Methods for inactivation of seafood Anisakis larvae and prevention of human anisakiasis: a mini-review.

Anisakiasis is an arising zoonosis induced by parasitic nematodes belonging to the family Anisakidae. Anisakiasis is often caused by the ingestion of larval nematodes in uncooked or minimally processed seafood dishes, which are regularly consumed by humans. Significant potential sources of infection are raw fish (e.g., sushi and sashimi) that can be found in traditional Japanese cuisine and can be part of the culinary tradition of consumption of raw or marinated fish that is particularly diffused in European countries. During the last five decades, the global prevalence of human anisakiasis has been rising, becoming an emergent major public health problem. Thus, there is an unmet need for well-defined and cost-effective methods aimed at killing Anisakis larvae, thus reducing the incidence of anisakiasis. In this mini-review, we discuss the clinical features of anisakiasis as well as the effectiveness and mechanisms of action of the main methods employed for increasing seafood safety and killing Anisakis larvae, including freezing, heating, use of high hydrostatic pressure, salting process, pepsin digestion method and use of garlic oil.

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae).

Introduction: Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized. Methods: Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis. Results and discussion: EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.

Anisakiasis Annual Incidence and Causative Species, Japan, 2018-2019.

Using data from 2018-2019 health insurance claims, we estimated the average annual incidence of anisakiasis in Japan to be 19,737 cases. Molecular identification of larvae revealed that most (88.4%) patients were infected with the species Anisakis simplex sensu stricto. Further insights into the pathogenesis of various anisakiasis forms are needed.

World-wide prevalence of Anisakis larvae in fish and its relationship to human allergic anisakiasis: a systematic review.

The infective stage of Anisakidae nematodes responsible for allergic reactions in humans is found in a variety of edible fish and cephalopods. The identification of geographical regions that are high risk for infected seafood may help prevent allergic reactions in humans. Despite an abundance of published literature which has identified anisakid larvae in an array of edible seafood as well as scattered reports of human allergic anisakiasis, the relationship between the two has not been fully explored. Therefore, a systematic spatio-temporal study was conducted to determine the prevalence of Anisakis spp. in fish from January 2000 to August 2020 firstly to explore the relationship between fish infection and cases of allergic anisakiasis and secondly to use fish infection data to map potential allergic anisakiasis 'hot spots'. A systematic literature search for original English text articles was conducted through search engines, Web of Science, Scopus, PubMed, Science Direct and Google Scholar. Out of 3228 articles which describe anisakid infection in fish, 264 were used for data extraction. Of 904 articles describing allergic anisakiasis, 37 were used for data extraction. A qualitative summary of the extracted data was performed using equal interval method (ArcMap software) in order to compare the global distribution of Anisakis-infected fish. Of the 152-identified fish hosts, five families were most commonly infected with Anisakis spp. These included Lophiidae (86.9%), Trichiuridae (77.05%), Zeidae (70.9%), Merlucciidae (67.8%) and Gadidae (56.8%). The hot spot areas for allergic anisakiasis were North and northeast of Atlantic Ocean, southwest of USA, west of Mexico, south of Chile, east of Argentina, Norway, UK and west of Iceland (confidence 99%). The highest rate of allergic anisakiasis was in Portugal and Norway with the prevalence rate of 18.45-22.50%. Allergologists should consider allergic anisakiasis as a public health issue particularly in high-risk countries where high prevalences in fish have been demonstrated.

Differential proteolytic activity in Anisakis simplex s.s. and Anisakis pegreffii, two sibling species from the complex Anisakis simplex s.l., major etiological agents of anisakiasis.

Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen-host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p < 0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p < 0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although, in L3-0h, the activity of these proteases was significantly higher (p < 0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites. This may be related to the significantly higher activity (p ≤ 0.05) of serine protease in all the larval stages of A. simplex studied at pH 6.0. Thus, there are interspecific differences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

Human anisakiasis in Italy: a retrospective epidemiological study over two decades.

