Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.
The liver fluke Opisthorchis viverrini, an important fish-borne parasite , and soil-transmitted nematodes, including Ascaris lumbricoides, Trichuris trichiura, and hookworms, were the major helminth species prevalent among Laotian people [2-5]. However, in 1991, Haplorchis taichui, an intestinal fluke, was reported in Laotian students . Since then, H. taichui and several other intestinal fluke species including Haplorchis pumilio, Haplorchis yokogawai, Phaneropsolus bonnei, and Prosthodendrium molenkampi have been added to the major helminths infecting Laotians, particularly among residents along the Mekong River [7-10].
There is strong locality specificity for helminth species. For example, soil-transmitted nematodes are highly prevalent in northern mountainous areas, including Luangprabang, Phongsaly, Huaphan, and Saysomboune Province, whereas foodborne trematodes, including O. viverrini, and H. taichui and other intestinal flukes, are prevalent along the Mekong River in the middle and southern areas, including Vientiane Municipality, Khammouane, Savannakhet, and Saravane Provinces [5,7-9]. In Vientiane Municipality, O. viverrini is dominant, whereas in Savannakhet Province, O. viverrini and H. taichui are half and half, and in Saravane and Khammouane Provinces, H. taichui is the dominant species .
When we  performed a national survey of intestinal parasites among primary schoolchildren in Lao PDR during 2000 and 2002, we found that the egg positive rate of O. viverrini or minute intestinal flukes (we designated these as small trematode eggs) were considerably higher in 4 villages (Phonxay, Tredsabane, Nathoune, and Phoxayneau) of the Khoua District, Phongsaly Province, the northernmost area of Lao PDR bordering southern China and northwest Vietnam (Fig. 1). The fecal examination results for 269 schoolchildren are shown in Table 1 (not shown in ). The prevalence of small trematode eggs was 27.6-57.4% (av. 48.7%) in addition to the high prevalence of soil-transmitted nematodes (Table 1). The egg positive rates were not significantly different (P > 0.01) between men and women, or between different age groups (data not shown). Unless adult worms are recovered, it is not possible to determine whether the eggs are of the liver fluke, intestinal fluke, or both, because their eggs are very similar in size and shape. Therefore, in the present study, we attempted to determine the kinds of flukes through recovery of adult worms from residents of Phonxay village, a mountainous area of Khoua District, which is located in the southern part of Phongsaly Province, Lao PDR (Fig. 1).
We visited Phonxay village, Khoua District, in December 2008 and explained our purpose of visit to the village leader. Ten volunteers, consisting of 8 men and 2 women aged 31-57 years who frequently consumed raw freshwater fish and experienced at times gastrointestinal troubles such as diarrhea and abdominal discomfort, were recruited randomly. After obtaining informed consent from each person, without preliminary fecal examination for helminth eggs, they were treated with a single oral dose of 40 mg/kg praziquantel (Distocide®, Shinpoong Pharm. Co., Seoul, Korea) and 10 mg/kg pyrantel pamoate (Hangzhou Minsheng Pharm. Group, Hangzhou, China), and then purged with 30 g magnesium sulfate. Whole diarrheic stools passed 4-5 times were pooled individually and processed as previously described . Worms were collected using a glass pipette and then washed in water. Some were fixed in 10% formalin under cover slip pressure, acetocarmine-stained, and morphologically identified using a light microscope. This study was performed under the agreement of Korea-Laos International Collaboration Project on Parasite Control in Lao PDR (2007-2011).
Eight of the 10 treated people expelled 1 or more species of intestinal flukes with or without intestinal nematodes, and 2 were negative for any helminth worms (Table 2). The worms recovered included 861 specimens of H. taichui (from 8 people), 59 of H. yokogawai (6), 1 of P. bonnei (1), 2 of Trichostrongylus sp. (2), 2 of A. lumbricoides (1), 11 of Enterobius vermicularis (3), and 1 strobila with scolex of Taenia saginata (1).
These results suggest that mountainous areas of Phongsaly Province, Laos are not endemic for the liver fluke but are endemic for intestinal flukes, in particular H. taichui and H. yokogawai. The possibility for failure of praziquantel treatment and magnesium salt purgation to expel O. viverrini worms within a short time (5-6 hr) after treatment may be raised. However, we think that such possibility is low because, in our previous studies, the same protocol was applied and substantial numbers of O. viverrini worms were expelled in the diarrheic stools [4,7-9]. In the present study, we could not collect even a single specimen of O. viverrini.
We regret that we were unable to examine freshwater fish caught from the streams nearby the village. To confirm that there is no O. viverrini in this area, surveys of snails and fish are essential. In addition, surveys of H. taichui and H. yokogawai infections in intermediate hosts in this area (in particular, freshwater fish, the source of human infections) are urgently needed to establish preventive and control strategies against these intestinal fluke infections.
The results of the present study again clearly demonstrated that fecal examination is not enough to understand the real status of trematode infections or to determine the species of trematodes endemic in certain areas. This issue has been pointed out in Vientiane Municipality and Saravane Province , Savannakhet Province , and Khammouane Province , Laos. In Vientiane Municipality, where small trematode egg positive rate of some riverside people was 53.3% , the worms expelled after chemotherapy and purgation were a mixture of several different species of trematodes, including O. viverrini, H. taichui, H. pumilio, H. yokogawai, Centrocestus caninus, P. molenkampi, and P. bonnei. Of the 1,656 fluke specimens recovered from 18 persons , O. viverrini comprised of 62.9% (1,041 worms) and 6 other species of trematodes comprised 37.1% (615 worms). Similar results were reported in Savannakhet Province, where people were mixed-infected with approximately 50% O. viverrini and 50% minute intestinal flukes [8,10].
In Saravane Province, where the small trematode egg positive rate of people was 70.8% , the worms expelled were predominantly intestinal flukes (155,207 worms). Of these, H. taichui (153,253 worms) and H. pumilio (1,125 worms) comprised over 99% of all flukes recovered. In Khammouane Province, over 90% (19,149 worms) of trematodes recovered were intestinal flukes and only a small proportion (1,377 worms) was O. viverrini .
The term 'small trematode eggs' should be used when trematode eggs of 20-30 µm in length are detected in fecal examinations of people in southeast Asia, including Laos. In Laos, eggs of O. viverrini (27.1 ± 2.6 µm), H. taichui (27.0 ± 1.7), H. pumilio (30.8 ± 0.8), H. yokogawai (28.6 ± 0.7), P. molenkampi (25.3 ± 1.6), and P. bonnei (26.4 ± 2.4) [11-14] should be included among the list of differential diagnosis. Worm collection after chemotherapy and purgation is essential to draw a precise diagnosis. However, it is tedious, not feasible, and impracticable as a routine procedure. Therefore, other diagnostic techniques, using ultrastructural, molecular, or genetic characteristics, should be developed for differential diagnosis of small trematode eggs in fecal samples.
We thank the staffs of the Luangprabang Provincial Health Department, Luangprabang, Lao PDR, for helping us with this study. This study was supported by BK21 Human Life Sciences, Ministry of Education, Republic of Korea.
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a H. taichui, Haplorchis taichui; H. yokogawai, Haplorchis yokogawai; P. bonnei, Phaneropsolus bonnei; T. trichiura, Trichuris trichiura; A. lumbricoides, Ascaris lumbricoides; E. vermicularis, Enterobius vermicularis; T. saginata, Taenia saginata.