Usefulness of the Nasal Allergen Provocation Test in the Diagnosis of Shellfish Allergy.

BACKGROUND AND OBJECTIVES: Shellfish allergy is a major cause of food allergy and anaphylaxis worldwide. Several allergenic proteins have been described in the last few years, but the only diagnostic tool that still enables discrimination between allergic and nonallergic sensitized persons is the oral food challenge (OFC). The aim of this study was to evaluate the usefulness of the nasal allergen provocation test (NAPT) as a diagnostic tool in shellfish allergy. METHODS: Forty-five patients with confirmed sensitization to shrimp by a positive skin prick test (SPT) result with a commercial shrimp extract were recruited and classified as sensitized-allergic or sensitized-nonallergic based on current tolerance to shrimp intake, the result of an OFC with a freeze-dried cooked shrimp mixture extract, or a recent history of anaphylaxis induced by shrimp ingestion. These patients and 10 controls not sensitized to shrimp underwent NAPT with a freeze-dried cooked shrimp mixture extract. The response was evaluated using acoustic rhinometry and a visual analog scale. RESULTS: Significant differences (P=.001) were found between the sensitized-allergic group (18/20 positive NAPT, 90%) and both the sensitized-nonallergic group (2/18 positive NAPT, 11.1%) and controls (0/10 positive NAPT). NAPT enables differentiation between allergic and nonallergic persons with a sensitivity of 90%, specificity of 89%, positive predictive value of 90%, and negative predictive value of 89%. CONCLUSIONS: Our results indicate that NAPT makes it possible to differentiate between sensitized symptomatic patients and sensitized tolerant patients and could be a valuable diagnostic tool when assessing shrimp allergy.

Shellfish Allergy: Unmet Needs in Diagnosis and Treatment.

Seafood is a major cause of food allergy and anaphylaxis worldwide. Shellfish is included among the "big eight" food groups, which are responsible for more than 90% of all cases of food allergy. Approximately 2.5% of the world's population has experienced an adverse reaction to seafood. Seafood allergy is one of the most frequent and lethal allergies that exist. The several allergenic proteins involved in allergic reactions that have been described in recent years include tropomyosin, arginine kinase, myosin light chain, and sarcoplasmic calcium-binding protein. Despite all the data reported in the last few years, shellfish allergy is still diagnosed and treated as it was 50 years ago. The only effective treatment to prevent allergic reactions to shellfish is avoidance. This review aims to update recently published data on shellfish allergy and to highlight those areas that have yet to be resolved.

Immunochemical and Physical Quantitation of Grass and Olive Pollen Allergens: Correlation With Asthma Admissions in Cáceres, Spain.

BACKGROUND AND OBJECTIVES: The association between pollen counts and allergen levels in the air is controversial. Objectives: The aims of the study were to quantify total and major allergen levels of Phleum pratense and Olea europaea and to analyze their correlation with grass and olive pollen counts and the number of asthma attacks attended at Complejo Hospitalario Universitario, Cáceres, Spain. MATERIAL AND METHODS: A volumetric air sampler and a Burkard spore trap were used for pollen and aeroallergen collection during April- June 2011. Filters were extracted, and major allergens were quantified using enzyme-linked immunosorbent assay. RESULTS: May was the main grass pollination period, with a maximum peak of 1362 grains/m3 (May 13). The main pollination period for olive was April 30-May 20, with a maximum peak of 851 grains/m3 (May 11). A moderate correlation was observed between asthma exacerbations and grass pollen counts or Phleum total allergen levels; this became stronger when a 3-day offset was introduced. A significant association was observed between asthma exacerbations and total olive allergen or olive pollen grain levels when a 1-day offset was introduced. The maximum correlation (moderate-high) was observed 4 days and 6 days away from the maximum olive pollen peak and the maximum Ole e 1 peak level, respectively. CONCLUSIONS: This study reveals a significant correlation between grass and olive pollination and an increase in the number of visits to the emergency room for asthma attacks. The aerobiological pattern of allergen levels in the air is similar to that of pollen counts during the grass and olive pollination periods.

