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

Abstract

Background: Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted.

Method: 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.

Results: 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.

Conclusion: 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.

Figure 1

Transverse sections of the Anisakis larva in the submucosa of the intestinal wall. Polymyarian muscle cells (thin arrows), separated into four quadrants by the chords, showing two wing-like distal lobes (arrow heads). Excretory cells are banana shaped (thick arrows) and situated ventrally to the intestine (H&E staining; original magnification ×100).

Figure 2

Alignment of the mtDNA cox2 (629 bp) sequences of Anisakis spp., by using BioEdit [42]. mtDNA cox2 (629 bp) sequence of the Anisakis pegreffii larva identified from the paraffin-embedded tissue of human intestinal anisakiasis (code: AEH), with respect to the other Anisakis spp. which have previously been sequenced [24] and deposited in GenBank. Accession numbers are reported in the text. Dots indicate identity.

Figure 3

Cox2 derived Maximum Parsimony (MP) and Neighbour-Joining (NJ) condensed consensus trees inferred by MEGA4.0 for the specimen of Anisakis pegreffii larva from the human intestinal case (code: AEH) sequenced for the mtDNA cox2 (629 bp). The MP tree was obtained by the bootstrap method with a heuristic search. There were 213 polymorphic sites and 178 parsimony-informative sites. Sequences of A. pegreffii from Engraulis encrasicolus are reported with the codes: AE71, AE65, AE72, AE70. Sequences of A. pegreffii from our previous studies are reported with the codes AE03 and AE02 [28]. Number of bootstrap replicates = 1000; bootstrap percentages of the clades ≥ 70 are shown at the nodes, with MP and NJ values are reported above and below the nodes, respectively. Sequences from the other Anisakis spp. are those previously analyzed by us [24,28] and deposited in Genbank. Accession numbers are reported in the text. Pseudoterranova decipiens (s.s.) was used as the outgroup.