CONCISE COMMUNICATION

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1 CONCISE COMMUNICATION Measurement of serum levels of eosinophil cationic protein in the diagnosis of acute gastrointestinal anisakiasis J. Domínguez-Ortega 1, J. C. Martínez-Alonso 1, A. Alonso-Llamazares 2, C. Argüelles-Grande 3, M. Chamorro 2, T. Robledo 2, R. Palacio 2 and C. Martínez-Cócera 2 1 Allergy Unit, Hospital Virgen de la Concha, Zamora, 2 Allergy Service, Hospital Clínico San Carlos and 3 Primary Care Service, Madrid, Spain Tel: þ jdort@mixmail.com Thirty-two patients with abdominal pain and/or intestinal pseudo-obstruction who had consumed raw or undercooked fish in the previous 72 h, were included in a study of anisakiasis, a parasitation of the human gastrointestinal tract by third stage Anisakis simplex larvae. Skin prick test (SPT) against A. simplex were positive in all the patients. High median eosinophil cationic protein (ECP) serum concentrations (> 15 mg/l) at day 0 with normal serum levels at day 30 and a rise of median total and specific IgE against A. simplex at day 30, were observed. We conclude that a raised serum level of ECP in the first 72 h from the onset of symptoms coinciding with a positive SPT against A. simplex and high total and specific immunoglobulin (IgE) in the first month after the parasitation, could be a useful tool in the diagnosis of gastrointestinal anisakiasis, even if the parasite cannot be isolated. Keywords Anisakiasis, Anisakis simplex, eosinophil cationic protein, IgE, intestinal obstruction Accepted 20 October 2002 Clin Microbiol Infect 2003; 9: INTRODUCTION Anisakiasis, a parasitation of the human gastrointestinal tract by third stage Anisakis simplex larvae, is an emerging disease, especially in areas where fish is often eaten raw or undercooked. Since 1960 when Van Thiel [1] identified this parasite in the Netherlands, a great number of cases have been reported in Japan [2], the country with the highest rate of fish consumption in the world, and more recently in Spain and other Mediterranean countries [3 5]. The parasitic infestation is characterized by the presence of symptoms that mimic other gastrointestinal disturbances such as gastric ulcer or intestinal motility disorders [6]. Unfortunately, the larva is not often isolated and the diagnosis depends essentially on clinical findings and indirect complementary diagnostic tools the presence of a skin prick test (SPT) such as to the parasite or raised serum levels of total immunoglobulin E (IgE) and specific IgE against A. simplex in the first 30 days after infection [7]. Eosinophilic infiltrations are present in the tissue surrounding the larvae [8]. However, eosinophilia of the peripheral blood is not often observed [9] and another diagnostic method to assess eosinophilic activation is required. Activated eosinophils release eosinophil granule proteins such as eosinophil cationic protein (ECP). ECP is released in response to any eosinophil activation [10]. Consequently, measurements of serum levels of ECP in the first hours from the onset of the symptoms, could provide additional information on the activated eosinophilic response when acute parasitation by A. simplex is being considered. METHODS Thirty-two patients (age range years), with no significant allergic background, who had come to the emergency service complaining of abdomß 2003 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 454 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003 inal pain and/or intestinal motility disturbance, were included in the study. All had eaten raw or undercooked fish in the previous 72 h. They were put on a fish-avoidance diet. SPT for A. simplex (IPI, Madrid, Spain) and implicated seafood extracts (CBF.SA. LETI, Barcelona, Spain) were performed on the volar side of the forearm. Saline solution (0.9%) and histamine hydrochloride (10 mg/ml) were used as negative and positive control tests, respectively. Immediate responses were evaluated at 15 min. A wheal diameter of at least 3 mm was considered positive according to the European Academy of Allergy and Clinical Immunology criteria. Serum levels of total IgE, specific IgE, and serum ECP concentration were determined within 72 h of the onset of symptoms (day 0) and after 1 month (day 30). Total serum IgE, specific IgE against A. simplex, Ascaris lumbricoides, Echinococcus granulosus and the implicated fish were assessed by the CAP radio-immunoassay technique according to the manufacturer s instructions. (Pharmacia- Upjohn, Uppsala, Sweden). CAP scores greater than 0.35 Ku/L were considered positive. The concentration of ECP was titrated with a double antibody fluoroimmunoassay according to the manufacturers (Pharmacia-Upjohn). Median values were calculated for ECP, total and specific IgE, and the Wilcoxon match-pairs signed rank test was used for comparison of ECP, total and specific IgE in the emergency room and at day 30. RESULTS Most of the patients had periumbilical abdominal cramps accompanied by nausea and, less frequently, vomiting without urticaria or angiedema. Symptoms had begun between 6 and 24 h prior to consultation and no signs of peritoneal irritation were observed on abdominal palpation. There were no significant findings in the rest of the physical examination. Leukocytosis was observed (in 23 of the patients), mild eosinophilia (12 patients), and increasing eosinophil counts (3 patients). Plain abdominal X-rays showed small intestine loop dilation and fluid-air levels. Ultrasonography showed intestinal wall thickening and free fluid. A previous intake of anchovies in vinegar sauce was implicated in 19 of the patients, and hake or herring in the other 13 patients. SPT with A. simplex extract were positive, and negative for fish allergens in all the patients. A fiberoptic gastroscopic examination was only performed in nine of the patients. Five of them had normal findings with a live nematode identified as an A. simplex larva in the others. Two patients suspected of having acute appendicitis were operated on, and the biopsies were reported as compatible with eosinophilic enteritis. Moreover, an A. simplex larva was identified in one of the biopsies [Figure 1]. Patients improved within 48 h as a result of fluid therapy and ranitidine. Median values of ECP, total and specific IgE against A. simplex at day 0 and day 30 can be seen Figure 1 Small intestine showing edema, congestion and polymorphonuclear leukocyte and eosinophil infiltrate. An Anisakis simplex larva is observed.

