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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, IgG 4  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, IgG 4  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 .

bmc infectious diseases

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