Anisakis allergy: an update

Revue française d’allergologie et d’immunologie clinique 45 (2005) 108–113 http://france.elsevier.com/direct/REVCLI/

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Anisakis allergy: an update Allergie à Anisakis A. Valls, C.Y. Pascual, M. Martín Esteban * Allergology Department, “La Paz” University Hospital, Castellana 261, 28046, Madrid, Spain Received 6 January 2005; accepted 10 January 2005

Abstract Anisakis simplex is a worldwide-distributed nematode that infects consumers of raw or under-cooked fish. The clinical signs of anisakiasis depend on the place in the digestive tract where the larva is deposited. Symptoms develop as a result of an inflammatory condition occurring in the gastric wall mucosa when the larva enters it. Many asymptomatic subjects show high levels of specific IgE against A. simplex. It is often complicated to diagnose an allergy to A. simplex, due to the cross-reactivity with other allergens. It is even more difficult to diagnose it in infants than in adults. Positive skin tests to A. simplex usually correspond to subjects with positive results to other allergens. The high cross-reactivity between this and other parasites more prevalent in infants would be responsible for this confusion when an accurate diagnosis is to be obtained. The secretory–excretory antigen is more specific for recognising patients who actually have parasites. This antigen could be used for indicating parasitation in the differential diagnosis of this type of sensitisation. To prevent this condition, raw fish not previously frozen for 48 h, or fresh fish not cooked for at least 20 min above 60 °C, should not be consumed. © 2005 Elsevier SAS. All rights reserved. Résumé Anisakis simplex est un nématode de distribution mondiale qui peut infecter les consommateurs de poisson cru ou mal cuit. Les signes cliniques de l’anisakiase dépendent de l’endroit du tube digestif où les larves se déposent. Les symptômes sont surtout une inflammation gastrique. Beaucoup de patients asymptomatiques présentent des taux élevés d’IgE dirigées contre A. simplex. Il est souvent difficile d’identifier l’allergie à Anisakis en raison de réactions croisées avec d’autres allergènes. Ce diagnostic est plus difficile chez l’enfant que chez l’adulte. Les tests cutanés positifs pour A. simplex correspondent habituellement aux patients ayant des tests positifs pour les autres allergènes. Ces réactions croisées importantes entre A. simplex et d’autres parasites, plus fréquentes chez les enfants, sont responsables de ces difficultés de diagnostic. L’antigène sécrétoire/excrétoire est le plus spécifique pour identifier les patients parasités. Pour prévenir l’anisakiase, les poissons crus, ou non réfrigérés pendant 48 heures, ou non cuits pendant au moins 20 min au-dessus de 60 °C ne doivent pas être consommés. © 2005 Elsevier SAS. All rights reserved. Keywords: Fish; Anisakis; Anisakiasis; Cross-reactivity; Food allergy Mots clés : Poisson ; Anisakis ; Anisakiase ; Réactions croisées ; Allergie alimentaire

1. Introduction Anisakis simplex is a worldwide-distributed nematode that, when an adult reproducer, parasites sea mammals and, at its different larva stages, fish and cephalopods. * Corresponding author. E-mail address: [email protected] (M. Martín Esteban). 0335-7457/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.allerg.2005.01.005

It is geographically located almost everywhere and infection is frequent. In the Mediterranean area, multiple parasited fish species have been described, including: scombriforms (frigate mackerel, largehead hairtail, mackerel, horse mackerel), gadiforms (European hake, blue whiting) and perciforms (common sea bream and bogue). In the North Atlantic it is mainly found in clupeiforms (herring), gadiforms (European hake, cod, blue whiting, whiting and haddock),

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scombriforms (mackerel, deep-sea rosefish), pleuronectiforms (halibut and turbot) and scorpeniforms (short-horn sculpin) [1]. A. simplex larvae have occasionally been found in freshwater fish [2], although this was attributed to the fact that they had been fed with infected, untreated sea waste. Salmon may be parasited through spending part of its cycle in the sea [2]. The adult parasites sea mammals (whales, seals, etc...). The eggs are excreted with the animal’s faeces and the first phase of larval stage (L1) develops in the water. These larvae are eaten by small plankton-type crustaceans (second larval stage, L2) that are in turn eaten by fish and cephalopods. The larvae cross their digestive barrier and migrate to the tissues, where the third larval phase (L3) develops, which is the one that parasites humans consuming these fish or cephalopods. Man is an incidental host and the L3 cannot complete its vital cycle in him. In the event of the parasited fish being swallowed by a sea mammal, the larva progresses to the fourth phase (L4) and then to the adult stage, and it completes the cycle (Fig. 1). The L3 can be seen rolled in a flat spiral on the muscle or viscera of the fish or cephalopods. Morphologically, the body of the A. simplex L3 is cylindrical, whitish and about 30 mm long, but when it is seen encapsulated on the muscles of the fish, its appearance changes and it sometimes becomes brown [3].