3 Concise Communication 455 Table 1 Median and interquartile ranges for serum eosinophil cationic protein (ECP), total and specific IgE against A. simplex at day 0 and day 30. Specific IgE augmented in 31 patients and decreased in one. No differences were observed between those patients with confirmed presence of the larva and the rest of the cohort Time ECP (mg/l) in Table 1. Serum-specific IgE levels to fish and other parasites were negative in 27 of the patients. Non-significant values (CAP class < 2) for specific IgE to Ascaris lumbricoides were found in the other five cases at day 30, and these values were always lower than those found for A. simplex-specific IgE. No parasites were found in either of the two stool samples analyzed per patient in these five cases. DISCUSSION Total IgE (ku/l) Specific IgE (ku/l) Day ( ) 83 ( ) 8.2 ( ) Day ( ) ( ) 93 ( ) A. simplex, a helminthic nematode parasite, is found in sea mammals, cephalopods and fish, the latter two as intermediary hosts. Humans can become accidental hosts when they consume raw or undercooked seafood containing the parasite. Although the existence of this fish parasite has been known for many years, anisakiasis is still an underdiagnosed disease [11]. In the last 5 years, many allergic type I reactions have been reported [12,13] and a new entity, gastro-allergic anisakiasis, has been proposed to describe those cases when the gastrointestinal symptoms are accompanied by allergic symptoms [14,15]. In this context, A. simplex is again being widely studied and new cases of gastrointestinal anisakiasis are continuously being reported. Unfortunately, the diagnosis of acute parasitation is not always easy because the larva is not often isolated even if an early gastroscopic examination is performed [2]. In our series, a fiberoptic gastroscopic examination was only performed in nine of the patients and the larva could only be observed in four. In Japan, where tremendous numbers of cases of anisakiasis have been reported because the Japanese diet consists of a large proportion of raw fish, parasitation by A. simplex is often borne in mind and endoscopic removal of the larva is frequently performed [16]. We are of the opinion that in Western countries this cause is not often considered in the emergency service and urgent gastroscopy is rarely performed in patients who have gastric symptoms after eating seafood. Furthermore, the diagnosis usually depends on clinical suspicion and indirect tools. There must be a previous intake of raw or undercooked fish to suggest this possibility. In our series, the intake of anchovies in vinegar sauce, a traditional Spanish dish in which the fish are not cooked but are marinated in vinegar, was implicated in most of the subjects, coinciding with earlier reports [4,17]. The clinical features described in our series coincide with those reported by other authors [2 6], mimicking an obstructive or acute peritoneal picture if the larva perforates the gastrointestinal mucosa. The leukocyte count in anisakiasis shows leukocytosis, neutrophilia or mild eosinophilia, or less frequently, increased eosinophil values 24 h after the onset of symptoms, as observed in our patients. The suspicion increased with the results of the skin test. Although there is a moderately high frequency of false positive results found in the normal population, 10% in some studies [18], a positive SPT against A. simplex and the presence of high levels of specific IgE to A. simplex, when accompanied by an unequivocal anamnesis, are valuable tools for the diagnosis of anisakiasis [19]. Moreover, the increase of total and specific IgE, 1 month later in 31 of the 32 studied subjects, could be considered as a further argument to suspect acute gastrointestinal anisakiasis [7]. Negative specific IgE against parasites in all the patients, and the analysis of the stools, helped to rule out a cross-sensitivity reaction to parasites [20,21]. Unfortunately, we could not perform an antigen-capture ELISA, although this procedure could provide additional information to confirm the diagnosis [22,23] It is well known that eosinophilic infiltrations are present in the tissue surrounding the larvae [8,9]. Eosinophils are thought to play a major role in the inflammatory response against parasites. Basic cationic proteins (ECP, Myeloperoxidase, etc.) are cytotoxic proteins released from the granule when eosinophils are activated [24]. In vitro studies have suggested that ECP is released from eosinophils in an IgE-independent way and that ECP could be a marker of eosinophil activation [25]. Some reports have established serum ECP levels in healthy controls to be between 3.38 and 8.2 mg/l, although there are slight differences among studies conducted around the world, a fact