Oral myeloid cells uptake allergoids coupled to mannan driving Th1/Treg responses upon sublingual delivery in mice.

BACKGROUND: Polymerized allergoids coupled to nonoxidized mannan (PM-allergoids) may represent novel vaccines targeting dendritic cells (DCs). PM-allergoids are better captured by DCs than native allergens and favor Th1/Treg cell responses upon subcutaneous injection. Herein we have studied in mice the in vivo immunogenicity of PM-allergoids administered sublingually in comparison with native allergens. METHODS: Three immunization protocols (4-8 weeks long) were used in Balb/c mice. Serum antibody levels were tested by ELISA. Cell responses (proliferation, cytokines, and Tregs) were assayed by flow cytometry in spleen and lymph nodes (LNs). Allergen uptake was measured by flow cytometry in myeloid sublingual cells. RESULTS: A quick antibody response and higher IgG2a/IgE ratio were observed with PM-allergoids. Moreover, stronger specific proliferative responses were seen in both submandibular LNs and spleen cells assayed in vitro. This was accompanied by a higher IFNγ/IL-4 ratio with a quick IL-10 production by submandibular LN cells. An increase in CD4+ CD25high FOXP3+ Treg cells was detected in LNs and spleen of mice treated with PM-allergoids. These allergoids were better captured than native allergens by antigen-presenting (CD45+ MHC-II+ ) cells obtained from the sublingual mucosa, including DCs (CD11b+ ) and macrophages (CD64+ ). Importantly, all the differential effects induced by PM-allergoids were abolished when using oxidized instead of nonoxidized PM-allergoids. CONCLUSION: Our results demonstrate for the first time that PM-allergoids administered through the sublingual route promote the generation of Th1 and FOXP3+ Treg cells in a greater extent than native allergens by mechanisms that might well involve their better uptake by oral antigen-presenting cells.

Allergenome characterization of the mosquito Aedes aegypti.

BACKGROUND: Saliva and muscle-derived mosquito allergens have been purified and characterized. However, the complete set of allergens remains to be elucidated. In this study, we identified and characterized IgE-binding proteins from the mosquito species Aedes aegypti. METHODS: Serum was obtained from 15 allergic individuals with asthma and/or rhinitis and sensitized to mosquito. IgE binding was determined by ELISA. Total proteins from freeze-dried bodies of A. aegypti were extracted and IgE-reactive proteins were identified by 2D gel electrophoresis, followed by Western blot with pooled or individual sera. IgE-reactive spots were further characterized by mass spectrometry. RESULTS: Twenty-five IgE-reactive spots were identified, corresponding to 10 different proteins, some of which appeared as different variants or isoforms. Heat-shock cognate 70 (HSC-70) and tropomyosin showed IgE reactivity with 60% of the sera, lysosomal aspartic protease, and "AAEL006070-PA" (Uniprot: Q177P3) with 40% and the other proteins with <33.3% of the sera. Different variants or isoforms of tropomyosin, arginine or creatine kinase, glyceraldehyde-3-phosphate dehydrogenase (GPDH), calcium-binding protein, and phosphoglycerate mutase were also identified. The mixture of three allergens (Aed a 6, Aed a 8, and Aed a 10) seems to identify more than 80% of A. aegypti-sensitized individuals, indicating that these allergens should be considered when designing of improved mosquito allergy diagnostic tools. CONCLUSIONS: The newly identified allergens may play a role in the pathophysiology of mosquito allergy in the tropics, and some of them might be important arthropod-related proteins involved in cross-reactivity between A. aegypti and other allergenic arthropods.

Safety of immunotherapy with glutaraldehyde modified allergen extracts in children and adults.

