In the age of globalization of infectious diseases, qualified personnel is needed for the diagnosis of parasitic diseases in the laboratory. This review aimed to introduce the methods for stool examination and identification of helminth eggs for the diagnosis of helminthic infections in laboratory and field surveys. The formalin-ether sedimentation technique (FEST) and the Kato-Katz egg counting technique (KKECT) are mainly described as representative stool examinations. The FEST is somewhat complicated and troublesome, but it is useful for differentiating small trematode eggs from opisthorchiid and heterophyid flukes. KKECT is useful in field surveys of large populations in areas endemic for soil-transmitted helminthiases.Helminth eggs are divided into four groups based on the presence or absence of the operculum and embryo. The eggs of Clonorchis sinensis and heterophyid flukes including Metagonimus spp. are relatively small and contain an operculum and an embryo (miracidium).Meanwhile, eggs of diphyllobothriid tapeworms, echinostomatid flukes, Paragonimus westermani, Fasciola hepatica, and Fasciolopsis buski are relatively larger, operculated, and contain germ cells and yolks instead of the embryo. The eggs of cyclophyllidian tapeworms, Taenia spp. and Hymenolepis spp., and blood flukes, Schistosoma spp., are embryonated but do not have an operculum. Nematode eggs have no operculum and embryo, but those of hookworms and pinworms sometimes have developed larvae inside. This review provides valuable insights into the methods of stool examination and helminth egg identification for the diagnosis of helminthic infections in the laboratory and field surveys.

Human anisakiasis (or anisakidosis) is a disease caused by the ingestion of marine fish or cephalopods infected with anisakid nematode larvae of the genera Anisakis, Pseudoterranova, Contracaecum, and Hysterothylacium. Anisakiasis is a clinically important disease that often manifests as an acute abdominal syndrome requiring emergency medical attention and care. In Korea, at least several thousand clinical cases have been diagnosed to date; however, only a small proportion of them have been reported in the literature (1971-2022). The most common etiological agents were Anisakis pegreffii (reported as Anisakis sp., Anisakis type I, or erroneously Anisakis simplex), followed by Pseudoterranova decipiens, Contracaecum sp., and Anisakis simplex sensu stricto (s.s.). Most cases involved the stomach and small or large intestine, with a few involving the oral cavity (oral mucosa, pharynx, and tonsils), esophagus, omentum, and mesocolic lymph nodes. Anisakis allergies and host immune responses have been studied in humans and experimental animals. Marine fish and cephalopods, including sea eel (Astroconger myriaster), squid (Todarodes pacificus), yellow corvina (Pseudosciaena manchurica), Japanese flounder (Paralichthys olivaceus), codfish (Gadus macrocephalus), yellowtail (Seriola quinquaradiata), and rockfish (Sebastes spp.), are the most common infection sources. Surveys were performed on anisakid nematode larvae in marine fish and cephalopods caught in the western, eastern, and southern seas of Korea. The larvae recovered from fish or cephalopods caught from the western and southern seas were predominantly A. pegreffii larvae; however, the larvae from the eastern sea were either A. pegreffii larvae (in the chub mackerel, Japanese flounder, and rockfish) or A. simplex s.s. (in the salmon and pollock; these fish migrate through the northern North Pacific Ocean and Bering Sea and come to Korea). Health education to avoid eating raw or improperly cooked marine fish and cephalopods (particularly the viscera) is crucial for preventing human anisakidosis in Korea.

This article analyzed the infection characteristics of metacercariae of Metagonimus spp. (MsMc) in fish from 9 major water systems in Korea. A total of 19,568 fish in 87 species were examined over a period of 10 years (2011–2020). MsMc were detected in fish from all 44 survey areas in 9 water systems. Most of the surveyed sites showed very low and low infection levels (66.7%), while 33.3% of the areas, such as Tamjin-gang and Seomjin-gang, revealed moderate and high infection levels. High endemicity depends on the abundance of susceptible fish species, especially sweet smelt (Plecoglosus altivelis). The susceptibility index (SI) with MsMc in index fish, Zacco spp., was very low and low levels in 62.0%, moderate in 28.0%, and high in 10.0% regions. The SI was highest in the following order: Yeongam-cheon (283.8), Hoeng-cheon (192.3), Togyo-jeosuji (131.2), Deokcheon-gang (119.1), and Joyang-gang (106.3). The recent infection status of MsMc in P. altivelis was analyzed by the survey localities. In addition, except for P. altivelis, 9 fish species were highly infected with MsMc in some survey areas, including Zacco platypus, Z. koreanus, Z. temminckii, Opsariichthys uncirostris, Rhynchocypris oxycephalus, Carassius auratus, Acheilognathus rhombeus, Onchorhynchus masou, and Tribolodon hakonensis. In Korea, 74 fish species (15 families) are collectively listed as second intermediate hosts of Metagonimus spp. This review provides several novel characteristics of MsMc infection and clarifies the fish species of second intermediate host of Metagonimus spp. in this country.

