheless, the clinical history is central to provide reasoning (prior probability) applicable to additional test selection and interpretation on a patient-specific basis, as will be reviewed further below.

Elimination Diets

Elimination diets, with removal of one or a few specific foods, is considered useful in diagnosing food allergy, especially for disorders with chronic symptoms, such as eosinophilic esophagitis (EoE), atopic dermatitis, and allergic proctocolitis (Boyce et al., 2010; Muraro et al., 2014). A

diagnostic elimination diet is different from a treatment elimination diet, where an identified food allergen is removed from the diet as a form of therapy. When a properly performed diagnostic elimination diet does not ameliorate the symptoms, food allergy to the eliminated food(s) is unlikely. If elimination does result in amelioration of symptoms, re-administration of the food, for example during an OFC, may be needed to prove a cause- and-effect relationship. However, experts have recognized that for some disorders, such as FPIES, a successful elimination diet in combination with a convincing history may be sufficient for diagnosis (Boyce et al., 2010; Sampson et al., 2014). The rationale for this decision is based on the concern that the OFC may provoke significant morbidity and may be better reserved for evaluating later resolution of the disorder.

Determining which foods should be eliminated is based on medical history, allergy testing, and/or the epidemiology of the illness considering common triggers. The results of the elimination diet are monitored and evaluated over a pre-specified period, such as 2 to 4 weeks. There are many caveats regarding the interpretation of a diagnostic elimination diet because chronic symptoms may vary for reasons other than ones related to foods (e.g., eczema flaring due to infection). Studies evaluating their diagnostic value are lacking, and malnutrition resulting from prolonged elimination diets that exclude multiple foods is a concern (Boyce et al., 2010).

Skin Prick Tests

Guidelines recommend using SPTs for assistance in diagnosing IgE-mediated food allergies, but the test results alone are not considered sufficient for diagnosis (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). The test can be done in any age group, although reactivity may be lower in infants and the elderly. The test involves puncturing the surface of the skin to introduce an allergen and evaluating the area for a wheal (small swelling) and flare (redness) response that can be measured. The test is applied to the forearm or back and the results of the allergen tests are compared with a negative saline and a positive histamine control test. The choice of tests is guided by the clinical history. Results are read at 15 or 20 minutes. A positive test correlates with the presence of specific IgE antibodies bound to the surface of cutaneous mast cells. The test is considered safe, because systemic allergic reactions are rare. In contrast, intradermal testing¹ with food is not recommended because it is overly sensitive and could induce systemic reactions (Boyce et al., 2010; Sampson et al., 2014).

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¹ Intradermal test consist of delivering the food into the dermis, the skin layer underneath the epidermis (which is the upper skin layer where an SPT is performed). The dermis is, on most places of the human body, only a few mm thick.

Various caveats have been identified regarding SPTs. Trained health care personnel are needed because of a risk of serious allergic reactions. Variables that can affect outcomes include the device used to introduce the allergen (a number of devices are on the market), operator error, the extract (not standardized), the manner of recording and reporting test results, and the timing of day, age, and sex of the patient, the patient’s use of any antihistamines, and anatomical site of testing (forearm versus back). Extracts may lack relevant allergens and testing using fresh extracts of food has been suggested for some circumstances, such as testing fruits and vegetables for pollen-food allergy syndrome. False negative tests (i.e., a skin test that is negative despite the fact that the patient experiences a reaction from ingesting the tested food) are possible, requiring caution if suspicion of allergy is high. The SPT reagents and methods have not been standardized. A systematic review and meta-analysis identified varying sensitivity and specificity according to the food evaluated, at a cut-off value of 3 mm wheal diameter in studies using OFCs as the diagnostic standard (Soares-Weiser et al., 2014) (see Table 4-1). Sensitivity is generally high, whereas specificity is lower.

These tests have a low positive predictive value for making a diagnosis of food allergy but high negative predictive value. Although a positive test is generally considered a wheal diameter equal to or greater than 3 mm, studies suggest that larger mean wheal diameters correlate with a higher likelihood of clinical reactivity (Pucar et al., 2001; Saarinen et al., 2001; Sporik et al., 2000; Verstege et al., 2005). A systematic review (Peters et al., 2012) evaluated studies reporting SPT wheal sizes that correspond to high predictive values for allergy (i.e., skin tests sizes above which allergy is almost certain). However, this review (Peters et al., 2012) noted that predictive values vary between studies, likely for numerous reasons including patient selection, food challenge protocols, reagents used for testing, and manner of reporting.

Food-Specific Serum IgE

Guidelines recommend using sIgE tests to identify foods that may provoke IgE-mediated reactions, but the test result alone is not considered sufficient for diagnosis (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). The choice of tests is guided by the clinical history. Modern tests use fluorescence enzyme-labeled assays and have replaced radioallergosorbent tests (RAST). The term “RAST” is therefore antiquated. In the United States, the Food and Drug Administration (FDA) has approved three automated systems to measure sIgE. Each system has slightly different methods for test development, and results from one system are not directly comparable to others (Hamilton and Williams, 2010; Hamilton et

TABLE 4-1 Sensitivity and Specificity of SPT for Selected Foods

NOTE: CI = confidence interval; SPT = skin prick test.

SOURCE: Soares-Weiser et al., 2014.

al., 2011; Wang et al., 2008). sIgE is not affected by antihistamine use, as SPTs are.

The sensitivity and specificity of SPT and sIgE were evaluated in a 2010 meta-analysis with a conclusion that neither test was statistically superior (Chafen et al., 2010). However, SPTs and sIgE tests do not always correlate, and so doing both tests can be advantageous, as can doing one followed by the other, if clinically warranted. A 2014 systematic review and meta-analysis (Soares-Weiser et al., 2014) considered mixed cut-off levels for sIgE but chose a >0.35 kU_A/L² value when possible. The sensitivities and specificities for various allergenic food are in Table 4-2.

Laboratory reports of undetectable sIgE concentrations occasionally occur in patients who go on to react to the food tested probably for reasons similar to the ones described above for SPT, so caution and additional evaluation is necessary in this circumstance if a history is highly suggestive of food allergy. In addition, different laboratories or test systems may report test results at different detection limits, for example <0.10 or <0.35 kU_A/L.

Studies have correlated increasing sIgE levels with increasing risk of clinical allergy. Some studies have calculated cut-off levels suggesting 95 percent predictive values for clinical reactivity (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). Although 95 percent predictive cutoff values have been calculated in specific studies, these values vary between studies, likely due to differences in patient selection, age, clinical disorders evaluated, and many other factors. The predictive values of certain cut-offs are dependent on the frequency of the food allergy and may therefore differ widely in different populations.