BACKGROUND: Glutaraldehyde-modified natural allergen extracts show significant reduction in the IgE-binding capacity and proteolytic activity. This allows the administration of higher doses in a shorter period of time, and to mix different allergen extracts. OBJECTIVE: Evaluate the safety of different concentrations and mixtures of glutaraldehyde-modified allergen extracts in a large group of paediatric and adult patients undergoing specific immunotherapy treatment. MATERIALS AND METHODS: 1855 patients (1156 adults and 699 children), suffering from rhinoconjunctivitis and/or asthma, participated in an observational multicentre cohort study, to evaluate the safety of immunotherapy using vaccines containing modified allergen extracts. Patients were monosensitised, or polysensitised, and received a therapeutic vaccine containing polymerised allergen extracts adsorbed onto aluminium hydroxide. Safety was assessed by recording all side reactions related to immunotherapy. RESULTS: The clinically relevant local reactions totalled 120, (90 immediate and 30 delayed) (1.02% of injections). Of them, 31 (0.26% of injections) occurred in children (26 immediate and 5 delayed) and 89 (0.76% of injections) in adults (64 immediate and 25 delayed). There were 38 systemic reactions. Eleven reactions were immediate (9 of grade 1 and 2 of grade 2) and 27 delayed (22 of grade 1 and 5 of grade 2). There were seven grade 2 systemic reactions (0.06% of the injections). No differences (P>0.05) in the number of reactions were observed between adults and children and between treatments were found in systemic reactions. All systemic reactions were mild and resolved spontaneously without the need of medication. CONCLUSION: Specific immunotherapy using natural modified allergen vaccines is safe to treat allergic patients, even at higher doses and in mixtures of unrelated allergen extracts. The percentage of adverse reactions detected is lower than those reported in the literature with native unmodified allergen extracts.

Mite hypersensitivity in patients with rhinitis and rhinosinusitis living in a tropical environment.

PURPOSE: Rhinitis and rhinosinusitis are major concerns in public health. Mites are important aetiological agents in the tropics. The present study investigated the in vivo response to mite allergens in patients with rhinitis and rhinosinusitis. METHODS: All patients with presumptive nasal allergy were included. Skin tests were done with inhalants and mite extracts. Patients were classified as allergic or non-allergic according to skin tests and history. RESULTS: Out of 229 patients, 175 (76.4%) showed positive skin tests. Allergic patients showed positivity to mites in 97.1% of cases, 51.4% to dog dander; 40.5% to cat dander; 36.5% to German cockroach; 22.8% to moulds; and 21.1% to grass pollens. Dermatophagoides farinae induced responses in 90.8% of patients, D. pteronyssinus in 90.1%, Blomia tropicalis in 74.8%, Glycyphagus domesticus in 62.2%, Chortoglyphus arcuatus in 58.2%, Acarus siro in 46.2%, Lepidoglyphus destructor in 35.4%, and Tyrophagus putrescentiae in 35.0%. Higher correlations were found between skin test diameters induced by mites from the same family. CONCLUSIONS: Sensitisation to inhalant allergens is present in 76% of allergy clinics' patients with rhinitis or rhinosinusitis. Our results confirm previous observations showing that mites constitute the most important cause of respiratory allergy in tropical settings and suggest that mite allergen cross-reactivity is responsible for the positivity of skin tests to mites not present in the patient's environment since the species Glycyphagus, Chortoglyphus, Acarus, Lepidoglyphus and Tyrophagus have not been found in Caracas house dust.

Occupational allergy to aquarium fish food: red midge larva, freshwater shrimp, and earthworm. A clinical and immunological study.

