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.

. Map showing the surveyed areas of Preah Vihear and Stung Treng province, Cambodia. Ten villages (small blue triangles) located along the tributaries of the Mekong River were included in this study.
Age-and sex-specific prevalence and EPG of Ov/MIF eggs. The prevalence and intensity of infection in EPG of Ov/MIF eggs showed a trend by age ( Fig. 2A, B). The average prevalence and EPG (both males and females) were the lowest in the age group 0-9 years, 33.8% (27.9-39.9%) and 408 (300-572), followed by the age groups 10-19 years, 57.5% (52. .2%) and 717 (602-863), 20-29 years, 72.6% (64.7-79.6%) and 730 (570-956), and 30-39 years, 77.8% (68.9-85.2%) and 1034 (770-1433), respectively. Then, the prevalence became steadily high showing a plateau, 78.9-80.0%, in the age groups between 40 and 49 and ≥ 60 years ( Fig. 2A). Meanwhile, the mean EPG showed the highest value at 40-49 years, 2273 (1505-3664), followed by a reduction in the ages 50-59 and ≥ 60 years groups (Fig. 2B). The age-prevalence and intensity of infection patterns were almost similar in males and females ( Fig. 2A Adult flukes and other helminths recovered from volunteers in Preah Vihear province. The adult worm recovery was successfully performed in 10 volunteers (5 men and 5 women; aged 21 to 45 years) who were positive for Ov/MIF eggs (Fig. 3), with EPG higher than 240. Eight kinds of helminths, including 2 trematode species, 5 nematode species, and 1 cestode species, were recovered from their diarrheic stools (Table 2). A total of 315 (4-98 per individual) adult specimens of O. viverrini were collected from 10 volunteers (Table 2; Fig. 4A), and a total of 103 adult specimens (1-31 per individual) of H. taichui (Fig. 4B) were collected from 7 of the 10 volunteers ( Table 2). The number of adult worms of O. viverinii and H. taichui recovered in each case showed a strong positive correlation (r = 0.85; P = 0.0019) with the EPG (eggs per gram of feces) (Ov/MIF eggs) of each case in the fecal examination (Table 2). EPG was measured by counting the total number of eggs on each Kato-Katz fecal smear, which was multiplied by 24, assuming that the volume of feces on each Kato-Kato smear was 41.7 mg 14 . In addition, 70 adult specimens of hookworms (64 Necator americanus and 6 Ancylostoma ceylanicum) were collected from 7 volunteers (reported separately by Chang et al. 15 ), 57 Enterobius vermicularis were from 6 volunteers, 9 Trichostrongylus sp. from 3 volunteers, 1 strobila of Taenia saginata from 1 volunteer (reported separately by Chang et al. 16 ), and 1 unknown nematode from 1 volunteer (Table 2).  A prominent age tendency is recognizable, but no significant difference is seen between males (n = 432) and females (n = 669).   The high prevalence of O. viverrini in the surveyed areas of Preah Vihear and Stung Treng province is comparable with those reported in Lao PDR, the most highly endemic country ever known [18][19][20][21][22] . The reported prevalence of Ov/MIF infection in Lao PDR was 43.8% around Pakse city (Champasak province) 18 , 67.1% in Savannakhet province 19 , 52.0-60.7% 20 or 81.1% 21 in Khammouane province, and 53.3% in Vientiane Municipality 22 . Thailand, formerly known as the most highly endemic country for O. viverrini infection, became a lower-grade endemic country owing to the active and sustained national control activities over 40 years since the 1980s 3 . The national prevalence in Thailand was 14.7% in 1980-1981, with the highest level of 34.6% in the northeast areas, but the national prevalence decreased to 11.8% in 1996 and 9.6% in 2001 3 . In northeast areas, the prevalence remained at 60.8% in Nakhon Phanom, 38.6% in Sisaket, and 32.6% in Amnat Charoen province by 2009; however, the prevalence in these areas also decreased substantially in 2014 with Nakhon Phanom remained the only province showing the prevalence over 20% 3 . After 2014, however, there were several publications reporting a considerably high prevalence (16.9-28.7%) in northeastern and central Thailand [23][24][25][26] . In Vietnam, Phu Yen province and some other mid-to southern provinces have been notified as endemic areas of O. viverrini, with the prevalence ranging from 0.3 to 36.9% 3 . A low-grade endemicity was recently reported in lower Myanmar with a 0.7% egg-positive rate 27 . Thus, the present survey areas in Cambodia appeared to be one of the most highly endemic areas of O. viverrini infection ever reported.