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² Kilounit allergen per liter.

TABLE 4-2 Sensitivity and Specificity of Food-Specific Serum IgE (sIgE) Test for Selected Foods

NOTE: CI = confidence interval.

SOURCE: Soares-Weiser et al., 2014.

Component Resolved Diagnostics

CRD, sometimes referred to as molecular testing, involves measuring sIgE against individual allergenic food proteins. This testing is available in single allergen formats and microarray. The comparative utility of the two approaches has not been extensively studied. Commercially available microarray provides semi-quantitative results that correlate with single allergen formats and may be more susceptible to antibody competition due to lack of allergen excess (Canonica et al., 2013). The aim of the test is to increase specificity, based on the understanding that some food proteins may be more potent for causing symptoms than others within the same food. For example, relevant proteins may resist digestion, and IgE immune responses against such proteins may have a greater diagnostic value for systemic allergy than immune responses against more labile proteins that degrade easily and are not systemically absorbed. The AAAAI Guidelines indicate that CRD can be considered for diagnosis, but is not routinely recommended because clinical utility is not fully elucidated (Sampson et al., 2014). Nonetheless, its utility in certain clinical scenarios is recognized. The EAACI Guidelines (Muraro et al., 2014) indicate that the test is promising and broadly studied, but that evidence from additional well-designed randomized controlled trials on the diagnostic test accuracy are required to assess its diagnostic value. A World Allergy Organization expert panel report suggests these tests as a third line approach following clinical history and extract-based testing, but that they may be included in second line testing for experienced users (Canonica et al., 2013). When SPT and sIgE are inconclusive, the EAACI Guidelines (Muraro et al., 2014) suggest that CRD, if available, provides additional information. The Japanese Guideline for Food Allergy (Urisu et al., 2014) describes advantages of using CRD for peanut, soy, and wheat allergies.

An accumulating number of studies have evaluated CRD for a variety

of foods; the best studied is CRD for peanut allergy. A systematic review (Klemans et al., 2015) found that sIgE testing to Ara h 2 had diagnostic superiority to other peanut protein components and to SPT and peanut-specific IgE using whole peanut extracts. The studies were primarily pediatric cohorts (21 of 22), and authors concluded that Ara h 2 testing should replace the other tests in clinical practice, especially in children. Although some disagreement may exist, various studies have determined that increasing levels of IgE against Ara h 2 correlates with risk of clinical reactivity (undetectable Ara h 2 does not exclude peanut allergy). Sensitivity and specificity of the test varies among studies, similar to the limitations described for sIgE and SPT, and some studies suggest geographic differences in correlation to clinical reactivity to different proteins (Agabriel et al., 2014; Ballmer-Weber et al., 2015; Beyer et al., 2015; Ebisawa et al., 2012; Eller and Bindslev-Jensen, 2013; Keet et al., 2013; Klemans et al., 2015; Kukkonen et al., 2015; Lieberman et al., 2013; Lopes de Oliveira et al., 2013). If sensitization to peanut is solely caused by Ara h 8 (the birch pollen–related protein in peanut) in regions with birch pollen exposure, systemic clinical allergy is unlikely (Asarnoj et al., 2012).

Numerous other foods have been less comprehensively evaluated by CRD. Sensitization to the hazelnut proteins Cor a 9 and Cor a 14 are associated with higher risk of food allergy to hazelnut and provide better diagnostic utility than the extract tests or other protein components (Beyer et al., 2015; Faber et al., 2014; Kattan et al., 2014; Masthoff et al., 2013). The soy proteins Gly m 4 and Gly m 5 (Berneder et al., 2013; Kattan and Sampson, 2015) appear relevant in soy allergy diagnostics. Literature on the utility of CRD testing on a number of foods is growing, including wheat, cashew, milk, egg, shrimp, carrot, and celery (Muraro et al., 2014; Savvatianos et al., 2015; Soares-Weiser et al., 2014). Sensitization to the cashew nut (Ana o 3, a protein belonging to the 2S albumin family of proteins) is highly predictive of cashew and pistachio allergy in Greek children (Savvatianos et al., 2015). Fruits typically induce mild oral allergic symptoms related to oral allergy syndrome induced by labile pollen-homologous fruit proteins. If IgE binds to stable fruit proteins, such as lipid transfer proteins, it may be associated with more severe reactions, but literature to characterize the role of component allergen testing in fruit and vegetable allergy is limited, and current studies show variable results (Lopez-Matas et al., 2015; Novembre et al., 2012; Tolkki et al., 2013; van Winkle and Chang, 2014; Vieira et al., 2014).

In summary, CRD is an emerging testing methodology in widespread use for select foods. They provide additional insights on diagnosis in specific circumstances. More studies are needed, however, to draw specific conclusions about their diagnostic utility. Component testing for peanut should be used when indicated (Dang et al., 2012; Klemans et al., 2015).

Like judicious use of the medical history, SPT and sIgE, CRD testing provides clinically useful results and can reduce the need for OFCs.

Oral Food Challenges

The OFC is a feeding test that typically involves a gradual, medically-supervised ingestion of increasingly larger doses of the food being tested as a possible food allergen. Guidelines recommend using OFCs to diagnose food allergy (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). Most OFCs are conducted with the food in its natural form; this is called an open OFC. Oral food challenges also can be performed in a single-blind protocol with the food masked from the patient’s perspective so less patient bias occurs because of anxiety. Bias is a concern with OFC because anticipation of a reaction can result in subjective symptoms (e.g., abdominal pain, nausea, or eczema flare) and possibly objective ones (e.g. hives). To address this concern a double-blind, placebo-controlled oral food challenge (DBPCOFC) can be conducted. This challenge, which is considered the “gold standard” for diagnosis of food allergy, involves masking the tested allergen and feeding it or indistinguishable placebo randomly without the patient or observer knowing if the allergen or placebo is being tested. However, the double-blind challenge is time-consuming and expensive, and is used more often for research, whereas open food challenges are routinely used in clinical settings. An open or single-blind OFC is considered reliable if no symptoms occur. An open feeding of a meal-sized portion of the food prepared in a usual fashion (e.g., scrambled egg, cooked fish) is also typically performed to confirm tolerance following a negative DBPCOFC with a smaller portion. If only subjective symptoms occur during a food challenge, a false impression of allergy is possible. If objective symptoms occur (e.g., urticaria, angioedema, or anaphylaxis) and the result correlates with medical history and laboratory tests, then the diagnosis is supported. Ambiguous results from an open or single-blind OFC can be evaluated by a DBPCOFC. This challenge also may be considered when patients have primary symptoms of chronic eczema or suspected anxiety.

