Molecular Diagnosis of Pseudoterranova decipiens Sensu Stricto Infections, South Korea, 2002‒2020 (original) (raw)

Hyemi Song1, Seungwan Ryoo1, Bong-Kwang Jung, Jaeeun Cho, Taehee Chang, Sooji Hong, Hyejoo Shin, Woon-Mok Sohn, and Jong-Yil ChaiComments to Author

Author affiliations: Korea Association of Health Promotion, Seoul, South Korea (H. Song, S. Ryoo, B.-K. Jung, J. Cho, T. Chang, S. Hong, H. Shin); Gyeongsang National University College of Medicine, Jinju, South Korea (W.-M. Sohn); Seoul National University College of Medicine, Seoul (J.-Y. Chai)

Human anisakiasis, which is caused by infection with larvae of the family Anisakidae after consuming infested marine fish or squids, is one of the most serious foodborne zoonotic diseases (1). Several species of Anisakis (A. simplex sensu stricto, A. physeteris, and A. pegreffii) (1–3), Pseudoterranova (P. decipiens sensu stricto, P. azarasi, and P. cattani) (4–6), and Contracecum (C. osculatum) (7) nematodes have been reported to cause human infections.

Human anisakiasis was reported in the Netherlands during 1960 and has been found to occur in various parts of the world, including Japan and South Korea (1). Most human case-patients were infected with larvae of A. simplex s.s. (1). However, after 1999, a considerable number of cases infected with A. pegreffii nematodes (a sibling species of A. simplex s.s.) were diagnosed in Italy, Japan, and South Korea on the basis of molecular analysis of the larvae (2,3). Compared with Anisakis spp. nematodes, human infections with Pseudoterranova spp. nematodes have been relatively rare in Asia (1,4–6). In South Korea, among 645 anisakidosis cases recorded after 1971 until 2015, only ≈11.8% were infected with Pseudoterranova larvae (8). However, all of these Pseudoterranova infections were diagnosed on the basis of only the morphology of the larvae (8).

Within the genus Pseudoterranova, 8 species have been validated on the basis of molecular and morphologic/biologic characteristics: P. decipiens s.s., P. kogiae, P. ceticola, P. azarasi, P. krabbei, P. bulbosa, P. decipiens E, and P. cattani (9). Among those, 6 species (P. decipiens s.s., P. krabbei, P. bulbosa, P. azarasi, P. decipiens E, and P. cattani) are morphologically and biologically related to each another and designated as the P. decipiens species complex or P. decipiens sensu lato (9). These species can be discriminated by allozyme or molecular genetic analyses (10).

In our study, 12 human pseudoterranoviasis cases were found among patients who visited health check-up centers or hospitals in South Korea during 2002–2020 because of vague abdominal discomfort. Larvae were extracted by using gastrointestinal endoscopy (11 case-patients) or colonoscopy (1 case-patient). The larvae were confirmed to be P. decipiens s.s. by sequence analysis of the mitochondrial cytochrome c oxidase 1 (_cox_1) and NADH dehydrogenase subunit 1 (_nd_1) genes.

The patients consisted of 5 men (41–55 years of age) and 7 women (29–59 years of age). A total of 12 larvae (1 larva from each patient) were collected from the stomach (11 patients) or cecum (1 patient) (Appendix Table 1) and were processed for sequencing of 2 mitochondrial genes (Appendix).

Sequences of the _cox_1 (141 bp) (samples nos. OK539788–OK539799) and _nd_1 (153 bp) genes (OK539800–OK539807) showed high homologies with the sequences of P. decipiens s.s. (GenBank accession no. NC_031645 for _cox_1 and _nd_1). The homology between samples from this study and P. decipiens s.s. was 99.3%–100% for _cox_1 and 97.4%–98.0% for _nd_1 (Appendix Tables 1–3).

Figure

Phylogenetic analyses of Pseudoterranova nematode larvae extracted from 12 health check-up patients in South Korea, 2002–2020 (black dots), in comparison with other anisakid species. A) mitochondrial cytochrome oxidase c gene sequences; B) mitochondrial NADH dehydrogenase gene sequences. Trees were constructed by using the neighbor-joining method based on the Kimura 2-parameter model of nucleotide substitution with 1,000 bootstrap replications and viewed by using MEGA-X (https://www.megasoftware.net). GenBank accession numbers and country of origin are provided for reference sequences. Details of patient information for the 12 samples from this study are provided in Appendix Table 1). Numbers along branches are bootstrap values. Scale bars indicate nucleotide substitutions/site.

Figure. Phylogenetic analyses of _Pseudoterranova_nematode larvae extracted from 12 health check-up patients in South Korea, 2002–2020 (black dots), in comparison with other anisakid species. A) mitochondrial cytochrome oxidase c...

