Standard- and large-sized eggs of Trichuris trichiura in the feces of schoolchildren in the Yangon Region, Myanmar: Morphological and molecular analyses.

Standard- and large-sized eggs of Trichuris trichiura were found in the feces of schoolchildren in Yangon, Myanmar during epidemiological surveys and mass deworming with albendazole in 2017-2019. The standard-sized eggs were identified as those of T. trichiura, but it was necessary to exclude the possibility of the large-sized eggs belonging to Trichuris vulpis, a dog whipworm. We conducted morphological and molecular studies to determine the species of the 2 types of Trichuris eggs. Individual eggs of both sizes were isolated from Kato-Katz fecal smears (n=20) and mechanically destroyed using a 23G injection needle. Nuclear DNA was extracted, and the 18S rRNA region was sequenced in 15 standard-sized eggs and 15 large-sized eggs. The average size of standard-sized eggs (T. trichiura) was 55.2×26.1 µm (range: 51.7-57.6×21.3-28.0 µm; n=97), whereas the size of large-sized eggs was 69.3×32.0 µm (range: 65.1-76.4×30.1-34.5 µm; n=20), slightly smaller than the known size of T. vulpis. Regarding standard-sized eggs, the 18S rRNA nucleotide sequences exhibited 100% homology with T. trichiura deposited in GenBank and 88.6-90.5% homology with T. vulpis. Regarding large-sized eggs, the nucleotide sequences showed 99.8-100% homology with T. trichiura in GenBank and 89.6-90.7% homology with T. vulpis. Both standard- and large-sized eggs of Trichuris spp. found in Myanmar schoolchildren during 2017-2019 were morphologically and molecularly confirmed to belong to T. trichiura. The conversion of eggs from smaller to large sizes might be due to anthelmintic treatments with albendazole.

Prevalence of head louse infestation among primary schoolchildren in the Republic of Korea: nationwide observation of trends in 2011-2019.

Head louse infestation is a significant public health problem across the world, particularly among preschool and primary schoolchildren. This study investigated the trends of head louse infestation in the Republic of Korea over a 9-year period (2011-2019), targeting primary schoolchildren in 3 areas of Seoul, 4 other large cities, and 9 provinces. A survey was administered annually by the health staff of each regional office (n= 16) of the Korea Association of Health Promotion (KAHP). The branch offices of KAHP examined a total of 51,508 primary schoolchildren, comprising 26,532 boys and 24,976 girls. Over the 9-year survey, a total of 1,107 (2.1%) schoolchildren tested positive for adults and/or nits of Pediculus humanus capitis. The prevalence was 2.8% (133/4,727) in 2011-2012 and gradually decreased to 0.8% (49/6,461) in 2019 (P< 0.05). Head lice were found more frequently in girls (3.0%; 746/24,976) than in boys (1.4%; 361/26,532) (P< 0.05). In terms of geographic localities, the highest infestation rate, 4.7% (average prevalence over 9 years), was observed in southern Seoul (Gangnam branch of KAHP), whereas the lowest infestation rate, 0.7%, was seen in Gyeongsang (north and south provinces) and western Seoul. Although the prevalence decreased significantly during the 9-year period, head louse infestation remains a health and hygiene issue among primary schoolchildren in the Republic of Korea. Regular surveys along with health education are needed to further improve children's hair hygiene.

Molecular Detection of Haplorchis pumilio Eggs in Schoolchildren, Kome Island, Lake Victoria, Tanzania.

A survey of intestinal helminths targeting 1,440 schoolchildren in 12 primary schools on Kome Island (Lake Victoria), Tanzania, revealed small trematode eggs in 19 children (1.3%), seemingly of a species of Haplorchis or Heterophyes. The eggs were molecularly confirmed to be Haplorchis pumilio on the basis of 18S and 28S rDNA sequences.

Green vegetable juice as a potential source of human fascioliasis in Korea.