2. Prevalence The most complete study on prevalence was performed in Japan on patients with urticaria or food allergy, and specific IgE against A. simplex was detected in 29.8% [4]. In that same country, with a high consumption of raw fish, another study detected specific IgE against A. simplex in 33% of the patients with atopic dermatitis, 75% of the patients with urticaria, and 10% of the healthy controls [5]. The incidence of allergic reactions to A. simplex is very different throughout

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the Spanish territory: cases in the southern and southeastern areas of Spain are infrequent, while there is a high incidence in the middle and north east of the peninsula [6]. In addition to the different sources of the fish consumed, based on the different fishing grounds of the fishing fleets, local customs in nutritional patterns can also have an influence [7]. The prevalence of sensitisation to A. simplex in adults in Spain varies, according to different studies, from 6% to 56% [8,9]. One study on prevalence in patients visiting an allergology clinic in Madrid shows a sensitisation of 16% and 21.8%, respectively, using the specific lgE skin test [10]. Specific IgE against A. simplex has also been shown in the general population with a frequency from 10% to 15% [5,11]. Asymptomatic sensitisations can be explained by the high consumption of fish and the high frequency of parasitation by A. simplex or the presence of cross-reactivity with other nematodes or arthropods.

3. Clinical issues and diagnoses In contrast to other nematode infections, the disease can be caused by one single parasite, though massive infections have been described [12]. On eating raw or under-cooked fish (smoked fish, fish in vinegar, ceviche, marinated fish, salted fish, pickled fish, etc.) parasited by A. simplex, humans can show different clinical signs [1,13]. Symptoms occur as a result of an inflammatory reaction when the larva’s head adheres to or penetrates the digestive tube mucosa. The clinical signs will depend on the area of the digestive tube where the larva is located. It is most often located in the stomach [14] or the intestine [15]. Gastric anisakiasis is characterised by a colic-type abdominal pain in the epigastrium that may be associated with nausea, vomiting or even changes in the intestinal rhythm if the small bowel is involved. In this case, the diagnosis can be confirmed using an endoscopy where the parasite can be seen. Taking it out will make the symptoms disappear. When the

Fig. 1. Biological cycle of A. simplex.

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condition is chronic, the formation of gastric or intestinal abscesses or granulomas [16] can mimic the symptoms of intestinal pseudo-obstruction or acute appendicitis [17,18]. Extra-digestive symptoms have been found related to the penetration of the larva into the digestive tube wall and its migration to the lung, liver or other organs [19]. The larva of A. simplex can also cause an immediate allergic reaction, resulting in systemic signs ranging from urticaria or angioedema to anaphylactic shock [20,21]. Cases of occupational disease (asthma and conjunctivitis) caused by sensitisation to A. simplex have been described [22,23], as well as contact dermatitis [24] and arthralgia after exposure to the parasite [25]. The most common clinical history in these patients is the presence of symptoms a few minutes or several hours after eating raw fresh or under-cooked fish. Likewise, they report a good tolerance to the fish involved other times. When the allergology study is performed, they show positive skin tests and specific IgE against A. simplex with a marked increase in total serum IgE [26,27]. Some patients have mixed symptoms of urticaria and/or angioedema (allergic symptoms) associated with abdominal pain (epigastralgia, vomiting, etc.), described as gastroallergic anisakiasis [28]. In this situation, the digestive symptoms usually occur before the allergic symptoms with a mean latency time of 3 and 5 h, respectively, from the intake of the parasited fish. The blood count is usually normal, though WBC count can show leukocytosis with neutrophilia and not very severe eosinophilia, in some cases with a later increase in eosinophil levels after 24 h from when the onset of the clinical condition. The biochemical measurements are not usually affected, unless they are secondary to complications such as pseudo-obstruction (repeated vomiting). The serum levels of eosinophil cationic protein can be very high, though this measurement is not commonly used [29]. Diagnostic imaging tests are not usually necessary. In intestinal conditions, ultrasounds would be of choice, though the signs observed are unspecific: thickening of the intestinal wall, free fluids, stenosis of the lumen and reduced peristalsis [29].