A retrospective analysis on human anisakiasis in Italy since its first description in 1996 was performed by conducting a literature search. Inclusion criteria based on the presence of a larva and on parasite identification were applied. Epidemiological data and clinical features were analysed. Particular attention was paid to the source of infection. In total, 73 cases were included in the analysis, while 34 were excluded. Cases were reported from eight Italian regions, most frequently Abruzzo, Apulia and Latium. The parasite was detected by endoscopy (51.4%) or laparotomy (48.6%). The site of infection was intestinal (42.5%), gastric (43.8%), oesophageal (1.4%) or ectopic (12.3%). Most of the parasites (71.0%) were identified as Anisakis sp. or A. simplex (s.l.). However, when molecular methods were used (21 cases), A. pegreffii was always identified. In most of the patients (65.7%), the source of infection was raw or undercooked anchovies, followed by "anchovies or sardines" (15.1%), generic "raw seafood" (15.1%), and sardines (1.4%). In only 2 cases (2.7%), the source was not available. This is the first systematic analysis of Italian cases of anisakiasis. The main conclusions derived from the results are: i) attention should be given to the history, in particular when raw marinated anchovies, proven to be the main source of human anisakiasis in Italy, are consumed; ii) in order to assess correct epidemiological data, a confirmed and specific etiological identification should always be sought.

Seafood-borne parasitic diseases in Australia: are they rare or underdiagnosed?

Australia is a multicultural country surrounded by water where seafood is regularly consumed. Literature suggests that some popular edible fish sold in fish markets may be infected with parasites transmissible to humans (notably, anisakids and other helminths); however the number of reported human cases due to these parasites is low. In this article we critically review topical publications to understand whether the low number of human infection is due to lack of expertise in Australia to identify and diagnose accurately seafood-borne parasitic infections. The risk these parasites pose to humans may be underestimated due to: (i) errors or inability of diagnosing these infections, primarily due to less sensitive and specific serological tests and misidentifying parasites without a taxonomist in the diagnostic team; and (ii) medical practitioners not being aware of these parasites or not considering them in the differential diagnosis even in patients with history of regular raw or undercooked seafood consumption.

A national retrospective survey of anisakidosis in France (2010-2014): decreasing incidence, female predominance, and emerging allergic potential.

A retrospective survey was carried out over the years 2010-2014 among all Parasitology laboratories of University hospitals in France (ANOFEL network). The objective was to estimate the incidence of anisakidosis in France as new culinary habits such as the consumption of raw fish (sushi) or undercooked fish are increasing. A total of 37 cases of anisakidosis were notified by all French laboratories: 7 proven cases with evidence of a worm, 12 possible cases with abdominal pain after consumption of raw fish with detection of anti-Anisakis precipitins, and 18 allergic cases defined as acute manifestations after consumption of fish, associated with specific IgE for Anisakis. The median age of affected individuals was 42 years (11-69) and there was a significant predominance of women (67%). Compared with previous surveys in France, this study indicates a decrease in clinical cases of anisakidosis and illustrates the emerging allergic potential of anisakids.

TITLE: Étude nationale rétrospective de l'anisakidose en France (2010-2014) : diminution de l'incidence, prédominance féminine et augmentation des allergies. ABSTRACT: Une enquête rétrospective a été réalisée au cours des années 2010-2014 auprès des laboratoires de parasitologie des hôpitaux universitaires de France (réseau ANOFEL). L'objectif était d'estimer l'incidence de l'anisakidose en France depuis l'émergence de nouvelles habitudes culinaires comme la consommation de poissons crus (sushi) ou insuffisamment cuits. Les laboratoires français de Parasitologie ont ainsi notifié 37 cas d'anisakidose: 7 cas certains avec présence du ver, 12 cas possibles se caractérisant par des douleurs abdominales après consommation de poisson cru et la présence de précipitines anti-Anisakis et 18 cas allergiques définis comme des manifestations aiguës après consommation de poisson associées à des IgE spécifiques anti-Anisakis. L'âge médian des cas était de 42 ans (11-69) et il y avait une prédominance féminine significative (67 %). Par rapport aux enquêtes précédentes en France, cette étude indique une diminution des cas cliniques d'anisakidose mais illustre le potentiel allergique émergent des anisakidés.