Fascioliasis, a food-borne helminthiasis, is primarily a disease of cattle and sheep that occasionally occurs in humans. The aquatic perennial herb water dropwort (Oenanthe javanica) has been identified as the primary source of human infections in Korea. Recently, a cluster of patients who had not ingested water dropwort, but had the use of a green vegetable delivery service in common was diagnosed with fascioliasis. Our study aimed to identify the association between the green vegetable juice delivery service and the occurrence of human fascioliasis. Patients with liver abscesses and eosinophilia were enrolled in this study. They were categorized into fascioliasis or non-fascioliasis groups according to serological test results, clinical manifestations, and computed tomography or magnetic resonance imaging findings. Patients were classified into the fascioliasis group when ova or the adult worms of the Fasciola species were detected or serological tests were positive, with compatible clinical and radiological findings. We included 30 patients in this study; 15 were assigned to the fascioliasis group and the remaining 15 to the non-fascioliasis group. The proportion of patients who utilized the juice delivery service was significantly higher in the fascioliasis group than in the non-fascioliasis group (53.3% vs. 0%, P < 0.01). Most of the other patients in the fascioliasis group had known risk factors and the intake of water dropwort or other raw vegetables. This study suggests that human fascioliasis could be transmitted by green vegetable juice produced and delivered by modern industrial systems. Further research on the product, industry and farm-level situations is required to validate these findings.

Exosomal miRNA-21 from Toxoplasma gondii-infected microglial cells induces the growth of U87 glioma cells by inhibiting tumor suppressor genes.

Toxoplasma gondii is an intracellular protozoan parasite that can modulate the microenvironment of infected hosts and is known to be associated with the incidence of brain tumor growth. In this study, we suggested that the exosomal microRNA-21 derived from Toxoplasma infection would contribute to the growth of brain tumors. Exosomes of BV2 microglial cells infected with Toxoplasma were characterized and confirmed internalization to U87 glioma cells. Exosomal miRNA expression profiles were analyzed using microRNA array and miR-21A-5p associated with Toxoplasma and tumor sorted. We also examined the mRNA level of tumor-associated genes in U87 glioma cells by changing the level of miR-21 within exosomes and the effects of exosomes on the proliferation of human U87 glioma cells. Expression of miRNA-21 was increased and anti-tumorigenic genes (FoxO1, PTEN, and PDCD4) were decreased in exosomes within T. gondii-infected U87 glioma cells. Toxoplasma-infected BV2-derived exosomes induced proliferation of U87 glioma cells. The exosomes induced the growth of U87 cells in a mouse tumor model. We suggest that the increased exosomal miR-21 from Toxoplasma-infected BV2 microglial cells may play an important role as a cell growth promotor of U87 glioma cells through a down-regulation of anti-tumorigenic genes.

Molecular Diagnosis of Pseudoterranova decipiens Sensu Stricto Infections, South Korea, 2002‒2020.

Human Pseudoterranova decipiens larval infections were diagnosed by molecular analysis of mitochondrial cox1 and nd1 genes in 12 health check-up patients in South Korea during 2002-2020. Based on high genetic identity (99.3%-100% for cox1 and 96.7%-98.0% for nd1), we identified all 12 larvae as P. decipiens sensu stricto.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) from Experimental Chicks Infected with Metacercariae Encysted in Brackish Water Clams in the Republic of Korea.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203-0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89-3.55) mm long and 0.68 (0.61-0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.

Enterobius vermicularis Infection among Preschool Children: A 12-Year (2008-2019) Survey in Large Cities and Provinces of the Republic of Korea.

