A seroepidemiological survey was conducted in Stung Treng province (Sdau) and Kratie province (Kompong Krabei, Kbal Chuor, Sambok, and Talous) during the first survey. We also conducted epidemiological studies in Rokakandal (Kratie province) and Chey Oudam (Rattanakiri province). Studies in these two villages are described in Sections 2) and 3), respectively. We collected blood samples using filter paper and brought them to Japan. The blood samples were examined with sodium metaperiodate (SMP)-ELISA in our laboratory at Dokkyo Medical University School of Medicine. Anti-schistosome antibodies were detected using SMP-ELISA with S. mekongi egg antigens (cut-off value = 0.2).

 The target population of the survey was schoolchildren. We collected a total of 104 blood specimens in Sdau, 126 in Kompong Krabei, 115 in Kbal Chuor, 150 in Sambok, and 135 in Talous. To estimate the incidence rate of schistosomiasis, we collected blood specimens preferentially from children who showed negative results in ELISA carried out in 2005 in Sdau, Kbal Chuor, and Talous. Therefore, it should be noted that there was a sampling bias among children in these three villages. Table 1 shows the result of ELISA with reference to the results of the survey in 2005. Figure 1 shows ELISA values as a scatter plot.

 Positive rates of children who were negative in 2005 were 10.5% (4/38) in Sdau, 43.2% (16/37) in Kbal Chuor, and 3.8% (3/79) in Talous. These positive rates indicate the risk of contact with schistosomes. In addition to the positive rate, a high risk of contracting the disease in Kbal Chuor was indicated based on the ELISA values (Figure 1). Median ELISA values were 0.170 in Kbal Chuor, but only 0.055 in Talous and 0.084 in Sdau. The number of individuals who had an ELISA value of greater than 0.3 in each village was nine in Kbal Chuor, one in Talous, and none in Sdau (Figure 1). A high incidence rate was also found in Kbal Chuor in 2005. Among the 256 children examined in the survey in Kbal Chuor in 2005, 52 had negative ELISA values in 2004. As a result of ELISA examinations in 2005, the positive rate of these 52 children was 30.8% (16/52). In contrast to Kbal Chuor, the endemicity of schistosomiasis in Sdau and Talous is nearly under control.

 Since the group of children who had not been examined in 2005 is considered to be random sampling, their positive rate indicates the degree of endemicity in each area. Positive rates of these children (not examined in 2005) were 43.7% (55/126) in Kompong Krabei, 62.9% (44/70) in Kbal Chuor, 32.7% (49/150) in Sambok, 12.5% (87/56) in Talous, and 18% (11/61) in Sdau. The positive rate and location of each village are shown on the map in Figure 2. These results suggest that schistosomiasis is still endemic in upstream areas in Kratie province, including Kbal Chuor.

 Stool examinations were performed in Kratie province among children who had not been treated with praziquantel during the mass treatment campaign in 2006. Six stool samples were collected in Kbal Chuor, and no schistosome eggs were found using the formalin-detergent technique. In Sdau, we requested stool samples from all the ELISA-targeted children, 92 samples were collected, and no schistosome eggs were detected.

　

 In Kratie province, many people live on houseboats on the Mekong River. Based on 1998 data, the houseboat population in Kratie province was estimated at 548. Generally, the residents are fishers and live close to the river water. Rokakandal is a village located near the town of Kratie. The Rokakandal population is divided into two groups, Rokakandal I and Rokakandal II. Rokakandal II consists of Cambodians. In contrast, Rokakandal I consists of Vietnamese who live in floating houses in what is called the "floating village." Residents of Rokakandal I also have houses on land, in addition to their houseboats. In our previous study in 1998, an ELISA survey was performed in two floating villages in Kratie province. They showed high positive rates of 94.1% (16/17) in Rokakandal and 75% (3/4) in Peam Te. Thereafter, the status of schistosome infection among floating villages was not investigated until 2006.

 In our first survey in April 2006, we conducted a survey in Rokakandal I. Sixty-five blood samples and three stool samples were collected and examined. The ELISA-positive rate was 49.2% (32/65), and S. mekongi eggs were detected in one specimen. Then, it was ascertained that people in the floating village of Rokakandal I are at risk of schistosome infection.

