New Perspectives on the Diagnosis of Allergy to Anisakis spp.

PURPOSE OF REVIEW: To compare the prevalence of sensitization in different countries based on specific IgE values and to evaluate the use of isolated native or recombinant allergens for diagnosis. RECENT FINDINGS: Isolated allergens help in the diagnosis of truly sensitized patients avoiding false positives due to cross-reactions. Their use is therefore highly recommended, especially when used as a combination of several relevant allergens. The use of purified allergens allows an accurate diagnosis and this has led to three important findings: (1) in addition to the digestive route of sensitization, occupational and non-digestive exposure seems to be clinically relevant. (2) The parasite appears as an important agent for chronic urticaria. And (3) in endemic countries, the amount of highly sensitized subjects in the general population could be as high as 7%. Adequate information to asymptomatic patients on fish consumption habits would avoid new contacts with parasite allergens and decrease their specific IgE levels and consequently the appearance of acute or chronic episodes induced by the parasite.

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii.

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI-TOF/TOF analysis or de novo sequencing. Seventy-five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty-eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662).

IgE sensitization to Thaumetopoea pityocampa : diagnostic utility of a setae extract, clinical picture and associated risk factors.

BACKGROUND: Setae from Thaumetopoea pityocampa larvae (the pine processionary moth or PPM) can induce hypersensitivity reactions, but their clinical role in IgE-mediated responses is still subject to discussion. The aim of this study was to evaluate a setae extract for in vivo and in vitro diagnosis in nonhospitalized patients with reactions to PPM. METHODS: Forty-eight adult patients presenting with PPM cutaneous reactions were studied by skin prick test (SPT) and specific IgE using setae and whole larval (WL) extracts. Biological standardized extracts were used for skin tests. RESULTS: A total of 47.9% patients had a positive SPT for PPM (70% to both extracts, 17% only to the WL extract and 13% only to the setae extract). IgE immunoblotting detected several reactive bands in 91% of the SPT-positive cases. In multivariate analysis, male sex, immediate latency (<1 h) and duration of skin symptoms (<24 h) were independent predictors of a positive SPT. CONCLUSIONS: IgE sensitization to PPM was found in 48% of the study patients, which was associated with immediate reactions and evanescent cutaneous lesions. Most of these patients reacted to both WL and setae extracts, but some reacted to only one of them. According to our data, skin and in vitro tests to PPM should be performed using both extracts.

Anisakis simplex recombinant allergens increase diagnosis specificity preserving high sensitivity.

BACKGROUND: So far, the frequency of Anisakis simplex-specific IgE antibodies has been determined by skin prick tests (SPTs) and the ImmunoCAP system. These commercial methods have good sensitivity, but their specificity is poor because they use complete parasite extracts. Our aim was to determine the frequency of sensitization to A. simplex using recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 and to evaluate these allergens for diagnosis, comparing their performance with the commercial methods. PATIENTS AND METHODS: We conducted a descriptive, cross-sectional validation study performed in an allergy outpatient hospital clinic. Patients without fish-related allergy (tolerant patients, n = 99), and A. simplex-allergic patients (n = 35) were studied by SPTs, ImmunoCAP assays and detection of specific IgE to A. simplex recombinant allergens by dot blotting. RESULTS: SPTs and ImmunoCAP assays were positive in 18 and 17% of tolerant patients, respectively. All A. simplex-allergic patients had positive SPTs and ImmunoCAP assays. Specific IgE against at least one of the A. simplex recombinant allergens tested was detected in 15% of sera from tolerant patients and in 100% of sera from A. simplex-allergic patients. Detection of at least one A. simplex recombinant allergen by dot blotting and ImmunoCAP assay using complete extract showed a diagnostic sensitivity of 100% with both methods. However, the specificity of dot blotting with A. simplex recombinant allergens was higher compared with ImmunoCAP (84.85 vs. 82.83%). CONCLUSIONS: There are 15% of tolerant patients with specific IgE against important A. simplex allergens. The recombinant allergens studied here increase the specificity of A. simplex diagnosis while keeping the highest sensitivity. A. simplex recombinant allergens should be included with A. simplex allergy diagnostic tests to improve their specificity.

Characterization of occupational sensitization by multiallergen immunoblotting in workers exposed to laboratory animals.

