Original Article

Anisakiasis, an Underestimated Infection:Effect on Intestinal Permeability

of Anisakis simplex—Sensitized Patients

Lorenzo Polimeno,1 Mariangela Loiacono,1 Barbara Pesetti,1 Rosanna Mallamaci,2

Maria Mastrodonato,3 Alessandro Azzarone,4 Emanuele Annoscia,1 Francesco Gatti,1

AnnaCinzia Amoruso,1 and Maria Teresa Ventura5

Abstract

Anisakis simplex is a parasite that, if present in uncooked and contaminated saltwater fish, can invade the humangut. Two different clinical situations are recognized: the first, known as a gastrointestinal disease, varying froman asymptomatic episode to vomiting and diarrhea, and the second, classified as an adverse reaction to food,characterized by a wide spectrum of allergic reactions like rhinitis, conjunctivitis, or even anaphylaxis causinghypotension and=or shock. The intestinal epithelium, the major defense system against external molecules, rep-resents an open gate for toxins and allergens if its protective function is compromised. Previous data havedemonstrated a strict relationship between an altered intestinal permeability (I.P.) and worsening of the clinicalmanifestations in patients with adverse reactions to the food. In this article we evaluated the sensitization toA. simplex among patients who referred clinical symptoms of allergy. All subjects underwent commonly usedalimentary skin prick test for food allergens, to which Ani s1, an A. simplex allergen, was added. In addition, inA. simplex–sensitized subjects, I.P. was determined upon their enrolment to the study (time 0) and after 6 monthsof consuming a raw fish-free diet (time 6). Five hundred and forty subjects were screened, and 170 had a positiveskin prick test, 87 (51.2%) of whom were positive to Ani s1. Increased I.P. was evidenced in A. simplex–sensitizedsubjects with worse clinical symptoms, which receded after 6 months’ elimination of raw seafood. With our datawe demonstrated that the alimentary habit to eat raw fish represents a high risk for the integrity of the intestinalmucosa, and we suggest that this pathological situation may constitute an ideal, under-estimated, open gate formolecules that predispose to other, more important pathologies.

Introduction

Anisakis simplex is a nematode parasite of which thethird-stage larvae, mainly located in the viscera and mus-

culature of adult sea fish, such as cod, tuna, salmon, pilchard,and anchovy (Audicana et al., 2002; Audicana and Kennedy,2008), accidentally infect humans when they eat raw or under-cooked seafood. The resulting disease, known as anisakiasisor anisakidosis, is usually an acute and transient infestation(Muraoka et al., 1996; Montalto et al., 2005; Audicana andKennedy, 2008). In humans the larvae usually infest the sto-mach and the intestine (Takei and Powell, 2007).

Eating raw or undercooked fish (such as sushi and sashimiin Japan, marinated anchovies in Italy, boquerones en vinagre

in Spain, and green herring in Germany) is becoming morecommon all over the word, causing a progressive increase ofAnisakis infestation (Gomez et al., 1998; Purello-D’Ambrosioet al., 2000). Between the 1960s and 1997 in Japan, about 32,300cases of anisakiasis were recorded, representing more than 90%of the cases recorded in all countries (Audicana and Kennedy,2008); for this reason, this parasitic disease is considered en-demic in Japan. The remaining 10% of the cases is distributedamong the Netherlands, Germany, France, Spain, and Italy.

The diagnosis of anisakiasis is obtained through anam-nestic data and needs to be considered especially in patientspresenting with abdominal pain who had consumed raw orundercooked seafood in the past 72 hours. Two different cli-nical situations of anisakiasis are recognized:

1Section of Gastroenterology, Department of Emergency and Organ Transplantation (DETO), University of Bari, Bari, Italy.2Pharmaco-Chemical Department, School of Pharmacy, and 3Department of Animal and Environmental Biology, University of Bari, Bari, Italy.4Unit of Gastroenterology, Ospedale ‘‘S. Paolo,’’ Bari, Italy.5Department of Internal Medicine, Immunology and Infectious Diseases (MIDIM), University of Bari, Bari, Italy.

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1. The patient presents with gastrointestinal symptoms thatmay vary from a mild gastroenteritis to sharp abdomi-nal pain, vomiting, and diarrhea, corresponding to acuteabdominal syndrome (Pellegrini et al., 2005).