 In the second survey conducted in December 2006, the epidemiological investigation focused on the village of Rokakandal. Surveys were performed in Rokakandal I, Rokakandal II, and Rokakandal School. Most of the children in Rokakandal School are from Rokakandal II, with a few from Rokakandal I. The results of ELISA are shown in Table 2 and Figure 3. As seen in Table 2, the residents of Rokakandal I (floating village) had a positive rate of 29.0% (20/69). In comparison with the 1998 data, the ELISA-positive rate had decreased.

 Stool examinations were conducted using two methods. About 40 mg of a specimen was examined using the Kato-Katz technique by CNM staff in the laboratory of the provincial hospital. Then, about 500 mg of stool sample was transferred to a plastic tube and treated with the formalin-detergent method. Those specimens were subsequently brought to Japan. A formalin-detergent solution was added to the specimen to make a volume of 1 ml, and 40 μl of suspension was observed twice under a　microscope in our laboratory. The results are shown in Table 3. Eggs of S. mekongi were detected in feces from four persons (9.3%) in Rokakandal I. All the schistosome egg-positive individuals also showed positive ELISA values.

 Among the four egg-positive feces samples, eggs were detected in two specimens only using the Kato-Katz method and from the other two specimens only using the formalin-detergent technique. The number of eggs detected in each sample was one egg in three specimens and three eggs in one specimen. These results indicate that the density of schistosome eggs in feces is too low for consistent detection in stool examinations.

 In comparing the two examination methods in detecting the positive rate of hookworm infection, the Kato-Katz method was less sensitive than the formalin-detergent technique (Table 3). This result agrees with our previous study (Report on the Schistosomiasis Control Program in Cambodia, Sasakawa Memorial Health Foundation, 2005). While the formalin-detergent technique has higher sensitivity than the Kato-Katz method, it requires more time, equipment, and labor. It is therefore important to select the appropriate method for each specific survey.

　

 While schistosome infection has not been reported in Rattanakiri province, we detected ELISA-positive children previously. Therefore, we conducted serological diagnosis, stool examinations, and a snail survey to confirm whether undetected infection had occurred.

 The survey in Rattanakiri province was conducted at Chey Oudam School in Lumphat. The Srepok River, a tributary of the Mekong River, runs along the southern boundary of Lumphat. Blood and feces samples were collected from children who showed positive ELISA values in 2005. As shown in Table 1, three of those five children had positive ELISA values, although they were comparatively low (less than 0.4). Stool examinations were performed among four children, and no schistosome eggs were detected.

 The snail survey was conducted in two rocky areas along the Srepok River at: 1) Ta Tay Resort, located 7 km upstream from the town of Lumphat; and 2) Than, located near Lumphat town. N. aperta was not detected in either area. Thus, we must consider the possibility that another factor affected the ELISA results.

　

4) Survey and collection of N. aperta

 A snail survey was conducted in Sdau (Stung Treng province), Krakor (Kratie province), and Lumphat (Rattanakiri province) to determine the distribution of the intermediate host snail N. aperta. Snail collection was performed in the first survey at Sdau and Krakor. The survey in Lumphat was described above (Section 3). In Sdau, we collected snails in a rocky area along the Sekong River. Although we collected approximately 1,000 snails in 2005, we only collected about 100 snails in 2006. We searched for the snails on the river banks around the main port of Sdau and detected a few N. aperta. In Krakor, we collected about 600 snails, which were brought to Japan and used for laboratory maintenance of S. mekongi.

 The snail survey was conducted in Krakor on 17 December during the second survey. In December, although the water level is decreasing after the rainy season from May to October, it is still higher than that in April. The search for the snails was conducted near the riverbed, and small snails resembling N. aperta were collected and brought back to Japan. However, based on the shape of the shell and teeth of the radula, those snails were not N. aperta. In regard to the life of N. aperta during the rainy season, Upatham et al. (1980)^* proposed the "egg survival" theory after observing the snail in the Mekong River in Ubol Ratchathani province, Thailand. In addition, Yasuraoka et al. (1994)^** reported a finding of adult N. aperta in Khong district, Lao PDR, in the high-water season. The bionomics of N. aperta in Cambodia throughout the year are not known.