BACKGROUND: Studies have estimated that 10% to 23% of workers exposed to laboratory animals report symptoms of laboratory animal allergy. OBJECTIVES: To determine the level of occupational sensitization in workers exposed to laboratory animals and to develop a diagnosis system based on a multiallergen IgE immunoblot. METHODS: A total of 75 workers exposed to laboratory animals were initially studied with skin prick tests performed with animal epithelia extracts. The workers with suspected occupational disease and positive skin prick test results were further studied with the ImmunoCAP system to determine specific IgE levels to urine and epithelia allergens and with multiallergen IgE immunoblotting to detect specific IgE levels to epithelia allergens and bovine serum albumin. RESULTS: Twenty of the 75 workers were studied with ImmunoCAP and multiallergen IgE immunoblotting. Nine were polysensitized and 3 were sensitized to only one animal. The results obtained by ImmunoCAP and multiallergen IgE immunoblotting were concordant except for in 3 workers, who had low or negative values of specific IgE determined by ImmunoCAP but positive allergen detections by immunoblotting. On the basis of the results of the study and the clinical symptoms related by workers, 16% were diagnosed as having occupational allergy. CONCLUSIONS: Multiallergen immunoblotting by means of a unique test offers a graphic representation of sensitization to the different animals to which workers are exposed, providing additional information on the clinical symptoms caused by the involved allergens. The results presented suggest that this system can improve the diagnosis of laboratory animal allergy by obtaining a sensitization profile for each exposed worker.

Setae from the pine processionary moth (Thaumetopoea pityocampa) contain several relevant allergens.

BACKGROUND: Pine processionary larvae produce urticating hairs (setae) that serve for protection against predators. Setae induce cutaneous reactions in animals and humans. The presence of toxic or allergic mechanisms is a matter of debate. OBJECTIVES: To detect the presence of allergens in setae and to characterize them. MATERIALS AND METHODS: Setae extracts were characterized by gel staining and immunoblot, with sera from patients with immediate reactions and positive prick test reactions, as well as a rabbit antiserum raised against setae. Setae proteins were fractionated by high-performance liquid chromatography. The most relevant allergen was analysed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and its sequence was deduced from an expressed sequence tag bank. Results. Setae contained at least seven different allergens. The most intense detection corresponded to a protein of MW ~ 14,000 that was similar to thaumetopoein, a previously described protein with mast cell-degranulating properties. MALDI-MS-based de novo sequencing provided a partial amino acid sequence different from that of the previously described allergen Tha p 1, and it was named Tha p 2. This allergen was detected in 61% of patients, and it is therefore a new major caterpillar allergen. CONCLUSIONS: Penetration of the setae from the pine processionary caterpillar delivers their allergenic content in addition to causing mechanical or toxic injury.

Prevalence of cutaneous reactions to the pine processionary moth (Thaumetopoea pityocampa) in an adult population.

BACKGROUND: Thaumetopoea pityocampa [pine processionary moth (PPM)] is one of the most important lepidopteran agents causing urticant cutaneous reactions in humans in Mediterranean countries. This species is also expanding northwards, because of global warming. OBJECTIVES: To investigate the prevalence, distribution by habitat group and possible risk factors of PPM cutaneous reactions in adults. METHODS: A randomly designed survey was carried out on 1224 adults. RESULTS: A point prevalence, estimated after corrections, of 8.7% was obtained (12% rural areas, 9.6% for semi-urban areas, and 4.4% for urban areas). The data showed a significantly higher risk of self-reported symptoms according to sex [p < 0.005; males, adjusted odds ratio (aOR) 1.84], habitat (p < 0.0005; rural, aOR 1.8; semi-urban, aOR 1.2), frequency of visits to pinewood areas (p < 0.005; daily exposure, aOR 2.1), and occupational exposure (p < 0.0001; aOR 5.04, 90% were males). Airborne contamination was the most important cause of reactions (83.3% of 48 participants who visited the hospital and fulfilled the criteria for a convincing reaction presented with symptoms after walking on/passing by pine tree areas). CONCLUSIONS: These findings show that PPM cutaneous reactions are common in this southern European population, including peripheral urban areas, and that the main risk is related to exposure to this insect.

Quantification of Anisakis simplex allergens in fresh, long-term frozen, and cooked fish muscle.