2. The clinical manifestation is mainly determined by IgE-mediated reactions (Ventura et al., 2008), wherebypatients, sensitized by previous contact with A. simplexantigens, present allergic reactions like rhinitis, asthma,or even anaphylaxis (Del Pozo et al., 1997; Daschneret al., 1998; Domınguez Ortega et al., 2000; Audicanaet al., 2002; Daschner and Pascual, 2005; Barbuzza et al.,2009). Usually it is diagnosed by skin prick test (SPT)that includes Ani s1 allergen (Lopez Penas et al., 2000),and serum-specific IgE determination against Ani s1(radioallergosorbent tests) (Arilla et al., 2008).

Of particular interest are the data reported by Armentia et al.(2006), who referred that subjects who are sensitive forA. simplex had allergic symptoms after the consumption ofchicken meat. They hypothesized that sensitization to A. sim-plex might be related to the presence of raw fishmeal in thechicken diet. Indeed, A. simplex–sensitized subjects who con-sumed chicken meat fed only with cereals did not refer al-lergic symptoms.

In various countries, specific studies have been performedto detect the relevance of anisakiasis, testing specific IgEagainst A. simplex. In Japan it has been shown that specific IgEagainst A. simplex is present in 33% of the patients with atopicdermatitis, 75% of the patients with urticaria, and 10% of thehealthy control subjects (Lindqvist et al., 1993).

In Spain studies carried out on a healthy population, eval-uating the prevalence of specific IgE antibodies against Ani s1,have reported conflicting data (elevated IgE serum levelsranging from 6.6% to 27.5% of subjects) (Del Pozo et al., 1996;Fernandez et al., 1997; Garcıa et al., 1997; Audicana et al., 2000;Purello-D’ambrosio et al., 2000).

Buendia and coworkers referred the presence of A. simplex–specific IgE in 23% of the healthy control subjects, in 36% ofthe patients with mixed symptoms like urticaria and=or an-gioedema, and in 56% of children with elevated total serumIgE (Buendia, 1997).

Food represents the major cause of intestinal antigenicchallenge during lifetime. The intestinal epithelium with ahealthy, full-grown mucosa and intact epithelial tight junc-tions represents the major defense organ against externalantigens, toxins, and macromolecules that reach the intestinallumen. When this defensive system is compromised, the in-testinal permeability (I.P.) becomes altered (Ventura et al.,2006).

In the early 1980s, Andre and collaborators (1987), focusingon I.P. evaluation in subjects reporting IgE-mediated reac-tions, reported an increase of I.P. after food allergen ingestion.We recently demonstrated that patients with allergic reactionsto food manifest an altered I.P. (Ventura et al., 2006).

To our knowledge, sporadic data are present in literaturereferring sensitization to A. simplex in Italy. Mainly the au-thors refer A. simplex clinical cases accidentally observed inthe operative room or during gastrointestinal endoscopy(Montalto et al., 2005; Foti et al., 2006; Biondi et al., 2008), andprobably some cases may not have been diagnosed becausethe infestation may have been asymptomatic or had healedspontaneously. For this reasons we believe that it could be

important to determine the prevalence of anisakiasis in a re-gion of Italy where the habit to eat raw seafood is common.

In this article we report the data obtained by evaluatingpatients with clinical symptoms of adverse reaction to food.A. simplex sensitization was determined by alimentary SPTand, when possible, serum-specific IgE levels were measured(Lopez Penas et al., 2000). In addition, the degree of I.P. inA. simplex–sensitized patients was determined before (time 0)and after a 6-month period of raw fish-free diet (time 6).

Materials and Methods

Subjects and study protocol

The study was carried out on 540 subjects who attended theHealth Centre of the Allergy Department of the University ofBari referring an adverse reaction to food. These patients weretested with common alimentary SPT to which Ani s1 (Alk-Abello, Horshomlm, Denmark) was added. Ani s1 is a majorallergen of A. simplex and indicates nematode infection and isuseful for the diagnosis of Anisakiasis (Moneo et al., 2000;Valls et al., 2003; Kobayashi et al., 2008). Specific IgE serumlevels determination, when possible, were evaluated (Del ReyMoreno et al., 2006; Arilla et al., 2008). The medical history ofthe allergic symptoms, the correlation with the consumptionof raw or undercooked seafood, the quantity of seafood in-gested, and the time between ingestion and symptom onsetwere examined.

Fifty healthy volunteers, matched for age and sex, selectedfrom the medical staff (19 men and 31 women; mean age40 years; range 18–70 years), with no symptoms or signs ofgastrointestinal disease, for at least 2 weeks, were included ascontrol. All subjects revealed negative allergic tests to food(alimentary SPT) and did not use raw or undercooked fish intheir diet.

The study protocol was approved by the Ethics Committeeof the Azienda Ospedaliero-Universitaria ‘‘Policlinico Hospi-tal’’ of Bari. All the participants provided their written in-formed consent.

