Laboratory tests for diagnosis of food allergy: advantages,disadvantages and future perspectives

Numerous biological tests point to the diagnosis of food sensitization: detection of specific IgEs by Rast techniques, multi-detection assays, immunoblotting, screening of basophil activation (BAT or FAST), assays for leukotriene LTC4 release (CAST), measurement of plasma histamine, serum tryptase, serum ECP, urinary EDN, completed by mannitol-lactulose test evaluating intestinal permeability, assay of fecal IgEs, Rast for specific IgG4. Primary screening for anti-food IgEs by multi-detection assays seeks justification from insufficient clinical data and false positive tests are common in patients sensitized to pollens or latex, on account of in vitro cross reactivities (CR). Multiple CR explain positive Rast to vegetal food allergens in such patients. Biological tests should not be performed as the first line of diagnosis. In vivo sensitisation is assessed by positive prick-tests, demonstrating the bivalence of allergens, as well as the affinity of specific IgEs, two conditions necessary to bridge membrane bound specific IgEs, leading to the release of mediators. Prick-tests are closer to clinical symptoms than biological tests. However, the diagnosis of food allergy is based on standardised oral challenges. Exceptions are high levels of specific IgEs to egg (> 6 kUl/l), peanut (> 15 kUl/l), fish (> 20 kUl/l) and milk (> 32 kUl/l), reaching a 95% predictive positive value. Rast inhibition tests are useful to identify masked allergens in foods. Research developments will have impact on the development of new diagnostic tools: allergen mixes reinforcing a food extract by associated recombinant major allergens, multiple combination of recombinant allergens (chips) or tests with synthetic epitopes aimed a the prediction of recovery. Laboratory tests take place in the decision free for the diagnosis for the food allergy and the follow-up of the levels specific IgEs is a tool to assess outcome and contributes to predict recovery or persistent allergy. Up to now the significance of positive laboratory tests showing the implication of IgEs is at the crossroads of the allergist's and biologist's expertise.     

Sublingual rush immunotherapy with latex extract in children

Latex allergy prevalence has increased notably in the last two decades. Latex ubiquity and its cross-reactivity with fruits make complete avoidance difficult to attain and studies have questioned long-term avoidance efficacy. Subcutaneous and sublingual routes of specific immunotherapy (SIT) to latex have been tested in double blind placebo controlled studies, with significant improvement in patients' tolerance to latex. We present our experience with a sublingual desensitization protocol in three latex allergic children. The build-up phase consisted of rush, with daily admission for 4 consecutive days, followed by a maintenance period at home. Only local reactions, with good response to antihistamines were observed during rush and no reactions have been reported with maintenance dose during 6, 5 and 2 months, respectively. The first patient has accomplished 6 months maintenance treatment. Allergological re-evaluation has shown a decrease in skin reactivity and serum specific IgEs to latex and cross-reactive fruits. Comparison of the results of specific IgEs measurement to a panel of latex recombinant allergens before rush and 6 months later clearly showed a decrease in Hev b 5 and Hev b 6.01, with no other detectable new sensitizations. Immunoblot was also performed before rush and 6 months later and shows a decrease in sensitization without appearance of new bands. Future enlargement of our series and prospective follow-up will help to clarify the real clinical efficacy of sublingual SIT. As this is a safe and easy to use protocol, it seems to be specially appropriate for children.     

Recombinant latex allergens

In the last few years many efforts have been made to reduce the incidence of latex allergy. The identification of the major latex allergens and their recombinant production are useful steps towards the understanding of latex allergy and its management. A clear advantage of the recombinant proteins in general is the possibility to produce large-scale quantities at highly reproducible quality. Recombinant allergens also facilitate the study of the molecular bases of the immune reactivity of the proteins. Nevertheless, they have to be validated against native proteins for equivalence in allergenicity before they can be more widely adapted for clinical use. Currently 13 latex allergens have been included in the latest nomenclature list of the International Nomenclature Committee of Allergens and assigned the official names Hev b 1–13. With the use of single native and/or recombinant allergens the determination of specific sensitization profiles for different patient groups dependent on their various routes of exposure is possible. The specific IgE-diagnostic was further improved significantly by addition of recombinant Hev b 5 to natural latex extract, which was underrepresented in the initial test extract.     