The OFC is generally indicated to demonstrate allergy or tolerance when the medical history and supporting tests are not sufficient to make a conclusion. This may include circumstances such as a suspected allergy with ambiguous test results, or with the expectation that a food allergy has resolved. The OFC also may be used for individuals with ongoing allergy to evaluate thresholds or response to therapy. As the generally accepted gold standard, the test is highly specific. However, patients uncommonly experience reactions on subsequent ingestion despite tolerance during the test; the rate of this occurrence may vary by dosing regimen (Caffarelli and Petroccione, 2001; Miceli Sopo et al., 2016; Niggemann et al., 2012).

The OFC is useful for evaluating food allergy whatever the underlying pathophysiology or time course of symptoms, and can be used for all age groups. The test carries a risk of allergic reactions and anaphylaxis, and so caution, content monitoring, experienced personnel and equipment, and medications for managing reactions are required. Feeding a small amount of the suspected allergen and gradually increasing the amount mitigates some risk. The test is stopped at the judgment of the supervising health professional due to the onset of symptoms or at the request of the patient. Immediate symptoms typically occur within 2 hours after ingestion, but increases in atopic dermatitis symptoms may occur over hours or days. Rigorous objective criteria for determining tolerance or reactivity, consistent application of procedures, and good record keeping and documentation are paramount. No universally accepted manner of dosing, scoring, and monitoring the OFC procedure has been established, and potential dosing regimens have not been compared prospectively. Various approaches have been suggested, and issues such as indications and contraindications have been summarized (Sampson et al., 2012, 2014). Standardized dosing protocols have been published but not validated (Muraro et al., 2014; Sampson et al., 2012, 2014). For infants, open OFCs with objective scoring criteria are generally sufficient to make or refute a diagnosis of food allergy. Application of the OFC to infants, and additional limitations of the test are additionally reviewed in Chapter 5, Methodological Limitations.

The OFC is usually undertaken with the goal of the patient ingesting an age-appropriate, meal-size portion of the food prepared in a manner that will be ingested in the future. Processing and cooking methods can alter its allergenic properties. For example baked egg or milk products are less allergenic than raw forms. The matrix in which the tested allergen is mixed also can affect outcomes, as absorption rates may vary. For example, fatty foods are absorbed more slowly than other foods (Grimshaw et al., 2003). Although foods could be freeze-dried and placed into opaque capsules to mask the taste as well as early signs of reaction involving the oral mucosa, this approach is not in favor due to alteration of proteins and lack of control of release of the food from the capsules. The initial dose is generally selected to be less than a likely threshold for a reaction, or significant reaction (e.g., less than 3 mg) if the patient is suspected of being highly sensitive (Rolinck-Werninghaus et al., 2012). If a threshold-determining OFC is being undertaken, a lower starting dose may be used. Doses are given at 15- to 30-minute intervals although adjustments can be made. If symptoms occur after several doses, it cannot be concluded that the “last dose” independently triggered a reaction, as symptoms could be caused by prior doses or a cumulative effect (Blumchen et al., 2014). Also, escalating dose OFCs are similar to certain immunotherapy protocols and may therefore result in a reaction at a higher dose than would be the case if this were

the first and only dose. The time of testing can vary but is typically 3 to 8 hours depending on the doses, symptoms, and challenge format. The test may be formatted differently for non-IgE-mediated food allergies, such as FPIES, where the feeding may be dosed more rapidly and the expectation of reaction is delayed, occurring approximately 2 hours later. The test is generally undertaken when the food has been excluded from the diet. In the case of suspected chronic symptoms, the time of exclusion is typically 2 to 8 weeks to obtain a baseline.

The risk of OFC tests includes an anaphylactic reaction. On the other hand, the test might have nutritional (when the food can be added back to the diet) social, emotional, and educational (learning which trigger foods must be avoided, providing safety, and learning about reaction characteristics, treatment, and threshold) benefits. Some evidence suggests that the OFC procedure does not increase long-term post-study anxiety and can improve quality of life whether the food is tolerated or not (Franxman et al., 2015; Knibb et al., 2012). Guidelines promoting the OFC as a recommended procedure use terminology of “positive” challenge test outcome to denote that the test elicited symptoms and a “negative” test outcome to indicate the food was tolerated. (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). This use of terms is deliberate to avoid terms such as “passed” and “failed” as outcomes, which carry negative implications of the patient having “failed” in some manner.

Patients may avoid having the procedure due to fear, disinterest in the food offered, or misunderstanding about risks or odds of tolerating the food. They might ingest the food on their own, against medical advice to undergo the procedure before reintroducing the food into the diet plan (Davis et al., 2015). Physicians may not offer the procedure due to patient safety risk, time constraints, lack of trained personnel, and poor reimbursement (Pongracic et al., 2012). Failure to reintroduce the food into the routine diet after tolerating the OFC has been noted, but the reasons not fully explored (Miceli Sopo et al., 2016; van Erp et al., 2014). Considering that OFC is often required to determine a definitive diagnosis of food allergy, it is clearly underused.

MODALITIES NOT RECOMMENDED FOR ROUTINE USE

Atopy Patch Test

The atopy patch test (APT) is performed in a manner similar to patch testing that is routinely used to evaluate allergic contact dermatitis, except that foods are used. The food, presented as a fresh extract or powder, is generally placed under an aluminum disc on the skin for 48 hours then removed and with the final test result determined at 72 hours after applica-

tion. Current guidelines do not recommend the APT for the routine diagnosis of food allergies (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014), based partly on a lack of standardized reagents, methods, and interpretation of results. The APT may have utility in evaluating non-IgE-mediated allergy in the context of atopic dermatitis and EoE. Its utility in the diagnosis of FPIES has not been substantiated (Jarvinen et al., 2012; Ruffner et al., 2013).

