Adult Opisthorchis viverrini flukes in humans, Takeo, Cambodia.

To the Editor: Opisthorchis viverrini and Clonorchis sinensis, the 2 major species of small liver flukes (family Opisthorchiidae), cause chronic inflammation in the bile duct, which leads to cholangitis and cirrhosis of the liver, and are a predisposing factor for cholangiocarcinoma (). Human infections with O. viverrini flukes are found along riverside areas of Indochina (Thailand, Lao People’s Democratic Republic [PDR], and Vietnam) ().

Small trematode eggs (length 20–32 μm) have been found in human fecal samples in Cambodia (,,). During 1981–1982, two of 102 Cambodian refugees in the United States were found to be positive for C. sinensis (likely O. viverrini) eggs (). Egg-positive cases were later detected in several provinces of Cambodia (,). Presence of O. viverrini flukes in Cambodia was verified by detection of metacercariae in freshwater fish in a lake on the border between Takeo and Kandal Provinces and by isolation of adult flukes in experimentally infected hamsters ().

In May 2010, we analyzed fecal samples from 1,993 persons in 3 villages (Ang Svay Chek, Kaw Poang, and Trartang Ang) in the Prey Kabas District, Takeo Province, Cambodia, ≈45 km south of Phnom Penh, to confirm the presence of O. viverrini flukes among humans. We found an egg-positive rate of 32.4% for small trematode eggs. Because these eggs may be those of Haplorchis spp. flukes (H. taichui, H. pumilio, and H. yokogawai) and lecithodendriid flukes (Prosthodendrium molenkampi and Phaneropsolus bonnei) (), we attempted to detect adult flukes that are responsible for these eggs.

Six of the small trematode egg–positive villagers, 1 man and 5 women (age range 16–72 years), who had occasional epigastric discomfort were selected for anthelmintic treatment, purgation, and recovery of adult worms. Fecal examination and anthelmintic treatment of villagers were approved by the Ministry of Health, Cambodia, under the agreement with the Korea–Cambodia International Collaboration on Intestinal Parasite Control in Cambodia (2006–2011). After obtaining informed consent, the villagers were treated with a single oral dose of praziquantel, 40 mg/kg (Shinpoong Pharmaceutical Co., Seoul, South Korea), and given a purgament (solution containing 30–40 g MgSO4). Feces was obtained 3 or 4 times in a 2–3-hour period after purgation, pooled individually, and processed as described (). Worms obtained were fixed with 10% formalin, stained with acetocarmine, and identified by morphologic features.

A total of 34 O. viverrini adult worms were obtained from the 6 villagers (14, 9, 5, 3, 2, and 1 from each villager, respectively). No other species of trematodes were obtained. Five worms were lanceolate and had a mean length of 9.5 mm (range 6.5–12.0 mm), a mean width of 1.5 mm (range 1.2–1.7 mm), and 2 characteristic 4–5-lobulated testes (Figure, panel A). Ten eggs in uteri were 27 μm long (range 25–29 μm) and 15 μm wide (range 13–16 μm).

Figure

A) Adult Opisthorchis viverrini liver fluke (length 12.0 mm) isolated from a human after chemotherapy and purgation in Takeo Province, Cambodia, showing the characteristic morphology of the two 4–5-lobulated testes. B) Metacercaria of O. viverrini fluke (diameter 0.22 mm) detected in a freshwater fish (Puntioplites proctozysron). C) Young adult O. viverrini fluke (length 5.5 mm) isolated 6 weeks after experimental infection of a hamster with metacercariae from P. proctozysron fish. Original magnification levels ×8 (A), ×120 (B), ×9 (C).

To detect the source of infection, 2 freshwater fish species, Puntioplites proctozysron (n = 5) and Cyclocheilichthys apagon (n = 10), were caught in nearby Ang Svay Chek village and examined for O. viverrini metacercariae by using a digestion technique (). A total of 50 metacercariae (Figure, panel B) were obtained from 5 P. proctozysron fish and fed to a hamster. Six weeks later, 42 young O. viverrini flukes (Figure, panel C) were isolated from the biliary tract of the hamster.

Our study identified only O. viverrini infections in humans in Cambodia. However, eggs of other hepatic and intestinal flukes also can be found in humans (). In Thailand, Vietnam, and Lao PDR, opisthorchiids (O. viverrini and C. sinensis), heterophyids (Haplorchis spp., Centrocestus formosanus, and Stellantchasmus falcatus), and lecithodendriids have been found in humans (,,). In several provinces in Lao PDR, mixed infections with O. viverrini and heterophyids or lecithodendriids were common (,), and the relative prevalence of each fluke species varied by locality. In Vientiane, Lao PDR, O. viverrini was the predominant species, whereas in Saravane Province, H. taichui predominated (). In a mountainous area of Phongsaly Province, H. taichui and H. yokogawai worms were obtained from 10 villagers; however, no O. viverrini worms were detected (). Thus, in Cambodia, the presence of human infections with intestinal flukes, including Haplorchis spp. and lecithodendriids, cannot be ruled out.