A 12-year nationwide survey (2008-2019) was performed to investigate the prevalence of Enterobius vermicularis infection among preschool children in Seoul, 4 large cites (Busan, Incheon, Daegu, and Ulsan), and 9 provinces (grouped into 5 localities) in the Republic of Korea (=Korea). The survey was carried out once a year by 16 regional offices of the Korea Association of Health Promotion. The cello-tape perianal swab method (1 smear per child) was applied to detect eggs of E. vermicularis and other helminths. According to the results, the egg positive rate of E. vermicularis infection in 2008-2009 was 1.8-2.0%, but it decreased gradually to 0.6% in 2019 (P<0.05). The prevalence was significantly higher in boys (0.7-5.0%, mean 1.8%) than in girls (0.5-2.8%, mean 1.3%) (P<0.05). The 2 most southern localities, Jejudo (Province) and Jeolla-do (inclusive of Jeollabuk-do and Jeollanam-do) and a mid-western province, Gyeonggi-do, revealed higher prevalences, whereas Seoul and Gangwon-do showed lower prevalences. The results indicate that a low-grade prevalence of E. vermicularis infection (less than 4%) has been maintained for the recent 12 years among preschool children in Korea. Continuous monitoring of enterobiasis in the child age group is necessary in Korea.

Genotypes of Blastocystis sp. among elderly health checkup people in South Korea with a questionnaire on risk factors.

Blastocystis sp. is a common zoonotic intestinal parasite of humans and animals, and has been classified into at least 17 distinct subtypes. Despite its potential impact on public health, the prevalence and subtype distribution of Blastocystis sp. have seldom been the study subject in South Korea. To determine the prevalence and subtype distribution of Blastocystis sp. and to obtain information on risk factors, we performed a cross-sectional study targeting elderly health checkup people, who visited Seoul Western Branch of the Korea Association of Health Promotion (KAHP) in October 2019. Stool samples were collected from 293 participants consisting of 128 males and 165 females with a mean age of 64.7 years (from 50 to 88 years) with a questionnaire on potential risk factors. All the samples were tested by PCR targeting the SSU rRNA gene of Blastocystis sp., and nucleotide sequences of positive samples were used to identify the subtypes. The overall prevalence of Blastocystis sp. was 9.2% (27/293). Among the positive samples, subtype 3 was predominant (59%; 16/27), followed by subtype 1 (41%; 11/27). No other subtypes were detected. In the univariable analysis, the age, sex, presence of digestive symptoms, source of drinking water, and history of drug intake were not significantly associated with Blastocystis sp. infection. Two parameters, including the Enterococcus hirae bacterial infection and the frequency of intake of cooked or boiled vegetables less than twice a week, showed statistical significance. However, the multivariable regression analysis revealed that only the latter parameter was statistically significant. The results suggested that subtypes 3 and 1 are the 2 major genotypes of Blastocystis sp. among elderly people in South Korea, and low frequency of consuming cooked or boiled vegetables is a potential risk factor.

Occurrence of a Hybrid Between Taenia saginata and Taenia asiatica Tapeworms in Cambodia.

Human infection with Taenia asiatica or a hybrid between Taenia saginata and T. asiatica has not been reported in Cambodia. We detected for the first time a hybrid form between T. saginata and T. asiatica in Preah Vihear Province, Cambodia. An adult tapeworm specimen, i.e., 75 cm long strobila without scolex, was expelled from a 27-year-old man after praziquantel medication and purging. It was morphologically indistinguishable between T. saginata and T. asiatica. Several proglottids were molecularly analyzed to confirm the tapeworm species. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (cox1) and nuclear genes encoding elongation factor-1α (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp) were sequenced, and a single-allele analysis was performed to confirm the haploid genotype. The results revealed that our sample showed a discrepancy between the mitochondrial and 2 nuclear genes. It possessed homozygous sequences typical of T. saginata at cox1 and ef1 loci. However, it was heterozygous at the elp locus, with 1 allele in T. asiatica (elpA) and 1 in T. saginata (elpC), which indicates that it is a hybrid between T. saginata and T. asiatica. The present results confirmed the presence of a hybrid between T. saginata and T. asiatica in Cambodia and strongly suggest the existence of also 'pure' T. asiatica in Cambodia.

Echinostoma miyagawai Ishii, 1932 (Echinostomatidae) from Ducks in Aceh Province, Indonesia with Special Reference to Its Synonymy with Echinostoma robustum Yamaguti, 1935.