In a systematic review and meta-analysis, three studies were identified that evaluated the diagnostic utility of the milk APT. Sensitivity was 53 percent (95% CI: 33%-72%) and specificity 88 percent (95% CI: 76%-95%) (Soares-Weiser et al., 2014). It is notable that despite a rather large number of studies, few meet criteria for meta-analysis (Isolauri and Turjanmaa, 1996; Keskin et al., 2005; Roehr et al., 2001). Several studies suggest poor utility of the APT (Alves et al., 2015; Caglayan Sozmen et al., 2015; Celakovska et al., 2010; Mehl et al., 2006). Other studies suggest some utility of APT for milk, especially for gastrointestinal symptoms or dermatitis (Boonyaviwat et al., 2015; Chung et al., 2010; Levy et al., 2012; Mowszet et al., 2014; Nocerino et al., 2013; Yang et al., 2014). The relevance of APT for EoE remains uncertain, but some studies suggest utility (Chadha et al., 2014; Rodriguez-Sanchez et al., 2014; Spergel et al., 2012). An updated expert panel report on EoE (Liacouras et al., 2011) summarized the results from seven studies, with negative predictive values of more than 90 percent and only 50 percent for milk, and variable positive predictive values. They suggested the APT (along with SPT and sIgE) can be used to identify foods associated with EoE, but alone the test is not sufficient to make a diagnosis of food-driven disease.

Total IgE

Guidelines recommend against the routine measurement of total IgE to diagnose food allergy (Boyce et al., 2010; Sampson et al., 2014). It is recognized that atopic persons may have elevated serum total IgE, but this does not provide guidance regarding the risk of specific food allergies. However, there is a notion that total IgE concentration may relate to sIgE (Federly et al., 2013) and that very high concentration of total IgE may influence the clinical relevance of sIgE for diagnostic purposes (Muraro et al., 2014).

Theoretically, the influence of total IgE on the clinical relevance of sIgE includes assay and in vivo effects due to competition for binding to allergen and effector cells (Hamilton and Williams, 2010). The FDA recommends that very low concentrations of sIgE antibodies should be evaluated with caution when total IgE values are above 1,000 kU/L (Merkel et al., 2015) (http://www.fda.gov/MedicalDevices/Safety/AlertsandNotices/TipsandArticlesonDeviceSafety/ucm109367.htm [accessed August 30, 2016]). One

of the few clinical studies (Mehl et al., 2005) evaluated 992 controlled OFCs performed in 501 children, looking at the utility of sIgE:total IgE ratio and found a correlation with challenge outcomes for milk, egg, and wheat, but not for soy. The diagnostic value of the ratio was not better than for sIgE alone. In contrast, another study looking at the ratio evaluated 195 OFCs among 161 children, and found that the ratio was more informative than sIgE alone for peanut, tree nuts, seeds, and shellfish but not milk, egg, wheat, or soy (Gupta et al., 2014). In contrast, the component specific to total IgE ratio did not improve peanut or hazelnut diagnosis (Grabenhenrich et al., 2016).

Although the NIAID/NIH-supported Guidelines and AAAAI Guidelines concluded that total IgE is not recommended for routine use in diagnosis (Boyce et al., 2010; Sampson et al., 2014), the EAACI Guidelines based on low-level evidence and expert opinion suggested that total IGE concentration may be useful in patients with severe eczema because a very high total IgE suggests that positive sIgE should be interpreted with care, as possibly representing asymptomatic sensitization (Muraro et al., 2014).

Basophil Activation Test

Basophils are allergy effector cells found in whole blood. Basophils degranulate upon cross-linking of sIgE, which is bound to the high affinity IgE cell surface receptors, and release mediators such as histamine. The granule marker, CD63, or CD203c, an activation marker, can be measured by flow cytometry and provide a measure of basophil activation. The basophil activation test (BAT) is conducted by exposing the basophil cells to various concentrations of the allergen to be tested, either an extract or individual component proteins in the test tube. The readout is the number of cells responding, or the concentration of allergen at which 50 percent of the cells respond. About 10 percent of people are BAT nonresponders, even though they are allergic and have positive skin tests. The test is a functional assay akin to a provocation test, such as a SPT.

Guidelines suggest not using the BAT clinically on the grounds that it is nonstandardized, but recognize its use as a research tool (Boyce et al., 2010). A position paper from a task force of the EAACI reviewed the BAT and made a number of recommendations in favor of using the test for diagnosis and monitoring of food allergy, and a recommendation to pursue standardization to make it available in diagnostic laboratories (Hoffmann et al., 2015). The EAACI task force evaluated diagnostic studies on peanut (N=4), hazelnut (N=2), peach (N=3), wheat (N=4), milk (N=2), egg (N=2), shellfish (N=1), and pollen-associated food allergy syndrome (PFAS) (N=5). The reported sensitivity ranged from 77 to 98 percent and specificity from 75 to 100 percent. In some studies BAT was more accurate than SPT or

sIgE. In a series of peanut allergy studies from one research group, which included a validation substudy, the BAT significantly improved diagnosis over SPT and sIgE, reducing the number of OFCs required for diagnosis (Santos et al., 2014) and provided predictive value for severity and threshold of reactivity (Santos et al., 2015). The position paper also reviewed the use of BAT to predict development of tolerance in food allergic children (N=4 studies), and to monitor responses to immunomodulatory therapy (N=11 studies). Overall, while the test is not available for widespread use, the potential utility is recognized and will require additional validation and standardization.

NONSTANDARDIZED AND UNPROVEN PROCEDURES

A number of tests have been referred to as “unproven,” “unconventional,” or “nonstandardized and unproven” by guidelines and are not recommended for food allergy diagnosis (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). These tests or procedures include: allergen-specific IgA, IgG or IgG₄, provocation neutralization, immune complexes, HLA screening, lymphocyte stimulation, facial thermography, gastric juice analysis, endoscopic allergen provocation, hair analysis, applied kinesiology, cytotoxic assays, electrodermal testing, mediator release assays, bio-resonance, and iridology. The rationale for not recommending these tests or procedures is the lack of evidence demonstrating the value of each method in diagnosis of food allergy. There is a concern that use of these methods may result in false positive or false negative diagnoses that may lead to unnecessary dietary restriction or may delay appropriate diagnostic evaluations.

For example, IgG₄ testing against foods as a diagnostic modality was reviewed in the 2008 EAACI Task Force report (Stapel et al., 2008). Many serum samples have positive IgG₄ results without corresponding clinical symptoms. The report noted a lack of convincing evidence for histamine-releasing properties of IgG₄, and a lack of controlled studies to determine diagnostic value. Conversely, evidence suggests that food-specific IgG₄ reflects exposure, and may indicate a state of immunological tolerance. The task force concluded that testing of IgG₄ to foods is irrelevant to the laboratory work-up for diagnosis of food allergy. It should be noted, however, that food-specific IgG and IgG₄ responses, when monitored during immune therapy with allergen exposure, is associated with clinical improvement in threshold. Thus, IgG and IgG₄ may be markers or mechanisms of desensitization and may have some role in diagnosis, especially during treatments, when considered along with other measurements, such as sIgE. Studies have begun to evaluate the diagnostic or prognostic potential of the IgE/IgG ratio or antibody classes. More studies are needed to validate these approaches,

as currently available data are conflicting (Ahrens et al., 2010; Caubet et al., 2012; Dannaeus and Inganas, 1981; Okamoto et al., 2012; Savilahti et al., 2012, 2014; Sverremark-Ekström et al., 2012; Tomicic et al., 2009).