Component-Resolved Diagnosis for Phleum Allergy: The Role of Recombinants

Background. Conventional diagnostic tests (such as radioallergosorbent test [RAST] and skin prick test [SPT]) use native raw pollen allergen extracts to establish allergy. However, recombinant allergens may offer important advantages compared with their natural counterparts. Objective. This study evaluated serum immunoglobulin E (IgE) in patients with grass-induced allergic rhinitis (AR) or AR with asthma (ARA), comparing assays with natural or recombinant grass allergens. Methods. Sixty patients (33 AR, 27 ARA) positive with SPT and serum IgE for Phleum pratense were enrolled in the study. Serum IgE specific for conventional and recombinant Phleum pratense: rPhl p 1, rPhl p 2, nPhl p 4, rPhl 5b, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, were measured by the IFMA procedure (ImmunoCAP, Phadia, Uppsala, Sweden). Data were expressed as the median (md) and percentiles. Recombinant allergen results were expressed also as the percentage of positive concentrations. The Wilcoxon test was used to compare samples. Because diagnosis is a binary variable (AR/ARA), logistic regression analysis was performed to identify possible correlates. Results. IgE concentrations assessed with recombinant allergens were significantly higher in ARA patients (p = .05) than in AR patients. A value >5.8 kU/L is the optimal cut-off to discriminate AR and ARA patients. Model specificity was 76%, sensitivity 78%, and efficiency 77%. Conclusion. This study shows that IgEs for natural and recombinant grass pollen allergens are significantly higher in patients with AR and asthma. Moreover, using recombinant allergens it is possible to define a prediction model for diagnosis with 77% efficiency. Therefore, this study may suggest that there are advantages of using recombinant or purified, native allergens over crude extracts.     

Appraisal of latex glove proteins in the induction of sensitivity to multiple latex allergens.

Six Hevea brasiliensis latex protein allergens, Hevb 1, Hev b 2, Hev b 3, Hev b 4, and two variants of Hev b 7 (7b and 7c), were purified from Hevea latex, while a seventh protein, Hev b 5, was prepared in recombinant form. The presence of these proteins in glove extracts was indicated by their respective antibodies in the serum of rabbits immunized against the extracts. The relative propensities of IgE binding to the individual latex allergens were compared using sera from latex-allergic patients. IgE recognition of Hev b 4, Hev b 7b, Hev b 5 and Hev b 2 was most frequently encountered, with 75, 61, 31 and 28%, respectively, of the patient sera reacting. Sensitivity to multiple latex proteins was common, and out of the 31 seropositive patients, 23 (74%/ ) had IgE against at least two latex allergens, while 12 (39%) had IgE specific for at least three allergens. Statistical analysis of the data suggested that many patients might have acquired sensitivity to Hev b 2, Hev b 4 and Hev b 7b from a common source. (e.g., from latex products). On the other hand, sensitivity to Hev b 5 and to Hev b 7c were interrelated. It is plausible that sensitivity to these two proteins might have been acquired from sources other than latex products (e.g., from certain foods).     

Adult food allergy

Adult food allergy is estimated at approximately 3.2% worldwide. The persistence of childhood food allergy is unusual, peanut allergies excepted. Once established in adults, food allergy is rarely cured. Factors favoring the acquisition of allergy could be sensitization to pollens, occupational sensitization by inhalation, drugs (such as tacrolimus), and sudden dietary changes. Severe anaphylaxis and oral allergy syndrome are frequent. The fatality risk is estimated at 1% in severe anaphylaxis. Risk factors for severe anaphylaxis are agents causing increased intestinal permeability, such as alcohol and aspirin. β-blockers, angiotensin-converting enzyme (ACE) inhibitors, and exercise are other factors. Gastrointestinal food allergy remains, to a large extent, undiagnosed in adults. Food allergens are mainly fruit and vegetable, related to pollen sensitizations, or to latex allergy. Wheat flour allergy is increasing. The diagnosis relies on prick skin tests, detection of specific IgEs, and standardized oral challenges. Strict avoidance diets are necessary. Specific immunotherapy to pollens may be efficient for cross-reactive food allergies.     