As stated by the European Academy of Allergology andClinical Immunology position paper on IgE-mediated foodallergy, the diagnosis of food allergy was confirmed by pos-itive SPT (European Academy of Allergology and ClinicalImmunology, 1989) and by radioallergosorbent tests (Paga-nelli et al., 1998), which were determined in patients upontheir enrolment to the study (time 0). In Ani s1þ SPT patients,I.P. was determined at the beginning of the study (time 0) and,when I.P. was altered, after 6 months of raw or undercookedfish-free diet (time 6). Further, all subjects from the controlgroup were tested for I.P. at the time of their enrolment to thestudy (time 0).

Allergological tests

Skin prick test. A positive control (histamine, 10 mg=L)and a negative control (glycerinated saline) with a panel offood allergens (tuna, salmon, mackerel, sardine, shrimp, andoctopus), inhalants (Dermatophagoides pteronyssinus, Acarussiro, Lepidoglyphus destructor, Tyrophagus putrescentia), andA. simplex, Ani s1, allergen (Alk-Abello) were tested and in-terpreted according to the European Academy of Allergologyand Clinical Immunology Subcommittee on Allergen Stan-dardization and Skin test. If the diameter of the reaction was

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more than 3 mm than the one provoked by the negative con-trol, the response was considered positive. SPT, similarly tospecific IgE serum level in vitro determination, is considered ahighly sensitive method for the diagnosis of A. simplex sen-sitization (Lorenzo et al., 2000).

Radioallergosorbent tests. UniCAP system (Pharmacia,Uppsala, Sweden) was used to detect specific IgE serum levelsagainst A. simplex, considering positive any value higher than0.35 kUA=L (Del Rey Moreno et al., 2006; Arilla et al., 2008).

I.P. test

I.P. has been determined as previously described in litera-ture (Generoso et al., 2003; Ventura et al., 2006). Briefly, thesubjects fasted overnight and then ingested 200 mL of tapwater containing 2 g of Mannitol (Ma) and 5 g of Lactulose(La). Urine samples were collected before ingestion of theprobes to ensure their endogenous absence, and during thefollowing 5 hours. Anion-exchange chromatography on eachurine sample was performed, the percentage of ingested Laand Ma evaluated, and their ratio (La=Ma) calculated. EachLa=Ma value has been multiplied by 100 (detected La=Mavalue�100). Each sample was analyzed three times.

Statistical analysis

The data of I.P. are been reported as arithmetic mean�standard deviation (AM� SD). Student’s t-test for unpairedvalues was used to compare the data obtained from the dif-ferent groups of subjects. The correlation between the severityof the symptoms and the La=Ma ratio levels was evaluated bythe rank correlation test for nonparametric data (Spearmantest). A p< 0.05 was considered statistically significant.

Results

Table 1 reports the alimentary SPT data. Five hundred andforty patients were screened, and the test result of 370 patientswas negative (SPT�). Among the 170 STP-positive subjects(SPTþ), the result of 87 (51.2%) was Ani s1þ and 83 (48.8%)Ani s1�.

Table 2 reports the detailed allergen positivity of 87 Ani s1þ

SPT patients. Sixty three subjects revealed the sensitizationonly to Ani s1, whereas 24 patients resulted sensitized byother food-derived allergens besides Ani s1. Fifteen, amongthe 24 multifood-derived allergen SPTþ patients, showed sen-sitization to seafood-derived allergens from intermediatesparatenic hosts of A. simplex life cycle.

In 38 of 87 A. simplex–sensitized patients specific IgE wereevaluated in serum samples drawn at the time of theirenrolment (time 0). In 21 subjects (55%) high levels of Ani s1–specific IgE (28.01� 7.84 kUA=L) were detected; the remain-ing 17 patients showed normal Ani s1–specific IgE values(0.15� 0.08 kUA=L).

Table 3 shows the characteristics and the clinical symptomsof Ani s1þ SPT patients. The mean age of patients was 44 years(range 14–75 years); male patients were almost half of thefemale patients. The clinical manifestations of these patientsrange from simple pruritus (Grade 1 group) to angioedemaand anaphylaxis (Grade 4 group).

Figure 1 reports the age distribution of the 87 A. simplex–sensitized patients collected by decades. Most of the subjectsaged 30–50 years, 6.89% aged less than 20 years, and morethan 35% were older than 50 years.