Adult echinostomes having 37 collar spines collected from the intestine of Pitalah ducks in Aceh Province, Indonesia in 2018 were morphologically and molecularly determined to be Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae). Among 20 ducks examined, 7 (35.0%) were found to be infected with this echinostome, and the number of flukes collected was 48 in total with average 6.9 (1-17) worms per duck. The adult flukes were 7.2 (6.1-8.5) mm in length and 1.2 (1.0-1.4) mm in width (pre-ovarian or testicular level) and characterized by having a head collar armed with 37 collar spines (dorsal spines arranged in 2 alternating rows), including 5 end group spines, and variable morphology of the testes, irregularly or deeply lobed (3-5 lobes) at times with horizontal extension. The eggs within the worm uterus were 93 (79-105) µm long and 62 (56-70) µm wide. These morphological features were consistent with both E. miyagawai and Echinostoma robustum, for which synonymy to each other has been raised. Sequencing of 2 mitochondrial genes, cox1 and nad1, revealed high homology with E. miyagawai (98.6-100% for cox1 and 99.0-99.8% for nad1) and also with E. robustum (99.3-99.8% for nad1) deposited in GenBank. We accepted the synonymy between the 2 species and diagnosed our flukes as E. miyagawai (syn. E. robustum) with redescription of its morphology. Further studies are required to determine the biological characteristics of E. miyagawai in Aceh Province, Indonesia, including the intermediate host and larval stage information.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea.

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) µm long and 101-213 (av. 157) µm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) µm in total length and 35-46 (av. 40) µm in width, with 69-92 (av. 85) µm long body and 39-54 (av. 49) µm long tail. The metacercariae were 210-250 (av. 231) µm in length and 170-195 (av. 185) µm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

Molecular Diagnosis of Taenia saginata Tapeworms from Two Residents of Northern Cambodia.

Taenia saginata infection has seldom been reported in Cambodia. In this study, we performed a survey of intestinal parasites in 1,156 residents of Preah Vihear and Stung Treng Provinces in 2018. The results revealed that 26 (2.4%) cases were positive for Taenia spp. eggs. In order to obtain the strobilae of the tapeworms, 2 patients in Preah Vihear were treated with praziquantel and purged with magnesium salts. The proglottids expelled after the medication were morphologically and molecularly analyzed to determine the species. The main uterine lateral braches in gravid proglottids were >15 in number suggesting that they are either T. saginata or Taenia asiatica. The sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and 2 nuclear loci, elongation factor-1 alpha (ef1) and ezrin-radixin-moesin-like protein (elp), were identical to the sequences of T. saginata available in GenBank but distant from Taenia solium, T. asiatica, and T. saginata-T. asiatica hybrid. This is the first report of the presence of T. saginata in the northern part of Cambodia bordering Lao PDR based on a molecular confirmation.

Echinostoma aegyptica (Trematoda: Echinostomatidae) Infection in Five Riparian People in Savannakhet Province, Lao PDR.

Human infection with Echinostoma aegyptica Khalil and Abaza, 1924 (Trematoda: Echinostomatidae) is extremely rare. In this study, we confirmed E. aegyptica infection in 5 riparian residents living along the Mekong River in Savannakhet Province, Lao PDR. The patients revealed eggs of Opisthorchis viverrini/minute intestinal flukes, echinostomes, and other parasites in fecal examinations using the Kato-Katz technique. Following treatment with praziquantel 30-40 mg/kg and pyrantel pamoate 10-15 mg/kg in a single dose and purging with magnesium salts, adult specimens of various helminth species were collected. Among the trematodes, echinostome flukes of 4.5-7.6 mm in length (n = 134; av. 22.3 specimens per case) were of taxonomic interest and subjected in this study. The flukes were morphologically characterized by having total 43-45 collar spines arranged in 2 alternating rows (corner spines usually 5 on each side) and compatible with previous descriptions of E. aegyptica. The patients were mixed-infected with other helminths, so specific clinical manifestations due to this echinostome fluke were difficult to determine. The present paper describes for the first time human E. aegyptica infections in Lao PDR. This is the second report of human infection (2nd-6th cases) with E. aegyptica in the world following the first one from China.