Fish-borne parasitic zoonoses such as Anisakiasis were once limited to people living in countries where raw or undercooked fish is traditionally consumed. Nowadays, several factors, such as the growing international markets, the improved transportation systems, the population movements, and the expansion of ethnic ways of cooking in developed countries, have increased the population exposed to these parasites. Improved diagnosis technology and a better knowledge of the symptoms by clinicians have increased the Anisakiasis cases worldwide. Dietary recommendations to Anisakis-sensitized patients include the consumption of frozen or well-cooked fish, but these probably do not defend sensitized patients from allergen exposure. The aim of our work was to develop a sensitive and specific method to detect and quantify Anisakis simplex allergens in fish muscle and its derivatives. Protein extraction was made in saline buffer followed by preparation under acid conditions. A. simplex antigens were detected by IgG immunoblot and quantified by dot blot. The allergenic properties of the extracts were assessed by IgE immunoblotting and basophil activation test. We were able to detect less than 1 ppm of A. simplex antigens, among them the allergen Ani s 4, in fish muscle with no cross-reactions and with a recovery rate of 82.5%. A. simplex antigens were detected in hakes and anchovies but not in sardines, red mullets, or shellfish. We detected A. simplex allergens in cooked hakes and also in hake stock. We proved that A. simplex allergens are preserved in long-term frozen storage (-20 degrees C +/- 2 degrees C for 11 months) of parasitized hakes. Basophil activation tests have proven the capability of the A. simplex-positive fish extracts to induce allergic symptoms.

Identification and allergenic characterisation of a new isoform of the A. simplex allergen Ani s 4.

Anisakis simplex hypersensitivity is a growing disease in developed countries. A positive diagnosis usually leads to the dietary recommendation of avoiding fish and seafood consumption. The protein Ani s 4 is a clinically relevant allergen due to its heat and pepsin resistant properties and its importance in the anaphylaxis process. The attempt of cloning Ani s 4 has led to the identification and characterisation of a new isoform that differs only in one amino acid with the previously published. This isoform was produced as an His tagged recombinant protein and its allergenic properties were tested by IgE immunoblot and by a flow cytometry basophil activation test. The results were compared to the allergenic properties of the isoform previously described. Both isoforms of Ani s 4 showed different capacities to bind IgE from sensitised patients and different potencies in the basophil activation test.

Cloning and expression of Ani s 9, a new Anisakis simplex allergen.

The larvae of the nematode Anisakis simplex parasitize seafood. When people eat raw or undercooked parasitized fish, they can suffer anisakiasis, an important immune human response to parasitic infection of the gastrointestinal tract. Even more, allergic manifestations like angioedema, urticaria or anaphylaxis can occur in sensitized patients. The aim of this work was to clone Ani s 9-cDNA and overproduce this recombinant allergen in Escherichia coli. The finding of this allergen was an unexpected result of a PCR using degenerate primers designed to amplify Ani s 5. The complete cDNA for Ani s 9 was obtained by RACE-PCR, cloned and sequenced. Expression of recombinant allergen was performed in E. coli. Immunodetection and immunoblot inhibition assays tests were carried out with sera from Anisakis allergic patients. The recombinant Ani s 9 (rAni s 9) is a protein of 147 amino acids. By immunoblot inhibition assay, it was located as a 14 kDa band present in a crude extract of the parasite. This new allergen is heat stable and is present in excretory/secretory products. Ani s 9 belongs to the SXP/RAL-2 family and shares amino acid sequence identity of 60% with As-14, an Ascaris suum allergen. Five of thirty-six Anisakis allergic patients (13.8%) were positive to rAni s 9 and natural Ani s 9 by immunodetection. In conclusion, Ani s 9 is a new allergen in Anisakis allergy and it has been cloned and successfully expressed in E. coli.

Analysis of basophil activation by flow cytometry in pediatric house dust mite allergy.

Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non-allergic children (n = 9). Allergen-induced basophil activation was detected as a CD63-upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut-off value of activated basophils discriminating mite allergic and non-allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite-sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97-1.01, p < 0.001] with a sensitivity and specificity of 96% for 16 microg/ml mite extract. Analysis of the data obtained with 1.6 microg/ml mite extract defined a cut-off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91-1.01; p < 0.001) with a sensitivity of 82% and specificity of 100%. Comparison between mite allergic and non-allergic children produced a cut-off of 8% activated basophils (AUC = 1.0) with 16 microg/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 microg/ml extract (AUC = 97%, 95% CI = 0.92-1.02; p < 0.001) but sensitivity decreased to 83%. Two atopic children showed negative skin prick and basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1-specific IgE (>100 kU/l). Cross-reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA-CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen-induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results.

