First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy

We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis.The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin.The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.Anisakiasis is a fish-borne zoonosis provoked by the larval ascaridoid nematodes of the genus Anisakis Dujardin, 1845. These nematodes have a complex life-cycle involving organisms at various levels of a trophic web in the marine ecosystem. Small crustaceans, mainly euphasiaceans, are the first intermediate hosts, whereas fish and squid are intermediate/paratenic hosts, and they reach the adult stage in the stomachs of marine mammals (mainly cetaceans) [1]. In fish, larvae are commonly localized on the surfaces of the visceral organs, body cavity, serous membranes and, occasionally, also in the muscle. Humans can accidentally be infected by the larvae when consuming raw, undercooked or improperly processed (e.g. marinated) parasi