Molecular Diagnosis of Cause of Anisakiasis in Humans, South Korea

Anisakiasis in humans in South Korea has been considered to be caused exclusively by the larvae of Anisakis simplex sensu stricto and Pseudoterranova decipiens. Recently, however, DNA sequencing of larvae from 15 of 16 anisakiasis patients confirmed the cause to be Anisakis pegreffii infection. Molecular analysis should be performed for all extracted larvae.

the basis of morphologic appearance (1,12). We performed molecular analyses of 26 Anisakis type I larvae recovered from 16 humans in South Korea by using DNA sequencing of the nuclear internal transcribed spacer (ITS) genes.

The Study
A total of 30 Anisakis type I larvae were removed from the stomach of 16 patients referred to the Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, Seoul, South Korea, from 2000 through 2013 (Table). Among them, 26 larvae were analyzed by DNA sequencing. All patients experienced acute gastric or abdominal discomfort, including epigastric pain and indigestion, and underwent gastroduodenoscopy. During the examinations, whitish nematode larvae were observed and extracted with biopsy forceps. Some larvae were preserved in 70% ethanol, and others were fixed in 10% formalin before being mounted on slides with glycerin jelly.
Total genomic DNA was extracted by using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany); nested PCR and nucleotide sequencing were performed on the ITS region (ITS1, 5.8S rRNA subunit, and ITS2) according to procedures reported previously (13). The PCR product was amplified by using the Cosmo Labopass X2 PCR Premix kit (Cosmo Genetech, Seoul, South Korea), and automated DNA sequencing was performed by Solgent Co., Ltd. (Daejeon, South Korea). Nucleotide sequences obtained were aligned by using the Geneious program, version 6.0.3 (Geneious Co., Wellington, New Zealand).
Of the 26 Anisakis larvae from 15 human patients, 25 showed 100% identity in the sequences of ITS region (244 bp, high-confidence variable positions) with those of the A. pegreffii sequence available in GenBank (accession no. AB277823), whereas their identity with A. simplex s.s. (accession no. AB277822) was 99.2% (Table) Human infection with A. pegreffii nematodes was first documented in Italy by use of PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis (6). The second case was reported from Japan (10), in which 1 of 100 anisakid larvae extracted from patients in Kyushu and Hokkaido was identified by PCR-RFLP analysis as A. pegreffii. Then, in 2009, A. pegreffii nematode infection was diagnosed for 2 women in Italy by PCR-RFLP and sequencing of the 28S gene (7). In 2011, A. pegreffii DNA was extracted from a paraffin-embedded granuloma from a man in Italy (8). Also in Italy, 8 more A. pegreffii nematode infections in humans were reported in 2013 (9). Thus, to date, including the 15 cases reported here, a total of 28 cases of A. pegreffii nematode infections in humans have been documented in the literature.
The markedly high proportion of A. pegreffii nematode infections identified among patients in South Korea (25/26 larvae from 15/16 patients) was not expected because in Japan (Kyushu and Hokkaido), which are geographically close to South Korea, Anisakis larvae from humans are mostly A. simplex s.s. (99/100 larvae from 84/85 patients); only 1 larva was identified as A. pegreffii (10). This remarkable discrepancy between South Korea and Japan remains to be further investigated. However, it is of note that the species of Anisakis larvae detected in fish varied according to the 2 large localities of Japan; from northern Japan to the Pacific sides and from the Sea of Japan to the eastern China Sea sides (14). The former locality, such as Hokkaido and eastern Japan, showed more A. simplex s.s. than A. pegreffii larvae, whereas the latter locality (southwestern Japan), including Kyushu and Fukuoka (close to South Korea), showed more A. pegreffii than A. simplex s.s larvae (14). This finding might partly explain the discrepancy between the Anisakis larvae species that infect humans in South Korea and Japan.
When the pathogenic potential of A. simplex s.s. larvae for human patients was compared with that of A. pegreffii (11), it was found that A. simplex s.s. larvae had greater potential than A. pegreffii larvae to survive acidic gastric juice and to penetrate the human stomach, small intestine, and colon. However, further studies are needed to elucidate this finding.
Another clinicopathologic concern associated with anisakiasis in humans is the potential for A. simplex s.s.  and A. pegreffii larvae to elicit gastroallergic reactions. These reactions are characterized by urticaria on the arms and abdomen and by angioedema or anaphylaxis when the live parasite attempts to penetrate the gastric mucosa (9). We did not notice such allergic reactions in the patients reported here. However, because of an increasing tendency toward Anisakis nematode allergy among patients in South Korea (15), attention should be paid to this clinical feature.
Our study demonstrates the predominance of A. pegreffii over A. simplex s.s. nematode infection among humans with anisakiasis in South Korea. The study highlights the need to perform molecular analysis for each larva extracted from human patients in this country.
Ms. Lim and Mr. Jung are research associates in the Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, Seoul, South Korea. Their major research interest is molecular studies of foodborne zoonotic parasites, including anisakids, heterophyids, gymnophallids, and Toxoplasma.