A new quantitative in vitro for the detection of latex-specific IgE antibodies

Immediate-type hypersensitivity to latex allergens has resulted in anaphylactic shock and death in numerous reported cases. The allergenic proteins of latex are contained within the natural rubber extract of Hevea brasiliensis and are eluted into the final product during the manufacturing process. The quantity and types of latex allergens found in different latex products depends on the manufacturing process. Not all of these allergens are available for use in the latex prick skin test, and as a result, such tests may not be conclusive. Furthermore, application of such allergens to the skin of undiagnosed hypersensitive individuals may have harmful effects on their health. Therefore, it is important to be able to utilize in vitro methods, which reliably identify latex allergy without placing hypersensitive individuals at risk. We have developed a relatively simple and new enzyme immuno-assay (EIA) method for the detection of latex allergy. This in vitro method is quantitative and allows for the classification of allergy to latex in a short time. In comparative studies, ninety-nine serum specimens with documented clinical history of latex allergy were tested by this method, and the results paralleled those of the skin prick test performed by an independent group. The data showed that the specificity and sensitivity of our assay approaches 97.5% and 100%, respectively. We conclude that, by using a simple assay, the detection of specific IgE to latex proteins may be valuable for screening individuals and for the diagnosis of allergy to latex.     

Prevalence of IgE antibodies to an extract from rubber tree (Hevea brasiliensis) latex and recombinant pollen allergens (Phl P 1, Phl P 2, Phl P 5, Bet v 1 and Bet v 2) in the sera of Italian atopic patients

BackgroundPrevious studies have reported cross-reactivity between latex and grass allergens. Inhibition studies have indicated cross-reactivity of IgE with latex and grass pollen proteins. A panel consisting of a few recombinant allergens, namely rPhI p 1, rPhl p 2, rPhl p 5 and profilin, was sufficient to diagnose grass pollen allergy in patients allergic to grass pollen.MethodsSerum samples from 528 consecutive outpatients with IgE antibodies towards at least one allergen (IgE level > 0.35 kAU/L) were selected for this retrospective study. Total and specific serum IgE to rPhl p 1, rPhl p 2, rPhl p 5, rBet v 1, rBet v 2, latex, birch, hazel, mugwort, wall pellitory, Dermatophagoides pteronissinus, Alternaria tenuis, cat and apple were measured by the immunoenzymatic capsulated hydrophilic carrier polymer (CAP) FEIA system (Pharmacia & Upjohn).ResultsOf 123 polysensitized patients with antilatex Ig1E, 12 (9.76%) had symptoms after latex exposure. Ten of 12 subjects monosensitized to latex had symptoms after latex exposure. Symptomatic patients had higher IgE levels to latex than symptomless patients (P = 0.046). A higher prevalence of antilatex IgE was seen in sera containing specific IgE to rPhl p 1, rPhl p 5 and rBet v 2. A good correlation (Spearman's r = 0.52; P = 0.001) between high levels of antilatex IgE and total serum IgE was found.ConclusionsThe findings of the present study may support the concept that a high proportion of sera containing IgE to rBet v 2, rPhl p 1 and rPhl p 5 simultaneously contain antilatex IgE. Therefore, patients with specific IgE to these recombinant allergens with no history of current latex exposure may need additional evaluation.     

Food allergy diagnosis

The diagnosis of food allergies (FA) relies upon the sequential use of different means and tools, according to a decision tree. Ten clinical characteristics point to a potential FA. A diary for food consumption during a week surveys all labellings, in order to detect masked food allergens. The second step is based on skin prick tests to natural foods, and epicutaneous tests to a few proteins (casein, gluten...). Biological tests using multi detection tests of specific IgEs to numerous allergens are not advised owing to the frequency of clinically not relevant in vitro cross-reactivity. Single determination of specific IgEs, have a 95% predictive positive value of high levels in cases of allergy to milk, egg, fish and peanut, and can spare oral challenges. The primary use of biological tests is not currently advised but may be of interest in litigious cases. Standardized oral challenges are the golden standard. Eviction regimens are an alternative used for cow's milk allergy in infancy, or for suspected digestive allergies in adults.     