Figure 2 reports La=Ma ratio determined in control subjectsand A. simplex–sensitized patients divided into four groupsfollowing the clinical manifestations as referred at time 0 andreported in Table 1. I.P. levels of normal control subjects(La=Ma¼ 1.73� 1.12, AM� SD) were similar to that alreadypresent in the literature (Generoso et al., 2003; Ventura et al.,2006). The data from the A. simplex–sensitized patients,stratified according to the clinical symptoms (Table 3), re-vealed a progressive increase in the La=Ma ratio, in the groups1 and 2 (La=Ma: 2.5� 0.9 and 2.43� 0.88, respectively,AM� SD), not statistically different when compared to thecontrol group value; on the other hand, I.P. from people ofgroups 3 and 4 was found to be statistically higher (AM� SD)when compared to normal control subjects (for group 3:

Table 1. Classification of Skin Prick Test–Positive

Patients According to Their Ani s1 Sensitization

Skin prick testNumber of

patients (%)

Alimentary allergensSPT-screened patients

540

Alimentary SPT� patients 370=540 (68.5)Alimentary SPTþ patients 170=540 (31.5)Anisakis simplex SPTþ 87=170 (51.2)A. simplex SPT� 83=170 (48.8)

Among the 170 alimentary STPþ patients, 87 resulted Ani s1 sen-sitized.

Table 2. Classification of Ani s1-Sensitized

Patients According to the Seafood-Related

or No-Seafood-Related Allergens Source

Tested allergensNumber of

patients (%)

Ani s1þ SPT patients 87 (100)Only Ani s1þ SPT patients 63=87 (72.4)Multifood-derived allergens SPTþ patients 24=87 (27.6)

Ani s1 and other seafood allergens(cod=tuna, shrimp=lobster, mussel=oyster)

15=24

Ani s1 and other no-seafood allergens 9=24

Among the 87 Ani s1-sensitized patients, 78 showed SPT positiveto Ani s1 alone or to other seafood-derived allergens, beside Ani s1.

SPT, skin prick test.

Table 3. Clinical Finding in Ani s1þ Skin Prick

Test Patients

Patients and clinical finding

Age, years (mean age; range) 44; 14–75Male:female (number of patients) 27:60Clinical symptoms of Ani S1þ SPT

patients (total number and %)87 (100%)

Grade 1: pruritus 18 (21%)Grade 2: pruritus and urticaria

and=or rhinitis28 (32%)

Grade 3: pruritus, generalized urticariaand diarrhea and=or asthma

28 (32%)

Grade 4: angioedema and=or anaphylaxis 13 (15%)

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La=Ma¼ 5.66� 1.06, p¼ 2.6�10�9, normal group vs. group 3;for group 4: La=Ma¼ 10.83� 8.6, p¼ 1.63�10�7, normal groupvs. group 4).

Figure 3 reports the correlation analysis between I.P. datafrom A. simplex–sensitized patients and the severity of theclinical symptoms referred by patients at time 0. The signifi-cant relationship (r¼ 0.7069) between these two parametersdemonstrates that worse allergic symptoms are strictly re-lated to higher I.P. levels.

In Figure 4, I.P. levels of A. simplex–sensitized subjects,evaluated at time 0 and after a 6-month period of raw fish-freediet (time 6), are reported. The data of patients from groups 3and 4 were contemporary considered since, at time 0, La=Mavalues were significantly higher than normal control subjects(Fig. 2). Taken together, the data obtained at time 0 (groups 3and 4: La=Ma¼ 7.3� 5.4; AM� SD) confirmed the statistical

difference ( p¼ 1.5�10�7), as compared to normal controlsubjects (La=Ma¼ 1.73� 1.12; AM� SD). I.P. evaluated in thesame patients at time 6 revealed a statistically significant( p¼ 6.5�10�8) reduction (La=Ma¼ 1.81� 0.99; AM� SD).

Discussion

In this article we (1) report the degree of A. simplex sensi-tization evaluated in a population of Italy, where the use ofraw or undercooked saltwater fish is frequent, and (2) dem-onstrate that A. simplex–sensitized subjects have altered I.P.integrity, which returns to normal values when seafood is notincluded in their diet.

The present analysis has been conducted evaluating thecommonly used allergens for alimentary SPT and Ani s1, the

FIG. 1. Anisakis simplex–sensitized patients. About 87 pa-tients (age range 14 to 75 years) positive to Ani s1 antigenwere grouped and divided by decades. The most represen-tative class was between 40 and 50 years old even if 6.89%was younger than 19 years and more than 33% older than 50.

FIG. 2. Levels of La=Ma urinary excretion of A. simplex–sensitized patients grouped by severity of clinical symptoms.The intestinal permeability (I.P.) of A. simplex–sensitizedpatients grouped according to the clinical symptoms wascompared to normal control group subjects. A significantalteration of La=Ma excretion ratio was detected in thosepatients who referred generalized urticaria, diarrhea, asthma(group 3), or angioedema and anaphylaxis (group 4).