Invaginación colocólica por anisakiasis, una causa infrecuente de obstrucción intestinal / Colonic intussusception caused by anisakiasis. A rare cause of obstruction

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Human gastric hyperinfection by Anisakis simplex: A severe and unusual presentation and a brief review.

Anisakiasis is an emerging marine food-borne zoonosis resulting from the accidental ingestion of Anisakis larvae, through the consumption of raw or undercooked infected seafood products. The first case of human gastric hyperinfection by Anisakis simplex with an unusual and severe presentation, occurring in a Portuguese woman, is described in this article. Over 140 anisakid larvae were removed by gastroscopy. Massive infection is uncommon in areas where the consumption of raw fish is not part of the traditional diet, as is the case in Portugal. The increased consumption of raw seafood products is considered a health determinant in the rise in cases of anisakiasis. However, clinicians should be aware of the emergence of these infections, not only because of the new dietary habits of the population, but also because of the high prevalence of Anisakis larvae in the different fish species usually consumed by the population, collected on the Portuguese coast.

Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay.

BACKGROUND: Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen. METHODS: Parasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied. RESULTS: According to the mtDNA cox2 and the EF1 α - 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases. In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like. CONCLUSION: The Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.

Prevalence and Mean Intensity of Anisakidae Parasite in Seafood Caught in the Mediterranean Sea Focusing on Fish Species at Risk of Being Raw-consumed. A Meta Analysis and Systematic Review.

OBJECTIVE: To assess the prevalence and mean intensity of anisakids in seafood caught in the Mediterranean Sea, focusing on fish species at risk of being raw-consumed. DESIGN: Systematic review and meta-analysis of studies published from 1960-2012. STUDY SELECTION: Main criteria for the inclusion of studies were as follows: Findings of anisakid larvae, in both muscles and viscera; fish species for human consumption caught in the Mediterranean Sea; prevalence and mean intensity data for each species; and sample size equal to or more than 40 fishes. RESULTS: Twelve studies were identified. Among these, four studies considered the following three fish species that are often consumed raw or preserved lightly, or not cooked thoroughly: anchovy, pilchard, and Atlantic mackerel. DATA SYNTHESIS: All pooled analyses were based on the random-effect model. Anisakids prevalence in fish muscle was 0.64% (P < 0.0001), in viscera it was 1.34% (P < 0.0001), and overall prevalence was 0.95% (P < 0.0001). Mean intensity in muscle was 2.31 (P = 0.0083), in viscera it was 1.55 (P = 0.0174), and overall it was 1.81 (P < 0.0005). Heterogeneity indices (I(2)) were significantly high with the exception of viscera mean intensity. CONCLUSIONS: Anchovy, pilchard, and Atlantic mackerel have a low prevalence and mean intensity of anisakidae larvae in both viscera and muscles. Mean Intensity was also low.

First report of human anisakidosis in Australia.

We present the first human case of anisakidosis acquired from eating locally caught fish in Australia. A 41-year-old woman experienced gastrointestinal pain, vomiting and diarrhoea of increasing severity over 3 weeks. All symptoms resolved spontaneously after a worm was passed in her faeces. Microscopic examination showed that it was a Contracaecum species larva of the family Anisakidae. Anisakidosis should be considered in patients with gastrointestinal symptoms who have recently eaten seafood.

Anisakiasis intestinal diagnosticada por enteroclisis / Intestinal anisakiasis diagnosed by enteroclysis

La anisakiasis es una parasitación frecuente en nuestro medio y que da lugar a cuadros de dolor abdominal que pueden simular otras patologías, incluyendo situaciones quirúrgicas (apendicitis o peritonitis), pero generalmente autolimitadas. Presentamos un caso de una paciente de 48 años con dolor abdominal agudo, febrícula y hallazgos patológicos en TAC (propios de un cuadro inflamatorio intestinal), en la que la realización de una enteroclisis fue capaz de identificar larvas de Anisakis. La paciente evolucionó favorablemente con tratamiento conservador y el patrón radiológico volvió a la normalidad. En nuestro caso, la posibilidad de contar con una técnica radiológica objetiva que sirvió de ayuda al diagnóstico proporcionó datos muy útiles en el manejo de la paciente. Se revisan las características clínicas y radiológicas de la enfermedad (AU)

