Invasive anisakiasis by the parasite Anisakis pegreffii Nematoda: Anisakidae: diagnosis by real-time PCR hydrolysis probe system and immunoblotting assayReportar como inadecuado




Invasive anisakiasis by the parasite Anisakis pegreffii Nematoda: Anisakidae: diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Infectious Diseases

, 17:530

Parasitological diseases

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.

KeywordsAnisakiasis Italy A. pegreffii Molecular diagnosis Real-time PCR Serodiagnosis Immunoblotting AbbreviationsEF1 α − 1Elongation factor gene

L3Third stage larvae

Mr.Relative mobility

mtDNA cox2Mitochondrial DNA cytochrome c oxidase subunit II gene

mtDNAMitochondrial DNA

nDNANuclear DNA

RT-PCRReal-time PCR

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Autor: Simonetta Mattiucci - Michela Paoletti - Alessandra Colantoni - Antonella Carbone - Raffaele Gaeta - Agnese Proietti - Stefa

Fuente: https://link.springer.com/







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