Allergenic properties and cuticle microstructure of Anisakis simplex L3 after freezing and pepsin digestion.

This article examines the viability of and the alterations to the larval cuticle and the pattern of the antigens released when live or frozen Anisakis simplex larvae were treated with acid and pepsin. The results showed that freezing did not greatly alter the larva body. If ruptures were observed, the antigen release to the incubation media was not enhanced, and most of the antigenic content was retained inside the bodies of the larvae. The immunoblotting assay demonstrated that most of the antigens released, including the allergen Ani s 4, were resistant to pepsin. Freezing killed the larvae, but their survival was not compromised by acid treatment or pepsin digestion when kept chilled. All these findings support recommendations about freezing fish for consumption raw or undercooked to prevent human infection by A. simplex larvae. However, our data show that the antigenicity of the larvae is preserved after freezing and may explain why some sensitized patients develop symptoms after ingestion of infested frozen fish.

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor.

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.

Occupational sensitization to fungal enzymes used in animal feed industry.

BACKGROUND: Industrial enzymes cause the increasing prevalence of occupational hypersensitivity. Our objective was to study workers occupationally exposed to fungal enzymes in 2 animal feed factories to determine if the sensitization originated in the enzymes or was caused by the microorganism used to produce the enzymes. METHODS: Eighty-six consenting workers were studied by skin prick tests with extracts from the enzymatic products handled in their factories. Positive workers were then studied by IgE immunoblotting and basophil activation was measured by flow cytometry. RESULTS: Eight of the 86 workers analysed (9%) tested positive and were more frequently sensitized to phytase from Trichoderma and Peniophora. Glucanase and alpha-amylase from Bacillus amyloliquefaciens did not cause sensitization in any worker. No cross-reactions were observed between Trichoderma and Peniophora sp. phytases. Workers were sensitized to the product that they handled. CONCLUSIONS: Fungal enzymes cause occupational hypersensitivity in animal feed industries. Immunoblotting and basophil activation are useful to evaluate the effects of handling enzymes as part of the medical surveillance of enzyme-exposed workers. We describe Peniophora sp. 6-phytase as a new allergen and enzymes from Trichoderma as strong sensitizers.

Changes over Time in IgE Sensitization to Allergens of the Fish Parasite Anisakis spp.

BACKGROUND: Sensitization to Anisakis spp. can produce allergic reactions after eating raw or undercooked parasitized fish. Specific IgE is detected long after the onset of symptoms, but the changes in specific IgE levels over a long follow-up period are unknown; furthermore, the influence of Anisakis spp. allergen exposure through consumption of fishery products is also unknown. OBJECTIVE: To analyse the changes in IgE sensitization to Anisakis spp. allergens over several years of follow-up and the influence of the consumption of fishery products in IgE sensitization. METHODS: Total IgE, Anisakis spp.-specific IgE, anti-Ani s 1 and anti-Ani s 4 IgE were repeatedly measured over a median follow-up duration of 49 months in 17 sensitized patients. RESULTS: Anisakis spp.-specific IgE was detected in 16/17 patients throughout the follow-up period. The comparison between baseline and last visit measurements showed significant decreases in both total IgE and specific IgE. The specific IgE values had an exponential or polynomial decay trend in 13/17 patients. In 4/17 patients, an increase in specific IgE level with the introduction of fish to the diet was observed. Three patients reported symptoms after eating aquaculture or previously frozen fish, and in two of those patients, symptom presentation was coincident with an increase in specific IgE level. CONCLUSIONS: IgE sensitization to Anisakis spp. allergens lasts for many years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level. CLINICAL RELEVANCE: Following sensitization to Anisakis spp. allergens, the absence of additional exposure to those allergens does not result in the loss of IgE sensitization. Exposure to Anisakis spp. allergens in fishery products can increase the specific IgE level in some sensitized patients.

Detection of Anisakis simplex-induced basophil activation by flow cytometry.

