Facultad de Medicina

Titres of parasite-specific IgE were investigated in 19 patients thought to have recurrent, acute mticaria caused by sensitization to Anisakis simplex (Dujardin, 1845), before and after they were placed on a fish-free diet. Patients with other allergic disease and those being treated with corticosteroids or antihistaminics were excluded. Skin-prick tests were carried out with A. simplex extract, and blue- and white-fish extracts. The CAP system (Pharmacia), a commercial test kit developed for the assay of food-specific IgE, was used to monitor serum concentrations of total IgE and antigen-specific IgE against Anisakis, Ascaris, Echinococcus, Toxocara, tuna, salmon, shrimp, mussel and cod. Before going on a fish-free diet, the 19 patients had CAP scores against A. simplex of 5 (three cases), 3 (seven) or 2 (nine). After a mean of 120 days on the diet, the scores against A. simplex were unchanged in 15 of the cases, reduced in three [from 5 to 4 (one case) or from 2 to O (two cases)] and increased in one (from 2 to 3). Most (16) of the patients no longer had any urticaria and the others reported significant reductions in the intensity and frequency of their symptoms.

The aim of this work was to study cross-reactivity in the diagnosis of two related ascaridosis. Nineteen patients diagnosed with recidivous acute urticaria (RAU) caused by Anisakis simplex and 26 patients diagnosed with visceral larva migrans (VLM) caused by Toxocara canis were studied employing commercial diagnostic kits and “in house” assay kits. Cross-reactivity observed was greater when using “in house” assay kits, suggesting that T. canis excretory–secretory antigens were not only recognized by antibodies from patients with RAU but with greater intensity compared to the A. simplex excretory–secretory antigens.

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.

Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-foodrelated allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections.

A majority of Kudoa spp. infects the somatic muscle of fish establishing cysts. Previously, elevated humoral responses were detected in BALB/c mice immunised with Kudoa sp. pseudocyst extracts and in BALB/c mice orally inoculated with Kudoa sp. pseudocysts, as well as the presence of anti-Kudoa sp. antibodies in human sera by enzyme-linked immunosorbent assay. The objective of this work was to test Kudoa sp. pseudocyst extracts by the skin prick test. Fifteen patients with gastroallergic and/or allergic symptoms related to fish ingestion were examined. Kudoa sp. pseudocyst extracts were administered (1 mg/ml) on the volar forearm skin. Four of the 15 selected patients were positive to Kudoa sp. extracts. The saline solution negative control did not induce any reaction.

Partial portal vein ligation is the experimental model most frequently used to study prehepatic portal hypertension. Different systemic and splanchnic biochemical and histological alterations in short-term (28–45 days) and long-term (12–14 months) evolutive phases which has been described in this experimental model suggest the existence of different pathophysiological mechanisms involved in their production. The enteropathy produced could develop in three phases: an early or acute phase with vasomotor hemodynamic alterations (ischemia-reperfusion associated with intestinal hyperemia, edema and oxidative stress); an intermediate phase with immunological alterations (mesenteric lymphadenopathy, increased mucosal infiltration by mast cells and the hepato-intestinal release of pro- and anti-inflammatory mediators); and a late or chronic phase with intestinal remodeling (vascular and epithelial). The alterations which are produced in these three evolutive phases make it possible to propose an inflammatory etiopathogeny for hypertensive portal enteropathy.