BACKGROUND: Chironomids seem to be the main cause of occupational allergy to aquarium fish food. OBJECTIVE: The aim of this study was to investigate the pattern of occupational sensitization to 3 different arthropod species used as components of aquarium fish food. METHODS: The study sample comprised 8 workers from a fish food packing department. The control group comprised 40 atopic patients (20 of whom were allergic to mites). We performed prick tests with extracts of red midge larva (Chironomus thummi), freshwater shrimp (Gammarus species), earthworm (Tubifex species), and other arthropod species and a battery of common inhalant allergens. We measured peak expiratory flow rate (PEFR) and specific immunoglobulin (Ig) E and performed a methacholine challenge test, nasal challenge test, and immunoblotting. Cross-reactivity analyses were completed using immunoblotting and CAP inhibition. RESULTS: Prick test results were positive to red midge larvae in 7 patients (87.5%), Gammarus in 5 (62.5%), Tubifex in 3 (37.5%), and mites in 6 (75%). In the mite-allergic controls, 30% had positive prick test results to red midge larvae. PEFR decreased > or = 20% during the packing process in all patients, and in 1 patient it indicated a dual asthmatic response. Methacholine challenge test results were positive in all participants. Nasal challenge tests were performed in 4 patients, and the results were positive. Specific IgE to red midge larvae was detected in 62.5%, Gammarus in 50%, and Tubifex in 16%. Bands of approximately 14-15 kDa and 31 kDa were observed in Gammarus and red midge larvae extracts. Cross-reactivity assays demonstrated that Gammarus totally inhibited red midge larvae, while Tubifex did so partially. Dermatophagoides pteronyssinus showed very low inhibitory capacity. CONCLUSIONS: Aquarium fish food arthropods are potent allergens with an elevated prevalence of sensitization and variable degree of crossreactivity. This is the first report of occupational allergy to Tubifex. More data are necessary to identify and characterize the responsible allergens.

Correlation between Alt a 1 levels and clinical symptoms in Alternaria alternata-monosensitized patients.

BACKGROUND: Alternaria alternata is a risk factor for developing asthma.Alt a 1, which has been described as the major allergen in A alternata, shows a good correlation with A alternata spores only when they have germinated. OBJECTIVES: The objective of this study was to determine the correlation between spore counts and clinical symptoms in patients with allergic asthma and/or rhinitis monosensitized to A alternata. METHODS: Two types of samplers were used to determine exposure: a Burkard spore trap to collect A alternata spores and a high-volume air sampler to collect airborne particles. A total of 366 air filters were collected. Alt a 1 levels were measured by monoclonal antibody-based enzyme-linked immunosorbent assay. Eighteen monosensitized patients were asked to record their daily symptoms throughout the year. RESULTS: A alternata spores were detected throughout the year, whereas Alt a 1 was detected only between March and December. Symptoms showed positive and significant correlations with spore counts (r=0.459, P<.001), and Alt a 1 levels (r=0.294, P<.001). The correlation between spores and Alt a 1 was low. The negative binomial model proved that an increase of 10 pg/m3 in Alt a 1 levels increased the number of symptoms at a 3-day lag by 5%. CONCLUSIONS: In patients who are allergic to A alternata, Alt a 1 levels can be considered an important marker for predicting the risk of respiratory symptoms.

Grass pollen, aeroallergens, and clinical symptoms in Ciudad Real, Spain.

BACKGROUND: In allergic individuals, onset of symptoms is related to atmospheric pollen grain counts and aeroallergen concentrations. However, this relationship is not always clear. OBJECTIVES: To analyze the correlation between grass pollen grain and aeroallergen concentrations in Ciudad Real, Spain, during the year 2004 and establish their association with symptoms in patients with allergic asthma, rhinitis, or both. METHODS: Two different samplers were used to assess allergen exposure: a Burkard spore trap to collect pollen grains and a high-volume air sampler to collect airborne particles. Individual filters were extracted daily in phosphate-buffered serum and analyzed by enzyme-linked immunosorbent assay based on serum containing high titers of specific immunoglobulin (Ig) E to grasses. The study population comprised 27 grass-allergic patients whose symptoms and medication were recorded daily. RESULTS: Grass pollens were detected between April 28 and July 18. There was a positive correlation between pollen grain counts and symptoms (r = 0.62; P > .001). Grass aeroallergens were detected not only during the grass pollination period, but also before and after this period. There was also a very significant correlation between aeroallergen levels and symptoms (r = 0.76; P < .0001). The threshold level for grass pollen was 35 grains/m3. CONCLUSIONS: Grass-related allergenic activity is present throughout the year, demonstrating the existence of aeroallergens outside the pollen season. Symptoms in allergic patients may be related to airborne particle concentrations. This fact should be taken into account in the clinical follow-up and management of allergic patients.