PREDICTION OF SEVERITY OR THRESHOLD OF REACTIONS

Severity of an allergy is typically defined by symptoms triggered during an allergic reaction, and threshold of exposure for a reaction refers to the dose of allergen that triggers symptoms. There is strong interest in, and need for, a test for severity or threshold. Dosing during OFC is generally stopped before severe symptoms, limiting the ability of this study design to predict severe reactions (Wainstein et al., 2010). No comprehensive reviews have been published on the prediction of severity or on simple tests to diagnose the severity of a reaction. One might surmise that increasing sIgE concentrations correlate with severity because they correlate with risk of clinical reactivity. Although a number of studies suggest this correlation, it has not been universally substantiated (Benhamou et al., 2008; Blumchen et al., 2014; Clark and Ewan, 2003; Neuman-Sunshine et al., 2012; Rolinck-Werninghaus et al., 2012; Summers et al., 2008; Ta et al., 2011; van der Zee et al., 2011; Wainstein et al., 2010). In addition, CRD could be considered a means to possibly diagnose severity of a reaction because, for example, isolated binding to Ara h 8 is associated with no or mild allergy (oral-pharyngeal symptoms, related to PFAS) while binding to Ara h 2 is associated with systemic peanut allergy. However, on an individual patient or research study participant basis, degree of binding to Ara h 2 does not appear to accurately predict severity (Astier et al., 2006; Klemans et al., 2013a,b; Leo et al., 2015; Peeters et al., 2007). Studies have suggested that modalities such as BAT (Homsak et al., 2013; Santos et al., 2015; Song et al., 2015) or analysis of epitope³ binding patterns (Flinterman et al., 2008; Shreffler et al., 2004) may hold promise for determining severity. Disparities in prediction of severity based on testing may have many methodological reasons, but on an individual basis, outside of studies that control for such variables, the tests may not or do not currently consider specific patient-circumstance variables, such as whether the individual with food allergy has asthma, is currently ill, exercising, or experiencing other factors that may cause increased sensitivity (i.e., eliciting factors, other factors regarding physiologic responses) (Summers et al., 2008; Vadas et al., 2008). A recent paper describes the lack of predictability, perceptions about severity, and the types of factors that may affect the severity of a reaction, including those related to a person’s behaviors (e.g., exercise) and other factors (e.g., infections) (Turner et al., 2016).

__________________

³ Epitopes are segments of a protein that are recognized by antibodies.

PROGNOSIS AND DISEASE MONITORING

The rate of allergy resolution varies based on the food, patient’s age, pathophysiology of the allergy, and other factors (Boyce et al., 2010; Sampson et al., 2014). Table 4-3 summarizes resolution rates of common food allergies (Savage et al., 2016). Most children with allergies to cow milk, egg, soy, and wheat will develop tolerance by adulthood, whereas resolution of peanut, tree nut, and seafood allergies is less likely (less than or equal to 20 percent) (Boyce et al., 2010; Sampson et al., 2014). Adults with food allergies may have experienced persistence from childhood or may have a new onset in adulthood, and these allergies tend to persist. The natural course of food allergy is not known for most foods. Periodic re-evaluation with testing is recommended and can be individualized based on patient characteristics, the food, and underlying food allergic disorder (Boyce et al., 2010; Sampson et al., 2014). In general, periodic re-evaluation is undertaken with history, SPT, sIgE, and OFC depending on the specific results of each test and history. This testing might be performed more frequently (e.g., yearly) for a young child with food allergies, and less frequently (e.g., every few years) for an adult with allergies to foods such as peanut, tree nuts, and seafood.

Unfortunately, no simple accurate prognostic tests exist. Having tests that could be performed early in life that reflect prognosis would be helpful in selecting the best periodicity of retesting, providing anticipatory guidance, and identifying which patients might benefit from interventional treatments (as these become available). Studies have suggested that higher compared to lower concentrations of sIgE or skin test size are a poor prognostic marker (Ho et al., 2008; Keet et al., 2009; Peters et al., 2013, 2015; Savage et al., 2007, 2010; Sicherer et al., 2014; Skripak et al., 2007; Wood et al., 2013). However, additional clinical factors are associated with prognosis, including severity of symptoms, threshold dose, family history, change in sIgE over time, ability to tolerate milk or egg in baked goods (for cow milk and egg allergy), comorbid asthma, and comorbid atopic dermatitis (including severity), and other factors (Cantani and Micera, 2004; Elizur et al., 2012; Ho et al., 2008; Peters et al., 2013, 2014, 2015; Savage et al., 2007; Shek et al., 2004; Sicherer et al., 2014; Skripak et al., 2007; Wood et al., 2013). Studies have used multivariate analysis to create predictive models using the variables with the greatest impact (especially sIgE levels), but validation is needed (Sicherer et al., 2014; Wood et al., 2013). Studies using newer in vitro tests, such as CRD and BAT, have not been extensively applied to develop prognostic algorithms. A 2013 systematic search and review on this topic identified 26 articles, noting heterogeneity and biases in the studies, and concluded that population-based, prospective studies are needed that use OFC—without bias of test results—to diagnose food allergy

TABLE 4-3 Natural Course of Food Allergy

SOURCE: Savage et al., 2016.

at baseline and then to follow up to develop thresholds for SPT and sIgE that predict the course of food allergy (Peters et al., 2013). Little is known about food allergy prognosis after diagnosis in adulthood.

Many of the modalities discussed here also have been evaluated during treatment studies, to identify markers that may indicate desensitization or tolerance of food(s) to which individuals are initially allergic, including sIgE, SPT, CRD, BAT, sIgE/total IgE ratio, sIgG₄, and ratio of sIgE to sIgG₄ (Nozawa et al., 2014; Savilahti et al., 2014; Thyagarajan et al., 2012; Vickery et al., 2013, 2014). Additional markers have been followed, including cytokines, regulatory T cells, T cell number and function, and B cell activity (Bedoret et al., 2012; Hoh et al., 2016; Syed et al., 2014; Varshney et al., 2011). However, biomarkers to confirm desensitization and tolerance without OFC remain to be found.