4. Antigens and allergens in A. simplex Three groups of antigenic molecules in A. simplex are known [30]: • Somatic antigens. These are the most abundant, with a molecular weight (MW) of between 13 and 150 kDa. They include proteins of the neoglucogenesis pathway and of the fatty acid synthesis. Some of these proteins, the biotinised ones, show cross-reactivity with other ascarid proteins. These antigens are obtained by homogenisation of the whole larvae and they contain all the parasite’s soluble proteins [31]. • Excretory–secretory (ES) antigen. This is synthesised in two structures, the dorsal oesophageal gland and the diges-

tive tract secretory cells, which are the largest source of histolytic enzymes (with proteolysis and hyaluronidase activity). These molecules help the parasite to penetrate the gastric mucosa and can degranulate mast cells in sensitised mice. The antibodies against these antigens are the first to appear [32]. This antigenic complex can be obtained by incubating the live L3 larvae in an adequate culture medium for long time periods, in which it is released by the larvae [31,33,34]. There are several MWs of the SE antigen proteins, but it has been demonstrated that low-MW proteins (14, 17 and 18 kDa) are only recognised by the serums of mice infected with the live larva of A. simplex. A possible explanation is that the low-MW antigens are only produced when the larva is alive [35]. In recent studies it has been shown that the larva L3 of A. simplex incubated in a diluted acid medium release a substantial amount of proteins [36]. • Surface antigen. This corresponds to molecules expressed in the parasite’s cuticle, that are also found in other nematodes. This antigen is expressed when the larva evolves from L3 to L4. Although it has been suggested that they are less antigenic and specific than the excretory–secretory antigen and the somatic antigen, recent studies have shown that they are a source of many proteins recognised by the antibodies of the infected mouse. These molecules can play an important role in the development of a chronic stimulus, such as in the case of granulomas [37]. Both in SDS-PAGE as in immunotransfer techniques, the surface and excretory–secretory antigens behave in a very different way, depending on whether they are in the third or fourth stage of the larval instar phase, suggesting a high stage specificity. On the contrary, the somatic antigen keeps a similar band pattern at both stages, which indicates that it is preserved during the development of the parasite [38]. At least four antigens have been identified with allergenicity (main allergens) [39] that includes products with a relatively low-MW. Ani s 1 has a MW of 24 kDa and has been found in the excretory gland, forming the abovementioned SE antigen. This protein is not recognised by the serum in asymptomatic patients with a positive skin and specific lgE test against the whole body antigen of A. simplex [40]. Ani s 2, also called paramyosin, is found in the larva’s body and has a MW of 97 kDa. Ani s 3 is a tropomyosin with a MW of 41 kDa. Ani s 4 is a low-MW allergen (9 kDa) from the body of the larva. It is resistant to heat and to pepsin, which could account for the symptoms appearing after eating wellcooked or canned fish.

5. Cross-reactivity with other allergens By means of studies on CAP inhibition and immunotransfer inhibition, the tropomyosin in A. simplex has been related to the cross-reactivity shown by this parasite with other arthropods such as Blatella germanica and Chironomus spp. Larvae [41]. It would also be related to cockroach, shrimp and

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mites, mostly Acarus siro and Tyrophagus putrescientae [42,43]. In different studies based on ELISA and immunotransfer techniques, the somatic antigen has been considered responsible for the existence of cross-reactivity between A. simplex and other nematodes like Ascaris suum, Toxocara canis, Hysterothylacium aduncum, Trichinella spiralis and Trichuris muris [44,45]. Enzymes with biotin group, contained in A. simplex somatic antigen extracts, are also present in other nematodes [46]. The carbohydrates from the A. simplex extract used for the diagnosis may account for the false positive diagnostic tests. The cross-reactivity existing between the whole body antigen of A. simplex and other allergens makes it necessary to use other antigens for performing skin tests and for measuring specific IgE to reduce the high rate of false positives. Previous treatment with periodate (a substance that destroys the carbohydrate structures) makes a mean MW band, recognised in immunotransfer by asymptomatic subjects, disappear [47]. Other authors, using monoclonal antibodies, have obtained the same results. On eliminating the o-glycan epitopes for monoclonal UA3, an antibody that recognises two proteins of A. simplex with MWs of 139 and 154 kDa, the problem of cross-reactivity almost disappears [48]. Pascual et al., through immunotransfer, compared the diagnostic value of the SE allergen of A. simplex in relation to the allergen of whole body in patients with specific IgE against A. simplex. They reported that only the IgE of patients with gastroallergic anisakiasis recognise the protein bands corresponding to the SE antigen [49]. These data show that the SE antigen is ideal for use in diagnosing allergy to A. simplex, increasing the specificity of the diagnostic technique. Serial measurements of total IgE and specific IgE against A. simplex can be useful for diagnosing acute parasitation. Daschner et al. [50] measured both parameters 24 h and 1 month after the reaction, and found an increase in the total IgE in 85.36% of the patients and in the specific IgE in 90.24% of them. In 89% of the patients with a positive gastroscopy, there was an increase of total and specific IgE (P = 0.02), and the latter was higher than in patients with non-parasite gastroscopy.