The phylogenetic tree for _cox_1 showed that the 12 study samples were tightly clustered with P. decipiens s.s. reported from Germany but separate from P. bulbosa from Canada, P. cattani from Chile, P. krabbei from Norway, and P. azarasi from Japan (Figure). The phylogenetic tree for _nd_1 showed that 8 study samples were closely aligned with P. decipiens s.s. reported from Germany but clearly separated from P. cattani from Chile, P. bulbosa from Canada, P. krabbei from Norway, and P. azarasi from Japan (Figure). We also determined genetic distances between the study specimens and P. decipiens, P. azarasi, P. bulbosa, P. cattani, and P. krabbei for cox1 (Appendix Table 2) and _nd_1 (Appendix Table 3).

For the specific diagnosis of anisakid larvae, analysis of the larval morphology is highly useful. However, extracting a fully intact larva from human patients for high-quality morphologic analysis is usually difficult. In such instances, molecular analysis of the larvae is helpful and essential for obtaining a specific diagnosis. Analyses of the internal transcribed spacer region and partial 28S rDNA could discriminate P. decipiens s.s. from P. bulbosa, P. krabbei, P. cattani, and possibly P. decipiens E (10). However, great sequence similarity was observed between P. decipiens s.s. and P. azarasi. Thus, it was difficult to distinguish them by using nuclear genes (10). Some investigators used mitochondrial genes, including _cox_1, cox_2, and nd_1, to distinguish them (4,5).

In our study, we used 2 mitochondrial genes, _cox_1 and _nd_1, to distinguish the species of Pseudoterranova. Our results showed that the nematode specimens from these patients nested within P. decipiens s.s. but were clearly separated from P. azarasi, P. bulbosa, P. cattani, and P. krabbei samples available in GenBank. Molecular analysis of larvae will be useful for obtaining specific diagnoses of infection.

Dr. Song is a research scientist at the Institute of Parasitic Diseases at Korea Association of Health Promotion, Seoul, South Korea. Her primary research interests are molecular aspects of parasites and parasitic diseases.

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We thank the endoscopy doctors and staff in the branch offices of the Korea Association of Health Promotion for assistance in collecting P. decipiens sensu stricto larval specimens from 12 health check-up patients, and members of the Institute of Parasitic Diseases, Korea Association of Health Promotion for assistance in processing the specimens for morphologic and molecular studies.

  1. Sohn WM, Chai JY. Anisakiosis (Anisakidosis). In: Palmer SR, Soulsby L, Torgerson PR, Brown DW, editors. Oxford textbook of zoonoses. Oxford (UK): Oxford University Press; 2011. p. 774–6.
  2. Lim H, Jung BK, Cho J, Yooyen T, Shin EH, Chai JY. Molecular diagnosis of cause of anisakiasis in humans, South Korea. Emerg Infect Dis. 2015;21:342–4. DOIPubMedGoogle Scholar
  3. Mattiucci S, Fazii P, De Rosa A, Paoletti M, Megna AS, Glielmo A, et al. Anisakiasis and gastroallergic reactions associated with Anisakis pegreffii infection, Italy. Emerg Infect Dis. 2013;19:496–9. DOIPubMedGoogle Scholar
  4. Nordholm A, Kurtzhals JAL, Karami AM, Kania PW, Buchmann K. Nasal localization of a Pseudoterranova decipiens larva in a Danish patient with suspected allergic rhinitis. J Helminthol. 2020;94(E187):
    e187
    . DOIPubMedGoogle Scholar
  5. Arizono N, Miura T, Yamada M, Tegoshi T, Onishi K. Human infection with Pseudoterranova azarasi roundworm. Emerg Infect Dis. 2011;17:555–6. DOIPubMedGoogle Scholar
  6. Weitzel T, Sugiyama H, Yamasaki H, Ramirez C, Rosas R, Mercado R. Human infections with Pseudoterranova cattani nematodes, Chile. Emerg Infect Dis. 2015;21:1874–5. DOIPubMedGoogle Scholar
  7. Schaum E, Müller W. [Heterocheilidiasis. Human infection with fish ascarides] . Dtsch Med Wochenschr. 1967;92:2230–3. DOIPubMedGoogle Scholar
  8. Sohn WM, Na BK, Kim TH, Park TJ. Anisakiasis: report of 15 gastric cases caused by Anisakis type I larvae and a brief review of Korean anisakiasis cases. Korean J Parasitol. 2015;53:465–70. DOIPubMedGoogle Scholar
  9. Mattiucci S, Nascetti G. Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host-parasite co-evolutionary processes. Adv Parasitol. 2008;66:47–148. DOIPubMedGoogle Scholar
  10. Nadler SA, D’Amelio S, Dailey MD, Paggi L, Siu S, Sakanari JA. Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals. J Parasitol. 2005;91:1413–29. DOIPubMedGoogle Scholar

1These authors contributed equally to this article.

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