Anisakiasis is a common infestation in our setting that produces abdominal pain that can simulate other diseases including surgical conditions (symptoms of acute abdomen, mimicking appendicitis or peritonitis), but it is generally a self-limiting process. We report a case of a 48-year-old female with acute abdominal pain, fever and pathological findings on the CT scan (inflammatory bow condition). Anisakis larvae could be identified by the enteroclysis. The patient evolved favorably with conservative treatment and radiologic intestinal pattern returned to normal. The possibility of a radiological technical to assist in the diagnosis provided very useful data in the management of our patient. The radiological and clinical features of the disease are reviewed (AU)

Risk factors for human Anisakis infection and association between the geographic origins of Scomber japonicus and anisakid nematodes.

It has been reported that nearly all cases of anisakiasis in Japan are caused by Anisakis simplex sensu stricto. To elucidate this further, we investigated the presence of Anisakis type I larvae and Pseudoterranova decipiens in 218 Scomber japonicus fish collected from the seas of Japan. Anisakis type I larvae were detected in 74.3% (162/218) of the fish, and 99.8% of the Anisakis type I larvae comprised A. simplex sensu stricto and Anisakis pegreffii. Molecular identification techniques were used for 7.5% (360/4806) of the Anisakis type I larvae. The larvae found in the fish of the Pacific stock (the Pacific coast of Japan) and the Tsushima Warm Current stock (the East China Sea and the Sea of Japan) were primarily A. simplex sensu stricto and A. pegreffii, respectively. In addition, for the first time in Japan, Anisakis simplex C and Anisakis ziphidarum were detected in the fish of the Pacific stock. The average number of A. pegreffii and A. simplex sensu stricto larvae per fish was 47 and 6, respectively. However, the average number (0.61 larvae) of A. simplex sensu stricto in the muscle per fish was 12 times the average number (0.05 larvae) of A. pegreffii. When fish on the purchased day were compared with those held at 4 degrees C and 20 degrees C for 20h, the penetration rates (ratio of the number of larvae detected in the muscle to the total number of larvae detected) of A. pegreffii and A. simplex sensu stricto were as high as 1.8% and 5.8%, respectively. In conclusion, we suggest that anisakiasis in Japan is mainly caused by A. simplex sensu stricto because it penetrates the muscle of the fish at a higher rate than A. pegreffii.

Anisakis simplex: the high prevalence in Madrid (Spain) and its relation with fish consumption.

Anisakiosis is a nematodosis with high prevalence in Spain. In this work we (a) investigated whether a recently introduced ELISA of Anisakis simplex-specific IgE in serum suffers from cross-reactivity with other common allergens; (b) used this assay to obtain an estimate of the prevalence of A. simplex-specific IgE in the population of Madrid; and (c) related positivity to fish consumption habits. No evidence of cross-reactivity between the ELISA and other allergens was found. The prevalence of positivity was 12.4% (11.7% among healthy subjects and 16% among patients with non-digestive non-allergic pathologies). All interviewed subjects reported consumption of uncooked fish (known to be the most likely source of infection); in addition, positivity was more prevalent among subjects who habitually consumed fresh and possibly undercooked fish than among those who generally consumed frozen fish or boiled or baked fish. These results are discussed in relation to the much lower prevalence observed in Galicia (N.W. Spain).

[Anisakiasis gastrointestinal manifestations: description of 42 cases]. / Manifestaciones digestivas de la anisakiasis: descripción de 42 casos.

