Abstract
BackgroundAnisakiasis 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.MethodsParasite 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.ResultsAccording 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.ConclusionThe 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.
Highlights
Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae
Molecular identification of CC1 and CC2 by direct DNA sequencing The Mitochondrial DNA (mtDNA) cox2 sequences obtained from the larval nematodes removed from the stomach in CC1 and the larva recovered in the intestine of CC2, showed 99 or 100% similarity with sequences of mtDNA cox-2 previously obtained for the species A. pegreffii [18] and retrievable from GenBank
These mtDNA cox2 sequences isolated from CC1 and CC2 were deposited in GenBank under the accession numbers: KY073403 e KY073404 (Fig. 2a)
Summary
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. The accidental ingestion of third stage larvae (L3) of Anisakis spp. by consuming infested raw, undercooked, or improperly processed (e.g. marinated) seafood, causes a parasitic zoonosis, known as anisakiasis. The “not invasive” form occurs when the larva is not able to penetrate the gastric or intestinal mucosa and it remains luminal; in this case, the patient could be asymptomatic and the larva could be eliminated with stools and/or vomit. Larva in the tissues, usually the gastric and intestinal mucosa In the latter case, severe gastrointestinal symptoms during the acute phase of infection, such as epigastralgia, nausea, vomiting, abdominal pain are described. The chronic infection of anisakiasis, is characterized by the Anisakis larvae deeply invading the gastric and intestinal walls, causing direct tissue damage, ulcers and, eventually, eosinophilic granulomas [1,2,3,4,5]
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