High Prevalence of Echinostoma mekongi Infection in Schoolchildren and Adults, Kandal Province, Cambodia.

A high prevalence of Echinostoma mekongi infection (13.9%; 260/1,876) was found among schoolchildren and adults in Kandal Province, Cambodia, by fecal examination, worm expulsion, and molecular analysis of cox1 and nd1 genes. The source of infection was consumption of Pila sp. snails, a finding confirmed morphologically and molecularly.

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.

High endemicity of Opisthorchis viverrini infection among people in northern Cambodia confirmed by adult worm expulsion.

Opisthorchis viverrini infection is an emerging disease in Cambodia, especially in central and southeastern areas. However, its status in northern areas bordering Lao PDR has been relatively unknown. The present study was performed to investigate the status of O. viverrini infection among people in Preah Vihear and Stung Treng provinces through fecal examination to detect eggs and recovery of adult flukes from some of the egg-positive cases. Fecal examinations were performed on a total of 1101 people from 10 villages in the 2 provinces using the Kato-Katz thick smear technique. For recovery of adult flukes and other helminth parasites 10 volunteers positive for eggs of Opisthorchis viverrini and/or minute intestinal flukes (Ov/MIF), in Kampong Sangkae village, Preah Vihear province, were administered a single oral dose of 40 mg/kg praziquantel plus 5-10 mg/kg of pyrantel pamoate and purged with 40-50 g magnesium salts. Adult trematodes, together with nematodes and cestodes expelled in diarrheic stools were collected under a stereomicroscope or with the naked eye. The proportion of egg-positive cases for overall liver and intestinal helminths was high but not notably different between the 2 provinces, 65.5% in Preah Vihear versus 64.7% in Stung Treng. The average proportion of Ov/MIF egg-positive cases was 59.8%. A total of 315 adult specimens of O. viverrini were recovered from the 10 volunteers (4-98 specimens per individual; mean, 32). A smaller number of Haplorchis taichui adults, an intestinal fluke, were found mixed-infected in 7 (103 specimens in total; 1-31 per individual; mean, 15) of the 10 volunteers. Adult specimens of hookworms, Enterobius vermicularis, Trichostrongylus sp., and a Taenia tapeworm strobila were recovered in some cases. Based on the results, it has been confirmed that the surveyed areas in Preah Vihear and Stung Treng provinces, Cambodia, are highly endemic areas of O. viverrini infection with a low-grade mixed infection with H. taichui.

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.

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.

Detection of Gnathostoma spinigerum Advanced 3rd-Stage Larvae in the Chinese Edible Frog, Hoplobatrachus rugulosus, from Local Markets in Phnom Penh, Cambodia.

The Chinese edible frogs, Hoplobatrachus rugulosus (n=20), and the striped snakehead fish, Channa striata (n=34), were purchased from local markets in 3 administrative regions of Cambodia (Phnom Penh, Pursat, and Takeo Provinces) from May 2017 to April 2019, and their infection status with Gnathostoma sp. larvae was investigated. The frogs and fish were transported to the laboratory with ice and examined using the artificial digestion method. Advanced 3rd-stage larvae (AdL3) of Gnathostoma spinigerum, 24 in total number (1-6 larvae/frog), were detected from 6 (60.0%) out of 10 frogs purchased from Phnom Penh. No gnathostome larvae were detected in 10 frogs purchased from Takeo Province and 34 snakeheads from Phnom Penh, Pursat, and Takeo Provinces. AdL3 isolated from the frogs were 2.55- 3.90 mm long and 0.31-0.36 mm wide. They had a characteristic head bulb (0.081×0.191 mm in average size) with 4 rows of hooklets, a muscular long esophagus (0.950-1.230 mm long), and 2 pairs of cervical sacs (0.530-0.890 mm long). The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. These features were consistent with G. spinigerum AdL3. By the present study, it has been first confirmed that the Chinese edible frog, H. rugulosus, from Phnom Penh serves as a second intermediate host for G. spinigerum, although their intensity of infection was not so high compared to other previously reported localities.

Prevalence and Intensity of Opisthorchis viverrini Metacercarial Infection in Fish from Phnom Penh, Takeo, and Kandal Provinces, Cambodia.

The prevalence and intensity of Opisthorchis viverrini metacercariae (OvMc) were investigated in fish from 3 southern administrative regions along the Mekong River in Cambodia, i.e., Phnom Penh, Takeo, and Kandal Provinces from 2017 to 2020. A total of 295 freshwater fish (24 species) were transported to our laboratory with ice and examined using the artificial digestion method. In Phnom Penh, among 4 fish species positive for OvMc, 9 (23.7%) of 38 specimens examined were infected, and their intensity of infection averaged 4.3 metacercariae per infected fish. In Takeo Province, among 10 fish species positive for OvMc, 24 (38.1%) out of 63 fish examined were infected, and their intensity of infection was av. 14.4 metacercariae per infected fish. In particular, all of 3 Osteochilus schlegelii fish examined were infected, and their infection intensity was high, 34.7 metacercariae per fish. In Kandal Province, among 6 fish species positive for OvMc, 46 (90.2%) out of 51 specimens examined were infected, and their infection intensity was 24.0 metacercaraie per infected fish. All fish of Systomus orphoides (n=17), Barbonymus altus (n=14), and Rasbora aurotaenia (n=2) were infected, and their intensity of infection averaged 37.7, 21.6, and 18.5 metacercariae per fish, respectively. Metacercariae of Haplochis yokogawai, Haplorchis taichui, and Centrocestus formosanus were detected in fish from Takeo and Kandal Provinces. From these results, it has been confirmed that a variety of fish species from Phnom Penh, Takeo, and Kandal Provinces are commonly infected with OvMc, and preventive measures to avoid human O. viverrini infection should be performed in Cambodia.

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.

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.

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.