Morphological and Molecular Confirmation of Parvatrema duboisi Metacercariae in the Manila Clam Ruditapes philippinarum from Gochang-gun, Korea.

Gymnophallid metacercariae found in the Manila clam Ruditapes philippinarum ('Banjirak' in Korean) from Gochang-gun, Jeollabuk-do, Korea were morphologically and molecularly confirmed to be Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974. The metacercariae were morphologically characterized by having a large oral sucker, small ventral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, which were all compatible with P. duboisi. Some of the metacercariae were experimentally fed to mice, and adult flukes were recovered at day 7 post-infection. The morphology of the adult flukes was basically the same as that of the metacercariae except for the presence of uterine eggs; the uterus was filled with up to 40 eggs. The nucleotide sequences (1,193 bp) from ITS regions (ITS1, 5.8S rDNA, and ITS2) of the metacercariae showed 99.7% identity with P. duboisi and 75.7% identity with Gymnophalloides seoi deposited in GenBank. These results confirmed the presence of P. duboisi metacercariae in the Manila clam R. philippinarum in an estuary region of Gochang-gun, Korea.

Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia.

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5-10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.

Echinochasmus caninus n. comb. (Trematoda: Echinostomatidae) Infection in Eleven Riparian People in Khammouane Province, Lao PDR.

Adult specimens of Echinochasmus caninus n. comb. (Verma, 1935) (Trematoda: Echinostomatidae) (syn. Episthmium caninum Yamaguti, 1958) were recovered from 11 riparian people who resided along the Mekong River in Khammouane Province, Lao PDR. In fecal examinations done by the Kato-Katz technique, the cases revealed eggs of Opisthorchis viverrini/minute intestinal flukes, hookworms, and in 2 cases echinostome eggs. To recover the adult helminths, praziquantel 30-40 mg/kg and pyrantel pamoate 10-15 mg/kg in a single dose were given and purged with magnesium salts. Various species of trematodes (including O. viverrini and Haplorchis spp.), cestodes, and nematodes were recovered from their diarrheic stools. Among the trematodes, small echinostome flukes (n=42; av. 3.8 specimens per case) of 0.7-1.2 mm in length are subjected in this study. They are morphologically characterized by having 24 collar spines interrupted dorsally and anterior extension of vitellaria from the cirrus sac or genital pore level to the posterior end of the body. Particularly based on this extensive distribution of vitellaria, the specific diagnosis was made as Echinochasmus caninus. The cases were co-infected with various other helminth parasites; thus, clinical manifestations specific for this echinostome infection were difficult to determine. The present paper describes for the first time human E. caninus infections in Lao PDR. Our cases marked the 4-14th human infections with this echinostome around the world following the 3 previous cases reported from Thailand.

Anisakid Larvae from Anchovies in the South Coast of Korea.

Anisakiasis (anisakidosis) refers to a foodborne zoonosis caused by ingesting raw or undercooked marine fish or cephalopods infected with anisakid larvae. The present study was performed to investigate the prevalence of anisakid larvae in anchovies (Engraulis japonica) purchased from 2 local markets in Gyeongsangnam-do, the Republic of Korea (=Korea), during 2018-2019. Anchovies were transported to our laboratory and examined by pepsin-HCl artificial digestion technique followed by microscopic observations and molecular analyses. The overall prevalence of anisakid larvae was 19.5% (39/200), from which a total of 51 larvae (av. 1.3 larvae/infected anchovy) were recovered. Sequencing of the larvae targeting the ITS region, including ITS1, 5.8S rRNA, and ITS2 genes confirmed the species of larvae as Anisakis pegreffii (54.9%; 28/51), Hysterothylacium sinense (23.5%; 12/51), and Hysterothylacium aduncum (21.5%; 11/51). The results suggested that anchovies could be a potential source of human anisakiasis in Korea.