There seem to be potential risk factors for the surveyed areas to appear as highly endemic areas of opisthorchiasis. One is the locality of the surveyed villages. These villages are located along the main stem or tributaries of the Mekong River. The 5 villages of Preah Vihear province locate just along the border between Cambodia and Lao PDR. In Lao PDR, O. viverrini infection has been known to be highly endemic, especially in Vientiane www.nature.com/scientificreports/ Municipality, Khammouane, Savannakhet, and Champasak provinces [18][19][20][21][22] . The 5 villages of Stung Treng province are located along the river and streams not far from the border area. Other risk factors include that most villagers are engaged in fisheries and are traditionally accustomed to eating raw or undercooked freshwater fish. This study showed a significant age tendency in the prevalence of Ov/MIF infection. The prevalence was the lowest among children under 9 years, followed by the age groups 10-19 years and 20-29 years. It approached a peak at ages 30-39 years and then maintained the peak up to the age groups 40-49, 50-59, and ≥ 60 years. This tendency was like those reported previously for liver fluke infections in other countries; O. viverrini in Lao PDR and Thailand [28][29][30] , O. felineus in Russia 31 , and C. sinensis in South Korea 32 . Also, in Cambodia (Takeo province), almost the same age-prevalence pattern of O. viverrini infection was observed among the riverside people 4,6 .
Sex difference in the incidence of liver fluke infections is known to be variable according to locality and human behavior 31,32 , although helminth infections are generally known to be heavier in males than in females in mammalian hosts 33 . In South Korea, males revealed a higher incidence of C. sinensis infection than females, related to some social customs in males, i.e., frequent consumption of raw or undercooked freshwater fish with liquor in social gatherings 32 . Similar trends were observed in China, where C. sinensis is endemic 32 . Reversely, in O. felineus infection, the greatest incidence was noted among housewives, i.e., females, in Russia 31 . However, in Thailand, where O. viverrini is prevalent, male preponderance or no significant sex difference has been reported 20,31,34 . In Cambodia, a male preponderance was reported in Takeo province 6 . However, in the present study, no significant sex difference was noted. One of the reasons may be that the major source of infection in the surveyed areas is traditional fish dishes popularly consumed by the people regardless of gender, such as 'plea tre' (fish salad), 'plea tre chou' (sour fish salad), and 'ma'am' (fermented fish, kept for 2-5 days).
There was a positive correlation between the number of adult O. viverrini and/or H. taichui specimens recovered and the number of EPGs in 10 volunteers, as presented in Table 2. Our results agreed with a previous report from Thailand where 231 residents of a northeast community positive for O. viverrini eggs were treated with praziquantel, and adult flukes were collected 35 . The pre-treatment egg counts (EPG) showed a strong linear relationship with the number of worms recovered after the treatment. It was also of note that the fecundity of O. viverrini appeared to be related to the degree of parasite aggregation within the host body and negatively associated with the total worm burden in each host individual 35,36 . In the present study, the number of cases in whom worm recovery was performed was small (only 10), and the density-dependent reduction in worm fecundity was difficult to analyze.
It is noteworthy that H. taichui was found mix-infected with O. viverrini among the volunteers in Preah Vihear province. To the best of our knowledge, this is the first report of human H. taichui infection in Cambodia. This combination of mixed infections was previously found in other countries, including Lao PDR 19,21,22,37,38 and Thailand 39,40 . One of the points that should be considered is the relative predominance of these fluke species. The average prevalence of S. mekongi appeared to be quite low, 0.8% in 10 villages surveyed, with a prevalence of 1.6% in Preah Vihear and 0.5% in Stung Treng province. These two provinces have been well known as the endemic areas of schistosomiasis mekongi, and long-time surveillance (1995-2010), as well as control efforts, have been implemented by the government 4 . The present findings seem to be the results of the governmental control activities against this trematode infection.