GENERAL DIAGNOSTIC ALGORITHMS

Guidelines and reviews have suggested general algorithms (i.e., panels) for diagnostic approaches (Greenhawt et al., 2013; Muraro et al., 2014; Sicherer, 2002; Urisu et al., 2014; Venter et al., 2013). Approaches typically begin with a medical history to identify the nature of the symptoms (whether likely reflecting food allergy or another disorder), the pathophysiology (IgE mediated or not), and the potential food triggers. Testing based on the initial impressions is conducted and interpreted based on the results of the history and suspected foods and related pathophysiology. This may include tests for IgE, elimination diets and/or OFCs, depending on the circumstances.

Different algorithms may fit specific disorders. For example, evaluation of food allergy in acute anaphylaxis, where symptoms come on quickly and are associated with sIgE antibodies, differs from evaluation of the role of food allergy in atopic dermatitis or EoE (Greenhawt et al., 2013; Sicherer,

2002; Urisu et al., 2014; Venter et al., 2013). No overarching approach has been universally accepted. However, because the sensitivity and specificity of individual tests are generally not 100 percent, using pretest probability obtained from one test (e.g., the medical history) is recognized as beneficial for interpreting the post-test probability of allergy following a second test (Muraro et al., 2014). Indiscriminately performing multiple tests is not recommended (Boyce et al., 2010), but a case can be made for using more than one test when additional diagnostic value may be obtained. Specific algorithms may, for example, consider diagnostic values of several tests performed in series to improve accuracy (Ben-Shoshan et al., 2010; Dang et al., 2012). Additionally, it may be possible to isolate a number of factors from the medical history and simple diagnostic tests to estimate the risk of an allergy, using a standardized approach, but this also needs validation (DunnGalvin et al., 2011). In summary, although no evidence-based, universally accepted overarching diagnostic algorithm exists, guidelines promote step-wise evaluations rather than solely depending upon single tests to conclude a diagnosis of food allergy in children (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). Information on adults is limited.

TESTING FOR SPECIFIC DISEASE STATES OTHER THAN ANAPHYLAXIS AND ATOPIC DERMATITIS

As indicated above, diagnostic approaches may vary depending upon the pathophysiology, epidemiology, and clinical characteristics of particular food-allergic disorders (Greenhawt et al., 2013; Muraro et al., 2014; Sicherer, 2002; Urisu et al., 2014; Venter et al., 2013).

Food Protein–Induced Enterocolitis Syndrome

FPIES and food protein-induced allergic proctocolitis are non-IgE-mediated disorders that lack current means of simple laboratory testing to identify causal foods or to confirm the diagnosis. Guidelines suggest using the medical history, resolution of symptoms during dietary elimination, and recurrence of symptoms upon exposure; for example, during a food challenge (although not typically necessary for proctocolitis), as a means of diagnosis (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). For FPIES, guidelines indicate that factors in the history may be so suggestive of the diagnosis that an OFC is not needed. For example, a patient may have experienced repeated reactions with typical symptoms or severe symptoms (Boyce et al., 2010; Sampson et al., 2014). It also is recognized that a subset of children may develop IgE antibodies (especially for cow milk) signifying prolonged course and possibly anaphylactic symptoms that can warrant periodic testing before using an OFC to evaluate for resolution

(Caubet et al., 2014; Sampson et al., 2014). The APT does not appear to be useful for diagnosing FPIES (Jarvinen et al., 2012; Ruffner et al., 2013). Endoscopy and biopsies are not typically needed for diagnosis (Boyce et al., 2010; Muraro et al., 2014). The OFC for evaluation of FPIES could induce severe symptoms (e.g., hypotension, methemoglobinemia [unexpected], acidemia) and requires caution.

Eosinophilic Gastrointestinal Diseases

Eosinophilic gastrointestinal diseases may have both a cellular and IgE antibody component. No specific diagnostic strategies other than elimination and OFC have been proposed for identifying the food-specific triggers in eosinophilic gastroenteritis, and no biomarkers to identify responses are currently available, making repeated endoscopy/biopsy necessary to identify responses to treatment. Guidelines suggest considering tests for food-specific IgE and APT to help identify causal foods, specifically for evaluating EoE (Boyce et al., 2010; Sampson et al., 2014). Testing for food-specific sIgE also derives from the observation that 15 to 43 percent of patients are diagnosed with typical IgE-mediated food allergies and up to 80 percent are sensitized to aeroallergens (Muraro et al., 2014). However, these tests are not to be depended on to identify causal foods, and the diagnosis of EoE also requires a trial of proton pump inhibitors, and evaluations to identify characteristic biopsy results for diagnosis (and to exclude other diagnoses) (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). Ultimately, trial elimination diets are needed, with follow-up biopsy to assess resolution of inflammation.

Pollen-Associated Food Allergy Syndrome

The best approaches for diagnostic testing for PFAS have not been systematically evaluated. A number of recommendations have been published (Sampson et al., 2014). The detailed medical history is important because the diagnosis should be considered in patients experiencing limited oropharyngeal symptoms when eating foods (raw) that have cross-reacting proteins with pollens; it may be noted that symptoms are increased during and just following the pollen season. Testing for sIgE to pollens is suggested, and performing SPT with fresh food (sometimes termed “prick-prick” testing which may be performed by pricking the raw fruit or some of its extracted juice with the skin test device and then pricking the skin) can also be used to aid diagnosis (Begin et al., 2011; Vlieg-Boerstra et al., 2013). Such testing is not standardized. The use of commercial extracts may be less useful because the responsible proteins are labile and may not be present. It is not understood why only some persons with pollen aller-

gies experience reactions, or why people with similar pollen allergies may have different patterns of reactions to different fruits and vegetables. Simple diagnostic tests lack the ability to differentiate or predict these variations (Crespo et al., 2002; Pastorello et al., 1994; Rodriguez et al., 2000; Ta et al., 2015). Variations in reactivity are noted even among cultivars of the same fruit, or with ripening or storage (Carnes et al., 2006; Sancho et al., 2006). Systemic reactions to the same foods that trigger PFAS can occur. The reason for systemic reactions could be explained by having reactivity to a higher dose of the labile allergen, a greater sensitivity to that allergen (possibly varying with cofactors such as exercise or illness), or having an immune response to proteins that are not labile (e.g., lipid transfer proteins) (Cudowska et al., 2008; Gomez et al., 2014; Pascal et al., 2012; Zuidmeer and van Ree, 2007). It is possible that CRD or BAT represent a means to evaluate this difference in risk, but studies have had mixed results (Asero, 2014; Ebo et al., 2010; Erdmann et al., 2005; Gamboa et al., 2009; Guhsl et al., 2015; Hofmann et al., 2013; Tolkki et al., 2013).

