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.

E chinostomiasis is a disease caused by infection with echinostome flukes (Echinostomatidae) and is characterized by intestinal inflammation accompanied by mucosal ulceration and bleeding (1,2).Echinostomiasis, a typical example of a foodborne helminthiasis, is contracted by consuming raw or improperly cooked snails, bivalves, fish, or amphibians (1,2).This disease has been neglected mainly because of underestimated prevalence and worm burden (global prevalence and burden unknown) as well as underrecognized clinical and public health significance.In South Korea and Japan, patients infected with the echinostome Isthmiophora hortensis reported gastrointestinal issues, and diagnosis was established after physicians extracted adult worms via gastrointestinal endoscopy (1).
Echinostoma mekongi was described as a new human-infecting echinostome that emerged in Kratie and Takeo Province, Cambodia, and identified through morphologic and molecular analyses (3).The adult flukes were recovered from persons residing along the Mekong River in these provinces, who reported abdominal discomfort, indigestion, and other gastrointestinal troubles (3).The metacercarial stage of E. mekongi was detected in freshwater snails, Filopaludina martensi cambodjensis, a popular food item in Pursat Province (4).We found a highly endemic area of E. mekongi infection in riverside villages of Kandal Province (surrounding Phnom Penh, the capital; population ≈1.27 million).Adult flukes were expelled after chemotherapy and purging and then analyzed morphologically and molecularly (cox1 and nd1 genes).Freshwater snails, Pila sp., were verified to be the source of infection, but the first intermediate host and the natural definitive host other than humans remain unknown.
We recruited 8 schoolchildren and 2 adult volunteers for the recovery of E. mekongi adult flukes (Table 2) and administered a single oral dose of 10-15 mg/ kg praziquantel (Shin Poong Pharm.Co., https:// shinpoong.co.kr/en/main/main.php),followed by purging with 20-30 g magnesium sulfate.We collected whole diarrheic stools 3 to 5 times and pooled them individually.We fixed adult flukes in 10% formalin, stained the samples with acetocarmine, cleared each in glycerin-alcohol, and mounted the samples in glycerin jelly.We kept some samples in 70%-80% ethanol for molecular analyses.
We recovered 48 adult and 38 juvenile specimens (86 in total) of E. mekongi flukes from the 10 volunteers (Table 2).Schoolchildren (n = 8) expelled a total of 64 worms (8 per child), and adults (n = 2) passed a total of 22 worms (11 per person) (Table 2).The adult flukes (Figure 1, panel B) were elongated and leaflike, with small head collars and small collar spines (37 in 2 alternating rows; 5 corner spines), globular or slightly lobed testes, vitelline follicles not merging near the posterior end, and 7.7-11.2(average 9.5) mm  We purchased Pila sp.snails (Figure 1, panels C and D) at a local market in Kandal Province and examined them for metacercariae by using the crushing method.We detected 10 metacercariae in 5 (7.1%) of 70 snails examined.The metacercariae (n = 5) were round, 165-188 (average 176) μm in diameter (Figure 1, panel E), and encysted with a thin, pinkish, refractile wall.The metacercariae were equipped with a total of 37 collar spines, oral and ventral suckers, excretory granules, and other internal organs.

Conclusions
Large trematode eggs, particularly, those of echinostomes, have been detected in various localities of Cambodia (7)(8)(9)(10)(11).In Pursat Province, echinostome eggs were found in 56 schoolchildren, and the worms expelled from 4 volunteers were assigned as E. revolutum by morphologic analysis (7).We think, however, that those worms might have been E. mekongi because E. mekongi and E. revolutum are morphologically close and almost indistinguishable (3).Molecular studies are necessary to draw a definite conclusion on the species of those echinostomes.In Oddar Meanchey Province, the eggs of echinostomes were detected in 13 persons, and the adult flukes expelled were confirmed to be Echinostoma ilocanum flukes, having 49-51 collar spines (8).Echinostome eggs were also detected in 71 persons in Kratie Province (9) and 52 persons in Takeo Province (10), and 6 volunteers were confirmed to be infected with E. mekongi flukes by morphologic and molecular analyses (3).
A previous study of persons in Kandal Province, Cambodia, found a high prevalence (46.5%; 106/228) of large trematode eggs (suggested to be Echinostoma spp.) among schoolchildren (5-18 years of age), but no adult worm recovery nor molecular analysis was performed (11).By the time of our study, it was confirmed that E. mekongi infection is highly prevalent among schoolchildren and adults in Kandal Province.The recovery of both juvenile and adult flukes may indicate the continuity of infection in this village.Freshwater snails of Pila sp. were proven to be the source of infection.It is speculated that E. mekongi infection might be prevalent not only in other localities of Cambodia but also in neighboring countries (Thailand, Laos, and Vietnam) along the Mekong River and its tributaries.Avoidance of consuming raw or undercooked Pila sp.snails is a preventive measure for this emerging parasitic infection in those areas.

Figure 1 .
Figure 1.Study area and specimens of Echinostoma mekongi flukes and Pila sp.snails for study of E. mekongi infection in schoolchildren and adults, Kandal Province, Cambodia.A) Study area in Cambodia.B) Adult specimen of E. mekongi fluke expelled from a volunteer after chemotherapy and purging.Scale bar = 1.2 mm.C, D) Pila sp.snails purchased from a local market in Kandal Province, showing variable sizes.The presence of metacercariae in these snails was confirmed.Scale bar in panel D = 3 cm.E) Metacercaria of E. mekongi encysted in the tissue of a Pila sp.snail, showing its characteristic structures, including 37 collar spines (arrows), oral sucker, ventral sucker, and excretory granules.Scale bar = 50 m.EG, excretory granules; OS, oral sucker; OV, ovary; T, testis; VS, ventral sucker.

Figure 2 .
Figure 2. Phylogenetic trees of cox1 (A) and nd1 (B) genes of Echinostoma mekongi adults (n = 6) extracted from volunteers and metacercaria (n = 1) extracted from Pila sp.snails for study of E. mekongi infection in schoolchildren and adults, Kandal Province, Cambodia.Sequences from this study (shades boxes) are shown in comparison with other 37-collar-spined Echinostoma spp.(outgroup;Opisthorchis viverrini).The trees were constructed using the maximum-likelihood method, employing the Tamura-Nei model of nucleotide substitution with 1,000 bootstrap replications and viewed in MEGA X (https://www.megasoftware.net).GenBank accession numbers are given for all sequences.Scale bars indicate substitutions per site.

Table 2 .
Worm expulsion after praziquantel treatment and purging from volunteers positive for Echinostoma mekongi eggs in fecal examinations in study of Echinostoma mekongi infection in schoolchildren and adults, Kandal Province, Cambodia* All case-patients were female.Fecal samples were collected individually 2-3 h after praziquantel administration and purging with MgSO4.†Eggs/g of feces; amount in a typical smear was assumed to be 41.7 mg.‡All recovered worms were adults that contained eggs except for 38 of 46 worms from schoolchildren case 1, which were juvenile or young adults containing no or only a few uterine eggs.§Adult specimens of Enterobius vermicularis (120 female worms in schoolchildren no.7 and 1 female worm in adult no. 2) were collected simultaneously *