Epigenetic writers including DNA and histone lysine methyltransferases (DNMT and HKMT, respectively) play an initiative role in the differentiation and development of eukaryotic organisms through the spatiotemporal regulation of functional gene expressions. However, the epigenetic mechanisms have long been suspected in helminth parasites lacking the major DNA methyltransferases DNMT1 and DNMT3a/3b. Very little information on the evolutionary status of the epigenetic tools and their role in regulating chromosomal genes is currently available in the parasitic trematodes. We previously suggested the probable role of a DNMT2-like protein (CsDNMT2) as a genuine epigenetic writer in a trematode parasite Clonorchis sinensis. Here, we analyzed the phylogeny of HKMT subfamily members in the liver fluke and other platyhelminth species. The platyhelminth genomes examined conserved genes for the most of SET domain-containing HKMT and Disruptor of Telomeric Silencing 1 subfamilies, while some genes were expanded specifically in certain platyhelminth genomes. Related to the high gene dosages for HKMT activities covering differential but somewhat overlapping substrate specificities, variously methylated histones were recognized throughout the tissues/organs of C. sinensis adults. The temporal expressions of genes involved in eggshell formation were gradually decreased to their lowest levels proportionally to aging, whereas those of some epigenetic tool genes were re-boosted in the later adult stages of the parasite. Furthermore, these expression levels were significantly affected by treatment with DNMT and HKMT inhibitors. Our data strongly suggest that methylated histones are potent epigenetic markers that modulate the spatiotemporal expressions of C. sinensis genes, especially those involved in sexual reproduction.

It is uncertain when the head collar and collar spines of Isthmiophora hortensis (Digenea: Echinostomatidae), a zoonotic echinostome species in Far Eastern Asia, develop during its larval stages. In this study, the appearance of the head collar and collar spines was studied using light and scanning electron microscopy in cercariae and metacercariae experimentally obtained from freshwater snails (Lymnaea pervia) and tadpoles (Rana nigromaculata), respectively. The cercariae were shed from the snail on day 30 after exposure to laboratory-hatched miracidia. Metacercariae were obtained from the experimental tadpoles at 3, 6, 12, 15, 20, 24, 26, and 30 h after exposure to the cercariae. The head collar was already visible in the cercarial stage, although its degree of development was weak. However, collar spines did not appear in the cercarial stage and even in the early metacercarial stage less than 24 h postinfection in tadpoles. Collar spines became visible in the metacercariae when they grew older than 24 h. It was concluded that the head collar of I. hortensis developed early in the cercarial stage, but the development of collar spines did not occur until the worms became 24-h-old metacercariae in our experimental setting. Counting the number of collar spines was concluded as an unfeasible diagnostic method for I. hortensis cercariae when they are shed from the snail host.

Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-μ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-μ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 μm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 μm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1μm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.

Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking731Leu–780 Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack.

The infection nature of Clonorchis sinensis metacercariae (CsMc) in freshwater fish hosts is closely related to the transmission of human clonorchiasis. This article reviewed the infection characteristics of CsMc in freshwater fish in the Republic of Korea (Korea). The status of CsMc infection was examined in a total of 17,792 cyprinid fish of 49 species in 9 water systems, which included Hantan-/Imjin-gang, Han-gang, Geum-gang, Mangyeong-gang, Yeongsan-gang, Tamjin-gang, Seomjin-gang, Nakdong-gang, and streams in the east coastal areas from 2010 to 2020. The infection status of CsMc was examined according to cyprinid fish species and water systems, after which analyzed by endemicity and susceptibility index. The high endemicity was shown in the cyprinid fish from 3 regions (6.1%) in the upper reaches of Nakdong-gang, such as Banbyeon-cheon (stream), Yongjeon-cheon, and Wi-cheon. The moderate levels were observed in fishes from 8 regions (16.3%), and low endemicity was shown in fishes from 20 regions (40.8%). No CsMc were detected in fish from 18 regions (36.7%). The susceptibility of CsMc in index fish, Puntungia herzi, was found to be a reliable index without examination of other fish species. CsMc infection rates were closely related to subfamily groups in the cyprinid fish hosts in a highly endemic area. In Korea, a total of 58 fish species in 10 families has been listed as the second intermediate hosts for C. sinensis. This review provides several novel features of CsMc infection and clarifies the species of second intermediate freshwater fish host in Korea.

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yongjeon-cheon (a branch of Nakdong-gang) located in Cheongsong-gun, Gyeongsangbuk-do, the Republic of Korea (Korea). A total of 750 fishes in 19 species were examined by the artificial digestion method for 2 years (2019 and 2020). CsMc were detected in 378 (51.4%) out of 735 fishes in 14 species (73.7%), and the infection intensity was 666 per fish infected. In 2019, CsMc were found in 172 (68.0%) out of 253 fishes in 10 species, and the infection intensity was 565 per fish infected. In 2020, CsMc were detected in 206 (62.2%) out of 331 fishes in 10 species, and the infection intensity was 751 per fish infected. The other zoonotic trematode, ie. Metagonimus spp., Centrocestus armatus, Echinostoma spp. and Clinostomum complanatum, metacercariae were also detected in fishes from the survey streams, but their endemicities were relatively low. Conclusively, it was first confirmed that CsMc are highly endemic in fishes from Yongjeon-cheon in Cheongsong-gun, Gyeongsangbuk-do, Korea.

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1-8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 µm in average diameter (163-190 µm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4-8.2)×1.4 (1.1-1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0-100% homology based on cox1 sequences and 98.9-99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.