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Summary: Background. Diagnosis of anisakis allergy (AA) is based on the skin prick test (SPT) and specific IgE (sIgE) determination. Anyway, false positivity cases are due to cross reactivity with numerous allergens. The aim of the study was to evaluate the reliability of a comprehensive diagnostic algorithm for the AA. Methods. An observational study was conducted on a sample of consecutive subjects accessing the allergology outpatient ambulatories of two hospitals located in Western Sicily. All the recruited outpatients were tested by Skin Prick Test performed using anisakis extracts by ALK-Abellò (Madrid, Spain). Specific IgE dosage for anisakis extracts was then performed by using ImmunoCAP250 (Immunodiagnostics Uppsala, Sweden). Consequently, outpatients who tested positive to first line tests underwent sIgE testing for ascaris and tropomyosin. Lastly, outpatients positive to the first line were invited to be further tested by basophil activation test (BAT) by using Flow CAST kit and anisakis commercial extract (Bühlmann Laboratories AG, Schönenbuch, Switzerland), as confirmatory analysis. Results. One hundred and eleven outpatients with an anamnesis suggestive of sensitization to anisakis (AS) and 466 subjects with chronic urticaria (CU) were recruited in the study. Of these, 22 with AS and 41 with CU showed a sensitization to anisakis allergens. The diagnostic algorithm revealed that 8.8% of outpatients who tested positive to sIgE determination were affected by CU, while 82.5% of all the sIgE positivity was related to cross-reactivity. Overall, a genuine anisakis seroprevalence of 2.3% was documented. Within a sub-sample of 15 subjects with clinical symptoms related to AA, n. 8 showed a real positivity after BAT. A greater response to A. pegreffii allergens as compared to A. simplex was reported. Conclusions. Our preliminary findings support the high clinical specificity of BAT for AA diagnosis, suggesting implementing this method in a comprehensive diagnostic algorithm.

A total of 1104 fish samples from markets of Sicily were analysed for the detection and species identification of Anisakidae nematodes. The preliminary analysis of the fish samples showed the presence of 2459 larvae. All the fish species revealed different prevalence of infestation, with a maximum of 100% for Lepidopus caudatus and a minimum of 4.5% in Sardina pilchardus. The 80% of the larvae examined by PCR-RFLP analysis belonged to Anisakis pegreffii species. The seasonal infestation trend of Anisakis was evaluated in all the fish sample examined. The results of the seasonal infestation trend showed a marked connection with the ecological aspects of the fish species examined. As far as we know, this work report for the first time important ecological aspects of Lepidopus caudatus specimens of South Mediterranean. This work could be useful to plan a seasonal fishing strategy aimed at reducing the health risks related to Anisakis .

We examined 151 European sea bass (Dicentrarchus labrax L.) samples from farms and fish markets of Sicily (Southern Italy) for Anisakidae larvae detection. All the samples were examined by visual inspection and modified chloro-peptic digestion. Two nematode larvae were found in the viscera of only one European sea bass sample from a farm located in Greece (FAO 37.3), giving a total prevalence of infestation of 0.7%. No other parasites were found after chloro-peptic digestion of the samples. The larvae were morphologically ascribed, at genus level, to morphotypes I and molecularly identified as Anisakis pegreffii. To the best if our knowledge, this is the first report on the presence of anisakid parasites in farmed European sea bass of Mediterranean Sea. Our findings suggest that the risk of exposure to Anisakidae nematodes in farmed European sea bass remains very low. However, further data on Mediterranean farms are needed to have a detailed risk analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this work 437 fish samples of species belonging to the families Myctophidae (Electrona risso and Diaphus metopoclampus) and Phosichthyidae (Vinciguerria attenuata) were examined for the presence of Anisakidae larvae. The study was performed with fishes in the central Mediterranean Sea, particularly in the Strait of Sicily and in the Strait of Messina. The visual inspection and chloro-peptic analysis revealed the presence of nematode parasites with prevalence values between 2.9% in Electrona risso samples and 5.4% in Vinciguerria attenuata samples. A positive correlation was found between standard length (SL) and prevalence of infestation in D. metopoclampus samples (p<0.05). The larvae examined were morphologically ascribed, at genus level, to Anisakis morphotypes I and II and molecularly identified as Anisakis pegreffii, Anisakis ziphidarum and Anisakis physeteris, in 67%, 9% and 24% of the fish samples examined. Overall, A. pegreffii and A. ziphidarum larvae were isolated in 14 and 2 specimens of D. metopoclampus respectively, A. physeteris larvae were found in 3 E. risso and 2 V. attenuata. A positive correlation was found between standard length and prevalence of infestation in D. metopoclampus samples (p<0.05). First information is provided on the presence of Anisakis spp. larvae of the myctophid fish species E. risso, D. metopoclampus and V. attenuata from the Central Mediterranean. It is also confirmed the role of lanternfishes (Myctophidae) as paratenic hosts for Anisakis spp., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005., (Copyright © 2017 Elsevier B.V. All rights reserved.)