Genetically engineered vaccines

The application of recombinant DNA technology to allergen research has provided the sequence information and genetic material to produce new types of allergy vaccines. One general strategy has been to use the knowledge to produce synthetic peptides that represent selected T-cell or B-cell epitopes. The production of genetically engineered allergens provides an alternative strategy to construct hypoallergenic vaccines, which can provide a better and less selected representation of the epitopes. Many strategies have been used to produce such hypoallergens, and their ability to reduce allergenicity has been amply demonstrated by skin and nasal provocation tests. The retention of T cell-stimulating activity has also been demonstrated, and a consistent feature of the vaccines has been, despite the reduced immunoglobulin E (IgE)-binding reactivity, the ability to induce anti-allergen IgG antibody. The lead hypoallergens have been polypeptide fragments and trimeric constructs of the birch allergen Bet v 1. A clinical trial with these medicaments has shown the ability to modify IgE and IgG antibody production, skin test reactivity, and symptom scores. This is the first trial of a recombinant allergy vaccine, and it has set a benchmark for further studies. A new generation of hypoallergens is now being produced based on the detailed knowledge of the tertiary structures of the allergens and of the T-cell and B-cell epitopes. The modifications have been made to change the topography of the allergens while retaining a stable, folding structure. In the case of Bet v 1, tertiary structures of hypoallergens have been determined. Structurally modeled hypoallergens have been produced for pollen, venom, food, and latex allergens, with promising characteristics from preclinical studies.     

Characterization of a mouse model of allergy to a major occupational latex glove allergen Hev b 5

Allergen-specific immunotherapy is a clinically proven effective treatment for many allergic diseases, including asthma; however, it is not currently available for latex allergy because of the high risk of anaphylaxis. There is, therefore, a crucial need for an animal model of latex allergy in which to develop effective immunotherapy. Previous mouse models of latex allergy either did not characterize the allergic pulmonary immune response or used crude latex extracts, making it difficult to quantify the contribution of individual proteins and limiting their usefulness for developing specific immunotherapy. We immunized mice with recombinant Hev b 5, a defined major latex allergen, or latex glove protein extract, representing the range of occupationally encountered processed latex allergens. The immune response was compared with that seen in ovalbumin-immunized mice. Immunization with Hev b 5 or glove extract elicits hallmarks of allergic pulmonary Th2-type immune responses, comparable to those for ovalbumin, including (1) serum antigen-specific IgE, (2) an eosinophilic inflammatory infiltrate in the lung, (3) increased interleukin-5 in lung bronchoalveolar lavage fluid, and (4) mucus hypersecretion by epithelial cells in the lung airways. This mouse model will aid the development of potentially curative treatments for latex-sensitized individuals, including those with occupational asthma.     

Latex allergy and fruit cross-reaction in subjects who are nonatopic.

Since the first case reported in 1927, latex allergy has attracted the attention of allergists including its capacity to cross-react with fruits. To evaluate the frequency of sensitivity to some fruit allergens shown to cross-react with latex, we evaluated 82 patients (43 men and 39 women, aged between 18 and 45 years) with latex allergy. All patients underwent skin tests with various fruit extracts that potentially cross-react with latex. Only patients with negative prick tests successively underwent prick-by-prick tests with fresh fruits. Thirty-nine of 82 patients (47.5%) were found to have positive skin tests. Prick tests with fruit extracts were positive in 28 patients (kiwi, 21 patients; banana, 17 patients; avocado, 8 patients; and papaya, 3 patients), and the prick-by-prick test had positive results in 11 patients (kiwi, 7 patients; banana, 4 patients; and avocado, 3 patients). In our experience patients with latex allergy are at a high risk of sensitization to some fruits and they often develop allergic reactions, even severe ones, after eating them; for this reason fruit sensitization should be taken into consideration when investigating patients allergic to natural rubber latex.     

Sublingual Immunotherapy: Recent Advances

The practice of administering sublingual immunotherapy for respiratory allergy is gaining more and more diffusion worldwide as a consequence of the robust demonstration of clinical efficacy and safety provided by recent high-powered and well-designed studies, confirming for individual seasonal allergens the results of previous metanalyses in adult and pediatric populations. Preliminary evidence derives from recent rigorous trials on perennial allergens, like house dust mites, and specifically designed studies addressed the benefits on asthma. Emerging research suggests that SLIT may have a future role in other allergic conditions such as atopic dermatitis, food, latex and venom allergy. Efforts to develop a safer and more effective SLIT for inhalant allergens have led to the development of allergoids, recombinant allergens and formulations with adjuvants and substances targeting antigens to dendritic cells that possess a crucial role in initiating immune responses. The high degree of variation in the evaluation of clinical effects and immunological changes requires further studies to identify the candidate patients to SLIT and biomarkers of short and long term efficacy. Appropriate management strategies are urgently needed to overcome the barriers to SLIT compliance.     