BASIS: Anisakiasis is an emerging parasitosis in our environment, although still scarcely known, mainly its gastrointestinal manifestations. PATIENTS AND METHODS: In this paper 42 cases of anisakiasis with gastrointestinal symptoms (with or without cutaneous allergic manifestations) are described; all cases were studied in our hospital between 1995 and May 2004. RESULTS: Thirty seven cases showed a gastric or gastroallergic form (6 confirmed with gastroscopy) and 5 suffered intestinal symptoms. In those in which endoscopy was not carried out, the diagnosis was done after clinical and biological findings. All patients were sensitized in the presence of Anisakis simplex. The 69% (29 cases) described raw anchovies ingestion or anchovies seasoned in vinegar ingestion some hours before the beginning of the clinical picture. The 59% showed allergic symptoms (gastroallergic anisakiasis); no patient with intestinal affectation showed allergic symptoms. Nausea (67%), vomiting (51%), and epigastralgia (50%) were the dominant symptoms in the group with gastric or gastroallergic anisakiasis without endoscopic confirmation; on the other hand, pain in right ileac fossa was the dominant symptom in the intestinal form. Fever was seen in two of the later patients (40%), but in no patient with gastric or gastroallergic anisakiasis. CONCLUSIONS: Gastrointestinal symptomatology after ingestion of fish suspiciously prepared should suggest anisakiasis, regardless if clinical picture includes allergic manifestations. In our study, pain in right ileac fossa, fever, and absence of allergic manifestations were typical of intestinal form.

Manifestaciones digestivas de la anisakiasis: descripción de 42 casos / Anisakiasis gastrointestinal manifestations: description of 42 cases

Fundamento. La anisakiasis es una parasitosis emergente en nuestro medio, aunque todavía escasamente conocida, principalmente en sus manifestaciones digestivas. Pacientes y métodos. Se describen 42 casos de anisakiasis con afectación digestiva (con o sin manifestaciones alérgicas cutáneas) estudiados en nuestro hospital entre 1995 y mayo de 2004. Resultados. Treinta y siete casos mostraban una forma gástrica o gastroalérgica (6 confirmados mediante gastroscopia) y 5 manifestaciones intestinales. En aquellos en que no se realizó endoscopia el diagnóstico se efectuó tras los hallazgos clínicos y biológicos. Todos los pacientes estaban sensibilizados frente a Anisakis simplex. El 69% (29 casos) referían ingesta unas horas antes de anchoas en vinagre o crudas. El 59% presentó clínica alérgica asociada (anisakiasis gastroalérgica), no presentando esta clínica ninguno de los casos con afectación intestinal. Las náuseas (67%), vómitos (51%) y epigastralgia (50%) fueron los síntomas dominantes en el grupo con anisakiasis gástrica o gastroalérgica sin confirmación endoscópica, mientras que el dolor en fosa ilíaca derecha lo fue en la forma intestinal. Dos de estos pacientes (40%) presentaron fiebre, aunque ninguno de los casos con anisakiasis gástrica o gastroalérgica. Conclusiones. La aparición de sintomatología digestiva después de la ingesta de pescado preparado de forma sospechosa debe hacer pensar en una posible anisakiasis, independientemente de que se asocien o no manifestaciones alérgicas. La presencia de dolor en fosa ilíaca derecha, fiebre y la ausencia de manifestaciones alérgicas fueron característicos de las formas intestinales

Basis. Anisakiasis is an emerging parasitosis in our environment, although still scarcely known, mainly its gastrointestinal manifestations. Patients and methods. In this paper 42 cases of anisakiasis with gastrointestinal symptoms (with or without cutaneous allergic manifestations) are described; all cases were studied in our hospital between 1995 and May 2004. Results. Thirty seven cases showed a gastric or gastroallergic form (6 confirmed with gastroscopy) and 5 suffered intestinal symptoms. In those in which endoscopy was not carried out, the diagnosis was done after clinical and biological findings. All patients were sensitized in the presence of Anisakis simplex. The 69% (29 cases) described raw anchovies ingestion or anchovies seasoned in vinegar ingestion some hours before the beginning of the clinical picture. The 59% showed allergic symptoms (gastroallergic anisakiasis); no patient with intestinal affectation showed allergic symptoms. Nausea (67%), vomiting (51%), and epigastralgia (50%) were the dominant symptoms in the group with gastric or gastroallergic anisakiasis without endoscopic confirmation; on the other hand, pain in right ileac fossa was the dominant symptom in the intestinal form. Fever was seen in two of the later patients (40%), but in no patient with gastric or gastroallergic anisakiasis. Conclusions. Gastrointestinal symptomatology after ingestion of fish suspiciously prepared should suggest anisakiasis, regardless if clinical picture includes allergic manifestations. In our study, pain in right ileac fossa, fever, and absence of allergic manifestations were typical of intestinal form