4 456 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003 which illustrate the importance of strict standardization of the procedure [26]. Eosinophils can also be activated in allergic diseases where a direct relation between the serum and sputum levels of ECP and airway inflammation has been observed [27,28]. However, none of the 32 studied subjects presented a significant allergic background or allergic symptoms coinciding with the gastrointestinal picture. Previous data suggested that allergic diseases are less frequent in patients in whom anisakiasis has been diagnozed [29]. Moreover, the median serum ECP drop at day 30 suggests a time-limited eosinophilic activation versus what happens in the case of allergic diseases in which the inflammation tends to be chronic [30]. In summary, we describe 32 patients with acute gastrointestinal symptoms in whom the suspicion of gastric anisakiasis seemed to be confirmed by the clinical picture and the results of SPT, total and specific IgE to A. simplex and serum ECP measurements, even though the parasite was only isolated in five of the patients. These findings suggest that parasitation by A. simplex should be considered when there is an intestinal pseudo-obstruction with a previous intake of raw or undercooked seafood. A raised ECP in this context, when a clinical suspicion of gastro-intestinal anisakiasis is being considered, could add information crucial for diagnosis, especially if a decrease of the serum levels is observed 30 days later. REFERENCES 1. Van Thiel PH, Kuipers FC, Roskam RTH. A nematode parasitic to herring causing acute abdominal syndromes in man. Trop Geogr Med 1960; 2: Sugimachi K, Inokuchi K, Ooiwa T, Fujino T, Ishii Y. Acute gastric anisakiasis. Analysis of 178 cases. JAMA 1985; 253: Del Olmo Escribano M, Cozar Ibáñez A, Martínez de Victoria JM, Ureña Tirao C. Anisakiasis at the ileal level. Rev Esp Enferm Dig 1998; 90: López-Penas D, Ramírez Ortiz LM, Del Rosal Palomeque López Rubio F, Fernández-Crehuet Navajas R, Mino Fugarolas G. Study of 13 cases of anisakiasis in the province of Cordoba. Med Clin 2000; 114: Maggi P, Caputi-Lambrenghi O, Scardini A, Scoppetta L, Saracino A, Valente M. Gastro intestinal infection due to Anisakis Simplex in Southern Italy. Eur J Epidemiol 2000; 16: Buendía E. Anisakis, anisakidosis and allergy to Anisakis. Allergy 1997; 52: Daschner A, Alonso-Gómez A, Caballero T, Suárez de Parga JM, López-Serrano MC. Usefulness of early serial measurement of specific and total immunoglobulin E in the diagnosis of gastroallergic anisakiasis. Clin Exp Allergy 1999; 29: Gómez B, Tabar AI, Larrinaga B, Alvárez MJ, García BE, Olaguibel JM. Eosinophilic gastroenteritis and Anisakis. Allergy 1998; 53: Domínguez Ortega J, Martínez-Cócera C. Guidelines in pathology induced by Anisakis. Alergol Inmunol Clin 2000; 15: Tomassini M, Tsicopoulos A, Tai PC, et al. Release of granule proteins by eosinophils from allergic and non allergic patients with eosinophilia on immunoglobulin- dependent activation. J Allergy Clin Immunol 1991; 88: Domínguez Ortega J, Cimarra M, Sevilla MC, Alonso Llamazares A, Moneo I, Robledo Echarren Martínez-Cócera C. Anisakis simplex: a cause of intestinal pseudo-obstruction. Rev Esp Enferm Dig 2000; 92: Moreno Ancillo A, Caballero T, Cabañas R, et al. Allergic reactions to Anisakis simplex parasitizing seafood. Ann Allergy Asthma Immunol 1997; 79: Domínguez Ortega J, Alonso Llamazares A, Rodríguez L, et al. Anaphylaxis due to hypersensitivity to Anisakis simplex. Int Arch Allergy Immunol 2001; 125: Daschner A, Alonso-Gómez A, Caballero MT, Barranco P, Suárez de Parga JM, López-Serrano MC. Gastro-allergic anisakiasis: an underestimated cause of acute urticaria and angioedema. Br J Dermatol 1998; 139: López Serrano MC, Alonso Gómez A, Daschner A, et al. Gastro-allergic anisakiasis: findings in 22 patients. J Gastroenterol Hepatol 2000; 15: Kakizoe S, Kakizoe H, Kakizoe K, et al. Endoscopic findings and clinical manifestations of gastric anisakiasis. Am J Gastroenterol 1995; 90: Gracia-Bara MT, Matheu V, Zubeldia JM, et al. Anisakis simplex-sensitised patients: should fish be excluded from their diet? Ann Allergy Asthma Immunol 2001; 86: Garcia M, Moneo I, Audicana MT, et al. The use of IgE immunoblotting as a diagnostic tool in Anisakis simplex allergy. J Allergy Clin Immunol 1997; 99: Kasuya S, Koga K. Significance of detection of specific IgE in Anisakis related diseases. Areugi 1992; 41: Akao N, Ohyama TA, Kondo K. Immunoblot analysis of serum IgG, IgA and IgE responses

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