FIG. 3. Correlation between La=Ma urinary excretion ofA. simplex–sensitized patients and clinical symptoms. Therank correlation test for nonparametric data applied to theLa=Ma urinary ratio from A. simplex–sensitized patientsshown to be strictly related to the severity of the clinicalsymptoms referred by the patients.

FIG. 4. Effect of raw-fish-free diet on La=Ma urinary ex-cretion of A. simplex–sensitized patients. I.P. was measuredafter 6 months of raw-fish-free diet (time 6) in A. simplex–sensitized patients who presented altered La=Ma urinaryexcretion at time 0. A statistically significant reduction wasobserved, reaching the normal value, showing a comeback ineach patient to the I.P. normality (hatched line).

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major allergen of A. simplex, an indicator of true nematodeinfestation and useful for the diagnosis of anisakiasis withoutcross reactions to other allergens (Moneo et al., 2000; Vallset al., 2003; Kobayashi et al., 2008).

The data reported indicate that 31.5% of the subjects testedby alimentary STP resulted positive; among these, 51.2% werepositive to Ani s1 (Table 1). More dramatic are the data re-porting that 63 (72.4%) of A. simplex–sensitized patientsshowed SPT positivity to Ani s1 allergen alone (Table 2).

Of interest are the data obtained by analyzing theA. simplex–sensitized patients grouped into decades: morethan one-third of the subjects were older than 50 years and,when questioned, did not know to be allergic to the A. simplex.It is known that A. simplex sensitization increases with age inpatients who consume frequently raw fish, but in our analysiswe believe that such patients in previous alimentary SPT testshad been diagnosed as ‘‘not allergic,’’ even though the an-amnestic clinical symptoms of allergy (pruritus, urticaria,and=or rhinitis) suggested for an allergic syndrome. We be-lieve that the presence of Ani s1 antigen in the SPT performedin the present study, and absent in the previous examinations,gave us the opportunity to reveal, for such patients, the causeof their clinical symptoms. In addition with our data we in-directly demonstrated that anisakiasis is, probably, an under-estimated infection.

As described in Figure 1, young people were also presentin the study, which is not surprising, considering the habit toconsume raw or undercooked seafood in this part of Italy.

A. simplex–sensitized patients were also evaluated for in-testinal mucosa integrity (Generoso et al., 2003; Ventura et al.,2006) at their enrolment to the study (time 0). Only the sub-jects referring worse clinical symptoms (Table 3, groups 3 and4) showed elevated I.P. values (Figs. 2 and 3). As alreadyreported in literature, other forms of adverse reactions to food(Ventura et al., 2006) showed a significant correlation betweenthe severity of the clinical symptoms and the La=Ma urinarylevels (Fig. 3). I.P. levels investigated in the same subjects after6 months of a raw or undercooked saltwater fish exclusionfrom the diet (time 6) revealed a statistically significant re-duction of La=Ma urinary ratio, reaching the normal-groupvalues (Fig. 4). This datum seems to be in contrast with thedemonstrated thermostability of A. simplex allergens (Audi-cana and Kennedy, 2008). Data from literature demonstratedthat Ani s1 allergen, in particular, seems to be highly resistantto heat (Caballero and Moneo, 2004; Shimakura et al., 2004)and that an A. simplex homologous allergen, present in As-caris, maintains its allergenicity even after high-temperaturedenaturation and consequent cooling (Christie et al., 1993;Kennedy et al., 1995; Xia et al., 2000). Many could be the in-terpretations of such apparent discordance with the literature:first of all, it is important to point out that most of the subjectswho underwent I.P. determination after the diet referred atotal elimination of seafood from their diet. On the other hand,those subjects who did eat seafood cooked it for more than10 minutes at 608C. It should be point out that I.P. becamenormal when the clinical symptoms were not present anylonger, as referred by the patients, suggesting that the re-covery of I.P. may be related to and determined by the samephysiological events still not totally investigated and, proba-bly more important, the proteins responsible for the allergenicsensitizations could be different of those which regulate theintestinal integrity.

In conclusion, our findings strongly support the necessityto eliminate raw or undercooked saltwater food from thediet, which, besides inducing allergenic reactions, may alsodetermine a leak in the intestinal mucosa, which often is un-recognized and it can predispose to other, more importantpathologies.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Lorenzo Polimeno, Ph.D.

Section of GastroenterologyDepartment of Emergency and Organ Transplantation (DETO)

University of BariPiazza G. Cesare, 11

70124 BariItaly

E-mail: l.polimeno@gastro.uniba.it

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