Natural rubber latex allergy and asthma

Allergic responses to natural rubber latex (NRL) continue to be reported. In adults, the major exposure is in the occupational setting, especially in relation to NRL glove use by health care workers. Issues addressed over the past year include improving diagnostic methods for NRL allergy and characterization of NRL allergens relevant to various exposure groups and evaluating strategies for prevention and early detection of NRL allergy. Assessment of in vitro tests show good intertest correlation but lower sensitivity compared with skin test responses. NRL allergens have been further characterized as reported in the past year. Development of recombinant Hev b 3, a major NRL allergen relevant to children with spina bifida, enhances the likelihood for improved diagnostic reagents. Preliminary reports of primary preventive strategies suggest that avoidance of high-protein, powdered gloves in health care facilities can be cost-effective and is associated with a decline in sensitized workers.     

Latex allergy: Review of recent advances

Latex allergy is an IgE-dependent immediate hypersensitivity reaction to latex proteins. Risk factors for latex allergy are contact with latex products and atopy. Children who undergo multiple surgical procedures and healthcare workers are the major groups at risk. Powdered latex gloves are an important source of sensitization. Preventive measures are leading to reduction in latex sensitization and allergic reactions. The prevalence of latex allergy in the general population may be as low as 0.1%, whereas the frequency of latex sensitization is reported to be 7%; this may be due to cross-reacting antipollen IgE. The most important latex allergens have been purified, and some have been cloned and sequenced. Many latex-allergic patients are also allergic to common plant-derived aeroallergens and foods. The structural and biologic relationships among plant-derived food allergens, including latex, explain these clinically important cross-reactions.     

Les allergènes recombinants : leur apport à l’allergologie en 2006

Advances in molecular biology techniques have led to the production of recombinant allergens, about thirty of them now being available for measurements (DIAGNOSIS?) in vitro. These recombinant allergens correspond to a precise molecular variant of a natural allergen, and their biological activity has to be evaluated in comparison with the corresponding natural allergen. The advantages of recombinant allergens are essentially the creation of allergenic preparations having constant pharmaceutic properties, which allows determination of specific IgE directed against different molecular components of an allergenic source, for example, pollen, mites, etc. The main consequences of these biological advances are the following: evaluation of sensitivities to allergen molecules in different populations (molecular epidemiology), improvement of extracts used for diagnosis by selection of the most pertinent allergenic sources and in quantifying their major allergen content, definition of the spectrum of recognition of specific IgE vis-à-vis different molecular components (spectrotype), quantitative evaluation of IgE responses, establishing the molecular basis of cross-reactions between different inhaled allergens, between different food allergens, and between inhaled allergens and food allergens. As regards allergy practice, this new diagnostic tool can lead to better interpretation of polysensitivities, observed by skin tests and in vitro tests. Some examples of particular clinical cases associated with specific sensitivities vis-à-vis certain recombinant allergens will be presented.     

Addressing Molecular Diagnosis of Occupational Allergies

Purpose of Review

Numerous clinically relevant allergenic molecules enhance the performance of specific (s) IgE tests and improve the specificity of allergy diagnosis. This review aimed to summarize our current knowledge of the high-molecular-weight allergens involved in the development of occupational asthma and rhinitis and to critically analyze the contribution of component-resolved diagnosis in the management of these conditions.

Recent Findings

There is a lack of standardization and validation for most available extracts of occupational agents, and assessment of sIgE reactivity to occupational allergen components has been poorly investigated, with the notable exception of natural rubber latex (NRL) and wheat flour. In the case of NRL, the application of recombinant single allergens and amplification of natural extracts with stable recombinant allergens improved the test sensitivity. IgE-sensitization profile in patients with baker’s asthma showed great interindividual variation, and extract-based diagnostic is still recommended. For other occupational allergens, it remains necessary to evaluate the relevance of single allergen molecules for the sensitization induced by occupational exposure.