Detection of allergen composition and in vivo immunogenicity of depigmented allergoids of Betula alba.

BACKGROUND: Chemical modification of allergen vaccines to reduce IgE binding improves safety while maintaining clinical efficacy. However, this also complicates the characterization of allergoids using techniques as for native allergen extracts. OBJECTIVES: The objective of this study was to analyse the molecular size of Betula alba depigmented allergoids, conservation of major allergens in the allergoids and in vivo antibody response to immunization. METHODS: The molecular size of depigmented allergoids was evaluated by high performance-size exclusion chromatography and light scattering techniques. Protein composition was compared with native extracts by capillary liquid chromatography-tandem mass spectrometry based peptide mapping. Rabbits were immunized with depigmented allergoid of Betula pollen adsorbed onto aluminium hydroxide (Depigoid). IgG antibodies against individual allergens were determined by ELISA and immunoblot. RESULTS: Depigmented allergoids contained a range of high molecular weight particles, approximately 60% of which had a molecular weight of 1-3 MDa. Peptide sequencing confirmed the preservation of five isoforms of Bet v 1, as well as Bet v 2, Bet v 6 and Bet v 7. Sera from immunized rabbits showed high levels of specific IgG to rBet v 1.0101 and rBet v 2. CONCLUSIONS: The mean protein content was 544+/-106 microg per mg of freeze-dried material for depigmented allergoids and 434+/-71 for native extracts. They retain the capacity to induce specific IgG antibodies against individual allergens present in the native extract. These findings confirm the immunogenicity of depigmented allergoids and may explain why patients treated with these vaccines are protected against the native allergens. Analysis of molecular size and allergen content may be useful techniques for characterization and standardization of allergoid products.

Cluster immunotherapy with a glutaraldehyde-modified mixture of grasses results in an improvement in specific nasal provocation tests in less than 2.5 months of treatment.

BACKGROUND: Cluster immunotherapy is becoming increasingly used. It allows for a rapid build up phase and the administration of higher doses of allergen in a shorter period of time. OBJECTIVES: To evaluate the effect of short-term pre-seasonal immunotherapy using a glutaraldeyde-modified allergen vaccine in reducing specific nasal hyperreactivity in nasal challenge tests. MATERIALS AND METHODS: Thirty-three patients were selected. All patients had a positive history of allergic rhinitis and skin tests to grass pollen, although most of them (72.7%) were sensitized to other allergens as well. The study was conducted outside of the pollen season and the patients did not receive any pharmacological treatment during this period of time. Two randomized groups of patients were established; Group A: 22 patients (13 females and nine males) and Group B, 11 control patients (seven females and four males). Patients in Group A received immunotherapy with a vaccine containing 50% of the wild grasses Trisetum paniceum and Dactylis glomerata. All patients underwent titrated nasal provocation tests (NPT) before and after completion of the study (2.3 and 2.8 months for Groups A and B, respectively). The administration schedule consisted of 0.1 and 0.2 mL at day 1, followed by 0.3 and 0.5 mL at day 7, 0.5 mL after 2 weeks followed by 0.5 mL monthly. A single vial was used containing an allergen concentration of 10 000 TU/mL (105 microg of total protein and 24.6 microg of Group 1 plus 5 allergens/mL). A mean of 6.5 injections were administered to Group A patients between NPTs. RESULTS: There were no significant differences between both groups at the beginning of the study (P=0.48). At the end, only Group A patients needed significant greater threshold concentrations for a positive NPT than at the beginning (P=0.002). CONCLUSIONS: A short-term cluster pre-seasonal inmunotherapy with a modified vaccine containing a mixture of grass pollen is effective as determined by an objective measure after only a mean 2.3 months of treatment.