6. Bases for prevention The allergenicity of A. simplex extracts subject to heat is reduced, but even those treated at 100 °C can show some capacity for binding IgE. Low-MW allergens are those best resisting high temperatures [51]. When the fish is frozen, the larva of A. simplex dies. Based on this, several authors have performed challenge tests with frozen fish on patients allergic to A. simplex, and they all had a good tolerance [52]. However, other authors report that up to 10% of the patients developed symptoms after eating frozen fish. Baeza et al. studied the 10-month evolution of 58 patients allergic to A. simplex, dividing them into three groups: one with a fish-free

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diet, another eating only frozen fish, and a third group that could eat as much fresh fish as they wanted. Sixteen percent and 5% of the patients on the fresh fish diet and frozen fish diet, respectively, developed symptoms of urticaria, though in the group of patients on a strict frozen fish diet, the difference was statistically significant from the group on a fishfree diet. Total IgE levels did not change in the group eating fresh fish, and these levels decreased by 57% and 45%, respectively, in the patients whose diet included frozen fish and in those not eating fish [37]. The type of diet that the patient allergic to A. simplex must follow is very important, for the fish-free diet may be problematic in patients with associated conditions, particularly those with dyslipidemia. Diet advice focuses on not eating raw or under-cooked fish, freezing the fish (–20 °C) for at least 48 h, and recommending frozen-at-sea or ultra-frozen fish for it is gutted early and the possibility of muscle parasitation is lower. The regulations applicable to fish products vary. Dutch health authorities established previous freezing of fish products for human consumption with no previous thermal treatment, obtaining a dramatic drop in the number of cases per year. This regulation is currently in force in the European Union, though it is not followed in many cases. The EU regulations [53] set out that fish to be consumed raw or almost raw must be frozen to –20 °C or less, throughout the entire product for at least 24 h. The larvae of Anisakis are sensitive to heat. They are inactivated in fish at 55 °C in a time period ranging from 10 to 60 s, and at 60 °C from 0.5 to 1 s [54]. In general, it is considered that a treatment of 60 °C in the centre of the product for 1 min is sufficient to kill the larvae, but it is recommended to reach at least 70 °C in the centre of the fish [54]. In Spain, before it is dispatched for human consumption, fish and fish products must be inspected visually for detecting and removing visible parasites, and products with parasites must not be placed on the market [53,55].

7. Anisakis in the children population As previously mentioned, age is a risk factor for sensitisation to A. simplex. There are few studies on the implications of A. simplex in the early years of life. Bernardini et al. performed a study in children referred to an allergy clinic, and a sensitisation of 6.1% was found. The existence of a positive skin test does not seem to indicate symptoms after eating fish with parasites. After adjusting for other variables, they found that sensitisation to A. simplex is significantly associated with skin tests that are positive to A. tennis, soy, seafood and mites [56]. In another study reporting a prevalence of 11% [57], it was concluded that sensitisation to A. simplex in children is generally asymptomatic, though there is a strong association with episodes of acute urticaria in those with a skin test positive to the parasite. The results of this study show the possi-

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bility of cross-reactivity with other nematodes such as Ascaris lumbricoides, as in seven of the patients with a skin test positive to A. simplex, specific IgE against this parasite was found. In a study on the prevalence of sensitisation to nematode allergens in paediatric patients, high values were surprisingly found [58]. Fifty-six percentage of the patients studied (41/73) had specific IgE against A. simplex and 65% against A. lumbricoides. The highest sensitisation rates to A. simplex were found in patients with immediate hypersensitivity to crustaceans, which could be due to their tropomyosin content.

8. Conclusions The condition caused by A. simplex affects a large part of the world population due to is wide geographical distribution, and to the fact that it involves a first-rate food product like fish. In children, the prevalence of anisakiasis is lower, possibly due to the fact that it is very uncommon for children to eat raw fish. The clinical signs caused by it show a wide range of symptoms that can be mild or lead the patient to an extremely serious situation. The excretory–secretory antigen has been shown to be the best antigen for diagnosis, as it shows no cross-reactivity with other allergens. In spite of the different criteria that still exist among the different groups studying this condition, freezing the fish seems to be the best alternative to prevent the appearance of symptoms. Finally, it must be stressed how important it is to adopt the necessary measures for compliance with the current regulations on the consumption of raw fish.

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