COMMON PITFALLS AND MISCONCEPTIONS IN DIAGNOSTICS

As indicated previously, diagnostic and monitoring tests have a variety of limitations that, if not appreciated, can result in over- or underdiagnosing food allergy in patients. Table 4-4 summarizes common misconceptions.

Sensitization Is Not Diagnostic of Clinical Allergy

Key among potential pitfalls is the fact that sensitization (demonstrated by a positive test) is not a sole indication for a diagnosis. Testing with panels (i.e., preselected lists) of foods without a consideration of the medical history can result in unnecessary concerns and is not recommended (Bernstein et al., 2008; Cox et al., 2008; Sampson et al., 2014; Sicherer and Wood, 2012). Physicians may not appreciate this test limitation (Gupta et al., 2010) and, as reviewed above, patients and clinicians may misinterpret test results with low values versus higher values as reflecting severity of the allergy.

Clinically Relevant and Nonrelevant Cross Reactivity

Another potential pitfall is recognizing the difference between cross reactivity identified on testing (sIgE or SPT) that may or may not be clinically relevant (Sampson et al., 2014; Sicherer, 2001). When food allergens share sufficient homology, antibodies may be detected to multiple allergen proteins, but the clinical relevance of the test finding can vary. For example, a large proportion of individuals with peanut allergy will test positive to

TABLE 4-4 Common Misconceptions About Food Allergy and Testing

other legumes, such as soy (up to 79 percent), but only a small proportion of patients (up to 5 percent) will experience allergic reactions to them. Although the test rate of cross reactivity is higher than the observed rate of clinical cross reactivity, studies on this topic are limited and likely reflect results that vary depending upon methodology, patient selection, and geographic influences, including pollen sensitization. Estimated rates of clinical cross reactivity among crustacean shellfish is 38 percent, among fish 30 to 75 percent, among tree nuts 12 to 37 percent (varies depending on the nuts; for example, walnut and pecan are more similar, cashew and pistachio are more similar), and between wheat and other grains 21 percent. An OFC is often needed to confirm tolerance if a potentially cross reactive food has not

already been tolerated in the diet. A serious pitfall can occur if a food tests positive in panels (and the patient removes it from the diet) when tolerance has already been proven by inclusion of the food in the diet.

Delayed Anaphylaxis Associated with Mammalian Meats

Although most pitfalls in food allergy diagnosis may occur from overdiagnosis related to misunderstanding of pathophysiology and test utility, a special case of under- or misdiagnosis involves mammalian meat allergy (beef, pork, lamb) attributed to sIgE antibodies against a sugar moiety, galactose-alpha-1,3-galactose (alpha-gal) (Commins et al., 2011, 2014; Hamsten et al., 2013; Kennedy et al., 2013). The syndrome is likely associated with initial sensitization to allergen in tick bites. In contrast to typical food anaphylaxis that occurs within minutes to 2 hours following ingestion of the trigger food, alpha-gal-related reactions to mammalian meat, with the same allergic symptoms, occur 3 to 6 hours after ingestion. Skin testing to the trigger foods may not be strongly positive but in vitro sIgE testing to alpha-gal is commercially available and can be used to confirm the diagnosis. The reason for the delay in onset of anaphylactic symptoms is not known with certainty.

ROLE OF ELICITING FACTORS

Eliciting factors, also referred to as cofactors and augmentation factors, are circumstances or ingestants that can alter threshold or severity of an allergy, resulting in more serious reactions or allowing clinical expression of a food allergic response to an otherwise tolerated food (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). These factors can include exercise, nonsteroidal anti-inflammatory drug (NSAID) agents, alcohol, body temperature, menstruation, infections, stress, and antacid medications (Niggemann and Beyer, 2014). These factors may influence absorption or immune responses. The best described entity is food-associated (dependent), exercise-induced anaphylaxis, where the food is tolerated when exercise does not occur, but reactions may occur when the food is ingested before exercise. Common food allergenic foods that trigger a reaction with exercise are wheat, shrimp, and celery, but numerous triggers have been reported (Romano et al., 2001).

The possibility that a cofactor is responsible for the expression of a food allergy is assessed by history, and assessment may include evaluation by SPT or sIgE of foods ingested before exercise or concomitant ingestion of alcohol or NSAIDs. A case can be made for evaluating specific allergens associated with these syndromes, such as gliadin and lipid transfer proteins in some settings, but the diagnostic utility is not fully understood (Muraro

et al., 2014; Romano et al., 2012; Urisu et al., 2014). The history and supporting test evidence may warrant the diagnosis, but OFC with exposure to the eliciting factor may be needed. The reliability of such testing is variable, and the symptoms can recur despite an OFC not triggering reactions. Many factors may confuse the diagnostic approach, such as the need for multiple different or a combination of augmenting factors to result in a reaction, various degrees of the factor (amount of food, exercise, alcohol), and testing methodology (Asaumi et al., 2016; Brockow et al., 2015; Jo et al., 2012; Medrala et al., 2014; Niggemann and Beyer, 2014).

FUTURE DIAGNOSTIC MODALITIES

Food allergy guidelines have recognized a large number of approaches under investigation to improve diagnosis and provide insights on prognosis and severity (Boyce et al., 2010; Muraro et al., 2014; Sampson et al., 2014). Many of these approaches have been reviewed above (CRD, BAT, and others). The diagnostic value of determining the pattern of IgE binding to synthetic sequential epitopes (binding segments) of allergens has been evaluated, with results suggesting that this testing can provide information on phenotype (i.e., ability to tolerate extensively heated milk in those with cow milk allergy), prognosis, and severity (e.g., diversity of binding associated with severity of reactions) (Cerecedo et al., 2008; Flinterman et al., 2008; Jarvinen et al, 2001, 2002; Lin et al., 2012; Shreffler et al., 2004; Wang et al., 2010).