BACKGROUND: Laboratory diagnosis of anisakidosis is based on specific serum IgE detection. Recently, detection of allergen-induced basophil activation by flow cytometry has been proposed as a valuable tool for allergy diagnosis. OBJECTIVE: To evaluate if detection of Anisakis-induced basophil activation by flow cytometry is a useful tool in the diagnosis of Anisakis allergy. METHODS: Patients with Anisakis allergy (A.s.+, n = 37), patients reporting chronic urticaria or abdominal pain unrelated to fish ingestion (A.s.-, n = 51), and healthy controls (n = 12) were studied. Specific IgE to Anisakis simplex (A. simplex) was quantified with CAP-FEIA method, and basophil activation test was performed with three different concentrations of an Anisakis crude extract. Basophil gating was performed with CD123 and HLA-DR, and cellular activation was measured with CD63. RESULTS: A.s.+ patients showed significantly higher age and total IgE levels than did the A.s.- patients. Specific IgE to A. simplex correlated with the activated basophil percentages obtained with 15 microg/mL (r = 0.80; P < 0.001), 1.5 microg/mL (r = 0.84; P < 0.001), and 0.15 microg/mL (r = 0.82; P < 0.001) of A. simplex crude extract. Nine individuals (3 in the A.s.+ group and 6 in the A.s.- group) were nonresponders to basophil stimulation with anti-IgE. Five A.s.- patients showed positive IgE values to A. simplex while the basophil activation test was negative. According to the receiver operating characteristics curves performed between A.s.+ vs. A.s.- and A.s.+ vs. healthy controls, the cutoff for a positive basophil activation test was >or=21% (specificity = 96%, sensitivity = 100%), and 16% (sensitivity and specificity of 100%) respectively. When nonresponders were included in the A.s.+ vs. A.s.- analysis, sensitivity decreased to 95%. Multivariate logistic analysis showed that the specific basophil activation was a factor independently associated with clinical symptoms of A. simplex allergy. CONCLUSIONS: Detection of A. simplex-induced basophil activation by flow cytometry is a useful laboratory technique for the diagnosis of anisakidosis, supplementing specific IgE determinations.

[Risk of sensitization to Anisakis simplex among the members of the families of patients with anisakiasis]. / Riesgo de sensibilización frente a Anisakis simplex entre familiares de pacientes con anisakiasis.

BACKGROUND AND OBJECTIVE: Infection by anisakid nematodes is associated with consumption of raw fish. The aim of this paper was to determine the prevalence of sensitization among members of families of patients with anisakiasis. INDIVIDUALS AND METHODS: Eleven asymptomatic members of families of patients diagnosed with anisakiasis were studied by means of IgE-immunoblotting. The frequency of sensitization was compared with the prevalence in a normal population from the same geographic area. RESULTS: A very high frequency of sensitization was observed among patients' family members (73%) as compared with the prevalence in normal population (12%) (p < 0.001). CONCLUSION: All members of the family of a patient diagnosed with anisakiasis should be studied to determine whether they are sensitized.

Identification of autoclave-resistant Anisakis simplex allergens.

Anisakis simplex is a fish parasite able to induce allergic reactions in humans infected when eating raw or undercooked fish parasitized with viable third-stage larvae. Some authors claim that exposure to nonviable Anisakis material can result in allergic symptoms in previously sensitized patients, indicating that parasite allergens are resistant to the thermal treatments of usual cooking procedures. Furthermore, some patients report symptoms after eating canned fish. The aim of this work was the analysis of parasite allergen stability in heating to 121 °C in an autoclave to simulate the thermal process applied to canned fish. Third-stage larvae were subjected to autoclaving for 20, 40, and 80 min, and parasite crude extracts were analyzed by electrophoresis, immunoblotting, and a flow-cytometric basophil activation test. Allergens resistant to autoclaving were separated by reversed-phase high-performance liquid chromatography and identified by ion trap mass spectrometry. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that autoclaving considerably reduced the number and intensity of identifiable protein bands in a time-dependent manner. Several allergens were detected by immunoblotting with a pool of A. simplex allergic patients' sera after autoclaving. Allergens of 9 and 14 kDa resistant to autoclaving were identified as Ani s 4 and Ani s 1 allergens, respectively. Functional analysis showed that allergens retain their capacity to activate basophils even after autoclaving for 80 min. In conclusion, some relevant A. simplex allergens retain their capacity to bind immunoglobulin E and activate basophils after being subjected to autoclaving, which is a method equivalent to that used in industrial canning processes.