The eggs of A. lumbricoides were not detected in this study. This finding denotes that the national control program against soil-transmitted helminthiases in Cambodia by the mass drug administration (MDA) using mebendazole and/or albendazole twice a year for over 20 years 4 has been highly successful. The prevalence of other soil-transmitted helminths appeared to be 12.2% for hookworms and 1.5% for T. trichiura; however, the prevalence of these parasites is also expected to decrease further so far as the national control program is continued. We regret that the prevalence of S. stercoralis, another soil-transmitted helminth species, could not be determined in this study because the Kato-Katz fecal examination method alone was applied. There were 6 people from whom female E. vermicularis adult nematodes were collected; among them, 5 people were egg-negative in Kato-Katz fecal examinations.
In conclusion, it has been confirmed for the first time in this study that the northernmost areas of Preah Vihear and Stung Treng provinces, Cambodia, bordering Lao PDR are highly endemic areas of O. viverrini infection, with a prevalence of 58.3 and 60.3% (59.8% in average), respectively. The adult flukes recovered from 10 volunteers in Kampong Sangkae village, Preah Vihear, were, in their majority, identified as O. viverrini. A substantial number of H. taichui adult specimens were also recovered from 7 of the 10 volunteers, together with a few or quite many specimens of adult nematodes (5 species) and cestodes (1 species

Methods
Ethical consideration and data processing. The National Ethics Committee for Health Research, Ministry of Health, Cambodia approved this study (approval no.: 099NECHR), and all the methods used were performed in accordance with the relevant guidelines and regulations. The CNM staff were informed of and oriented on the proposed project, explained about this project to the participants, and then the informed consent form was obtained from each participant for fecal examination and from each volunteer for adult worm recovery. The collected data were analyzed in Excel 2016 (Microsoft, Redmond, WA, USA) and were statistically evaluated using the chi-square test. The level of statistical significance was set at P < 0.05.

Survey areas and participants.
To determine the endemicity of O. viverrini and/or MIF among riparian people in the northernmost parts of Cambodia, a cross-sectional community-based survey was performed in April-May 2018 in 10 villages of Preah Vihear and Stung Treng province bordering Lao PDR (Fig. 1). The inclusion criteria for volunteers were those showing > 240 EPG of Ov/MIF eggs, and the exclusion criteria included pregnant women, children younger than 15 years, and elderly people over 61 years. The adult worm recovery was performed in a regional health center within the village. The volunteers were provided with informed consent and were given orally 40 mg/kg single dose of praziquantel (Shinpoong Pharmaceutical Co., Seoul, Korea) plus 5-10 mg/kg pyrantel pamoate (Hangzhou Minsheng Pharm. Co., Hangzhou, China) followed by 40-50 g of magnesium sulfate for purging. The whole consecutive diarrheic stool passed 4-5 times after the medication was collected and washed several times with > 10 volumes of water and re-suspended in water. After 10 min, the clean upper layer was discarded, and the dark lower layer was examined under the naked eye or placed under a stereomicroscope (Olympus, Japan) and explored for worm parasites. The number of worms collected was counted for each helminth species. Some of the adult fluke specimens were fixed in 10% formalin under a coverslip pressure, stained with Semichon's acetocarmine, dehydrated in a graded series of ethanol, cleared in xylene, and morphologically identified using a light microscope.
Statistical tests. Statistical  Ethics approval. The study protocols were approved by the National Ethics Committee for Health Research, Ministry of Health, Cambodia (approval no.: 099NECHR). Informed consent was obtained from all the participants for fecal examinations and adult worm collection following chemotherapy.

Data availability
The datasets used and/or analyzed during the present study are available from the corresponding author.