As reviewed above, a number of cellular markers are being evaluated to improve diagnosis and prognosis, including cytokines, regulatory T cells, T cell number and function, B cell activity, and epitope binding (Bedoret et al., 2012; Hoh et al., 2016; Syed et al., 2014; Varshney et al., 2011). One study suggests value in determining deoxyribonucleic acid (DNA) methylation signatures (Martino et al., 2015). Martino et al. performed genome-wide DNA methylation profiling on subjects who had undergone OFC, concurrent SPTs, and specific IgE tests (Martino et al., 2015). Fifty-eight were food-sensitized patients (ages 11 to 15 months), half of whom were clinically reactive, and 13 were nonallergic control subjects. Reproducibility was assessed in another 48 samples from an independent population of patients with food allergy. This study revealed a methylation signature consisting of 96 CpG sites that predict clinical outcomes. This methylation signature was superior to allergen-specific IgE and SPTs for predicting OFC outcomes. Therefore, in addition to elucidating mechanisms involved in the epigenetic regulation of food allergies and the interplay between genetic and environment, this evidence can be used to develop novel, practical, and improved diagnostic assays. Bioinformatics approaches that take into consideration multiple variables should support improved diagnostics (Lin

et al., 2012). These approaches, which could include data from numerous biologic markers such as genomic, transcriptomic, proteomic, metabolomics, microbiome, and various laboratory tests, will allow for assessment of billions of variables (Chen et al., 2012).

OVERALL CONCLUSIONS

Currently, no simple diagnostic tests exist for food allergy. Selection and interpretation of tests depend on the disorder being considered (epidemiology, pathophysiology) and the individual medical history. A common pitfall in diagnosis results from performing tests for sIgE without considering the medical history, resulting in unnecessary avoidance or removal of tolerated foods from the diet (a positive test alone may not indicate a clinical allergy). The gold standard test, the OFC, carries risk and expense, and is underused. The history and available test results can often suggest a likelihood of a food allergy, presenting a reasonable pretest probability for deciding upon the need for an OFC. Understanding how the size of skin tests, concentration of sIgE, and the clinical history can provide pretest probability estimations for providing a diagnosis at this point or proceeding to other tests, including the OFC is key. CRD is currently providing improved diagnosis in some circumstances. Developing “calculators” that evaluate these currently available parameters is promising. The BAT shows promising preliminary data, but validation and commercialization are needed. Sorely missing are simple tests that would indicate, for an individual with current possible allergy symptoms, degree of severity or threshold or both, as well as prognosis.

As reviewed in the discussion above, food allergy testing strategies (history, diagnostic elimination diet, OFC, SPT, sIgE, CRD, APT) are generally not well standardized, including the various factors involved with the history, elimination diets, and food challenge. Many methodologic issues are involved in evaluating test utility, and comparisons of diagnostic utility of specific tests among different populations often show some level of disparity. Regarding SPTs, extracts are not uniformly standardized and the individual allergenic protein content may vary (Hefle et al., 1995). The FDA has approved three automated systems to determine sIgE. Each system uses slightly different methods and results from one system are not directly comparable to others (Hamilton and Williams, 2010; Hamilton et al., 2011; Wang et al., 2008). The manner of reporting SPT skin test sizes varies (e.g., reporting greatest wheal diameter, mean wheal diameter, size in relation to controls), as does the representation of sIgE levels from serum tests (e.g., classes versus concentration, kU_A/L). Different OFC regimens have been proposed in the literature as well as different means to report

results. Attention to these issues affects research approaches as well as clinical care. Studies are under way to improve standardization.

Additional standardization and validation would require extensive study in different patient populations (e.g., ages, illnesses, geographic regions) and consideration of the role of eliciting factors, and circumstances where interventions are being applied to the patient (immunotherapeutic strategies as they become available). This is similarly the case for emerging diagnostics, such as epitope analysis.

Education is needed for patients and physicians to understand the meaning and limitations of commonly used food allergy test results, to know about unconventional and unproven tests, and to understand how to effectively use existing tests (or when to refer from primary care to specialist care). No comprehensive studies on the cost effectiveness of testing and misdiagnosis have been conducted. Studies on diagnostics have been primarily focused on children, and more studies of adults or comparison of adults and children are needed. Numerous potential diagnostic tests are in development. At this point, they are labor-intensive or expensive, but they may identify novel factors of use in the future.

RECOMMENDATIONS

RESEARCH NEEDS

Diagnosis of food allergy is complex, currently requiring expertise in assessing the medical history, understanding allergen cross-reactivity, understanding eliciting factors that may alter reactivity, selecting and interpreting imperfect tests, and possibly conducting a medically supervised OFC test. The OFC is currently the best diagnostic test to confirm an allergy, but it is time consuming, expensive, carries risks (e.g., the risk of triggering an allergic reaction), and is often deferred due to patient and physician concerns. Therefore, the OFC is underused. In addition, commonly available simple allergy tests (sIgE antibody tests or SPT) have limitations that can result in misdiagnosis, primarily overdiagnosis, requiring procedures such as OFCs to confirm a proper diagnosis. For example, currently available, simple diagnostic tests that are often used to diagnose IgE-mediated food allergies, the sIgE test and SPT, actually diagnose sensitization, not food allergy. A variety of diagnostic tests, such as CRD, the basophil activation test, and many others, are emerging or under study and may better inform diagnosis, prognosis, severity, and threshold.

To fill gaps in knowledge in this area, studies should be conducted to accomplish the following objectives:

• Optimize the currently available diagnostic tests and validate methods, such as OFC (including in special contexts, such as OFC in infants and young children), as well as pursue additional novel tests to improve diagnosis, prognosis, determination of severity of disease, and assessment of antigen thresholds, and to monitor host responses. These tests will be valuable in assessing the effectiveness and durability of interventions, such as immunotherapy. These studies should include all affected patient populations (ages, sexes, ethnicities, co-morbidities, socioeconomic strata, should consider the role of eliciting factors (such as exercise and infections), and also should be assessed in those circumstances where interventions are being applied to the patient (immunotherapeutic strategies as they become available).
• Comprehensively examine the utility, cost-effectiveness of, and barriers to testing, especially regarding the OFC, with a goal of maximizing the use of appropriate tests.
• Examine and assess educational approaches and tools to improve physician and health care provider education about both the natu-

• ral history of food allergies and the appropriate approaches to use to diagnose food allergies.

• Study the utility of emerging technologies in the area of “omics” methodologies (e.g., genomics, epigenomics, metabolomics). In particular, identify reliable and clinically useful biomarkers for the following important goals:
  • Assessing the severity of a food allergy (e.g., to identify those at high risk for anaphylaxis),
  • Evaluating and monitoring responses to therapy (e.g., immunotherapy),
  • Predicting prognosis (e.g., predicting severity),
  • Identifying populations at risk of developing a food allergy so that they can be included when conducting research on prevention and management strategies and on public health guidelines, and
  • Diagnosing food allergy in individuals and populations (e.g., for collecting data on prevalence).

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