Sensitization to tomato peel and pulp extracts in the Mediterranean Coast of Spain: prevalence and co-sensitization with aeroallergens.

BACKGROUND: Tomatoes (Lycopersicon esculentum) are consumed world-wide. The prevalence of sensitization to tomatoes remains unknown. OBJECTIVE: To determine the prevalence of skin test reactivity to tomato and to describe the characteristics of tomato-sensitized subjects. METHODS: Individuals attending for the first time during the period of the study to six Allergy centres, located along the Mediterranean coast of Spain, reporting respiratory and/or cutaneous symptoms, were included. All patients were skin prick tested with a battery of inhalant allergens and with peel and pulp of Canary tomato extracts. RESULTS: The study included 1734 individuals (757 males, 977 females; 31.9+/-17.8 years old). The prevalence of sensitization to tomato was 6.52% (113 patients; 65 males, 48 females; 29.5+/-13 years old). The peel extract was positive in 110 patients and the pulp extract in 47 patients; three patients were positive exclusively to pulp. Only 1.8% of individuals reported symptoms with tomato; 44% of them had skin test negative to both extracts. Among tomato-sensitized subjects, 16% reported symptoms with tomato, 97% were sensitized to inhalant aeroallergens, including 84% to pollens (mainly Artemisia vulgaris and Platanus hybrida), with differences between Northern and Southern centres. CONCLUSIONS: The prevalence found of skin test sensitivity to tomato is high. Peel extracts detected most of the sensitized subjects. Most of the sensitized subjects were asymptomatic and some patients reported symptoms without skin test sensitivity. Positive subjects were very frequently sensitized to pollens, suggesting allergen cross-reactivity. Regional differences may exist, possibly related to the pattern of sensitization to cross-reacting pollens.

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Usefulness of the Nasal Allergen Provocation Test in the Diagnosis of Shellfish Allergy

Background: Shellfish allergy is a major cause of food allergy and anaphylaxis worldwide. Several allergenic proteins have been described in the last few years, but the only diagnostic tool that still enables discrimination between allergic and nonallergic sensitized persons is the oral food challenge (OFC). Objective: The aim of this study was to evaluate the usefulness of the nasal allergen provocation test (NAPT) as a diagnostic tool in shellfish allergy. Methods: Forty-five patients with confirmed sensitization to shrimp by a positive skin prick test (SPT) result with a commercial shrimp extract were recruited and classified as sensitized-allergic or sensitized-nonallergic based on current tolerance to shrimp intake, the result of an OFC with a freeze-dried cooked shrimp mixture extract, or a recent history of anaphylaxis induced by shrimp ingestion. These patients and 10 controls not sensitized to shrimp underwent NAPT with a freeze-dried cooked shrimp mixture extract. The response was evaluated using acoustic rhinometry and a visual analog scale. Results: Significant differences (P=.001) were found between the sensitized-allergic group (18/20 positive NAPT, 90%) and both the sensitized-nonallergic group (2/18 positive NAPT, 11.1%) and controls (0/10 positive NAPT). NAPT enables differentiation between allergic and nonallergic persons with a sensitivity of 90%, specificity of 89%, positive predictive value of 90%, and negative predictive value of 89%. Conclusions: Our results indicate that NAPT makes it possible to differentiate between sensitized symptomatic patients and sensitized tolerant patients and could be a valuable diagnostic tool when assessing shrimp allergy (AU)