Summary

Progress has been made to characterize occupational allergens especially NRL and wheat, although there is still an unmet need to increase the knowledge of occupational allergens, to include standardized tools into routine diagnostic, and to evaluate their usefulness in clinical practice.

     

Latex protein: a hidden "food" allergen?

Avoidance of latex allergens is the primary method to prevent adverse reactions. Natural rubber latex is found in many different products in both the health care industry and in modern society, and consequently results in unexpected exposures of sensitized individuals. The use of latex gloves by food handlers provides one potential route for inadvertent exposure to latex allergens. In this study we have used two immunological methods to determine whether latex proteins are transferred to foods following contact with latex gloves. Direct transfer of latex protein to cheese was visualized using a modified immunoblot method. Sliced cheese was touched with a gloved finger. A nitrocellulose membrane was applied to lift the potential fingerprints and a rabbit anti-latex antiserum was used to visualize the transfer of any latex finger-prints. After handling lettuce with gloves, transferred protein was recovered by extracting the lettuce and quantified using an inhibition ELISA for latex proteins. Fingerprints of latex protein were readily detectable on cheese after contact with powdered latex gloves, but not with vinyl gloves. Furthermore, powdered latex glove use resulted in measurable amounts of latex protein on lettuce with an exposure-dependent increase in the latex protein levels. Lettuce alone or lettuce handled with vinyl gloves was negative for latex protein. The use of latex gloves by food handlers is the source of an indirect food additive in the form of latex proteins. It is recommended that food handlers avoid the use of latex gloves to eliminate inadvertent exposure of latex-sensitive individuals.     

Biology of tree pollen allergens

More than 25% of the population suffer from type I allergy. Pollens from trees of the Fagales, Oleaceae, and Cupressaceae belong to the most potent and frequent allergen sources. During the past 15 years, the nature of the most important allergens has been identified by molecular biological techniques, and recombinant allergens equivalent to the natural allergens have been produced. These advances provide insight into the biological functions of important allergens and allow the development of novel forms of diagnosis and therapy. In this review, we focus on Fagales allergens to illustrate the impact of recombinant allergens on diagnosis and therapy. We discuss structural similarities as a molecular basis for crossreactivities and develop diagnostic concepts by using speciesspecific marker allergens as well as highly cross-reactive allergens. The identification of the allergen recognition profiles of patients with recombinant allergens allows a more precise selection of patients for available forms of allergy treatment. Moreover, we describe novel recombinant allergen-based forms of specific immunotherapy.     

Correlation between inhalant allergen-specific IgE and pulmonary function in children with asthma

Sensitization to aeroallergens is associated with diminished lung function in adults. Little has been studied on the relationship between the inhalant allergen-specific IgE and pulmonary function in asthmatic children. This study was focused on four major inhalant allergens found in Korea, including Dermatophagoides pteronyssinus (Der p.), Dermatophagoides farinae (Der f.), and Alternaria- and German cockroach-specific IgEs, with evaluation of pulmonary function in relation to the amount of allergens. The parents or legal guardians of participants enrolled in this study gave informed consent. Fifty-five asthmatic patients and 48 nonasthmatic children were included. The amounts of specific IgE for the four specified inhalant allergens were determined by employing the CAP system FEIA. Forced expiratory volume in 1 sec (FEV(1))/forced vital capacity (FVC), FEV(1), and forced expiratory flow between 25% and 75% of FVC (FEF(25-75)) of subjects were evaluated through pulmonary function tests. In the asthmatic group, FEV(1), FEV(1)/FVC, and FEF(25-75) were significantly reduced (P < 0.05): reduction in FEV(1) (r = -0.44) and FEF(25-75) (r = -0.33) in association with the Der f.-specific allergen, and reduction in FEV(1) (r = -0.37) and FEF(25-75) (r = -0.34) in association with the Der p.-specific allergen, were observed. However, there was no significant correlation with German cockroach and Alternaria allergen. In the control group, no significant correlation was detectable between the allergen-specific IgE titers and the results of pulmonary function tests. In asthmatic patients, Der p.- and Der f.-specific IgEs, and not German cockroach and Alternaria, seem to play a considerable role in reduced pulmonary function among asthmatic children.