Re-Examination of Opisthorchis viverrini in Nakhon Ratchasima Province, Northeastern Thailand

The Opisthorchis viverrini; carcinogenic liver fluke, is an endemic in the Lower Mekong Basin, including Thailand, Lao People’s Democratic Republic, Cambodia and central Vietnam (Sripa et al., 2010). The underestimate of infections are considered, more than 10 million people are infected with O. viverrini in Thailand and Lao PDR (Sithithaworn et al., 2012). In Thailand, it is estimated that 6 million people are infected with the O. viverrini (Jongsuksuntikul and Imsomboon, 2003). This figure indicated that it is a serious public health problem in Thailand, particularly in northeastern and northern region (Kaewpitoon et al., 2008; Sripa et al., 2010). The O.viverrini infection is associated with hepatobiliary diseases including hepatomegaly, cholangitis, cholecystitis, and gallstones (Harinasuta and


Introduction
The Opisthorchis viverrini; carcinogenic liver fluke, is an endemic in the Lower Mekong Basin, including Thailand, Lao People's Democratic Republic, Cambodia and central Vietnam (Sripa et al., 2010). The underestimate of infections are considered, more than 10 million people are infected with O. viverrini in Thailand and Lao PDR (Sithithaworn et al., 2012). In Thailand, it is estimated that 6 million people are infected with the O. viverrini (Jongsuksuntikul and Imsomboon, 2003). This figure indicated that it is a serious public health problem in Thailand, particularly in northeastern and northern region (Kaewpitoon et al., 2008;Sripa et al., 2010). The O.viverrini infection is associated with hepatobiliary diseases including hepatomegaly, cholangitis, cholecystitis, and gallstones (Harinasuta and Vajrasthira 1960;Thamavit et al., 1978;Harinasuta et al., 1984). Recently, O. viverrini has been classified as Type 1 carcinogens by the International Agency for Research on Cancer, World Health Organization (WHO) (IARC, 1994).
A community-level health education campaign been conducted since late 1950s. O. viverrini control has been started as a small scale helminthiasis control program in some high risk areas. A large scale has been started, the program is operated in some provinces of the central and all provinces of the northeast and north of Thailand. The main strategies for liver fluke control comprise three interrelated approaches, namely stool examination and treatment of positive cases with praziquantel for eliminating human host reservoir, health education for a promotion of cooked fish consumption to prevent infection, and improvement of hygienic defecation for the interruption of disease transmission (Jongsuksantikul and Imsomboon, 2003;Sithithaworn et al., 2012).
The O.viverrini infection in Thailand was the first reported in 1955 (Sadun) and many strategies has been operated over period 1955-2000, the national prevalence of O. viverrini infection had fallen from 63.6% to 9.6% but the high prevalence rate is still found in the rural communities of provinces, Northeast (Sithithaworn et al., 2012). In addition, the high mortality rate of CCA was reported in the northeast areas where found frequently of O. viverrini infection (Sripa et al., 2010). Mortality rate of liver cancer and O. viverrini infection rate in different regions of Thailand has been reported and found that Nakhon Ratchasima province has 13.67-16.2 per 100,000 populations. Eradication of the fluke and identification of high-risk populations are urgently needed (Sripa and pairojkul, 2008). In addition, the distribution O. viverrini infection in Nakahon Ratchasima province has been reported, the prevalence of survey in 2009 was 4.6% (Sitthithaworn et al., 2012). A total of 1,168 stool samples were obtained from 516 males and 652 females, aged 5-90 years. Stool examination showed that 2.48% were infected with O. viverrini. (Kaewpitoon et al., 2012c). Furthermore, a total of 640 Cyprinidae family fish including 5 species were collected from different study sites of Nakhon Ratchasima province, and investigated for O. viverrini metacercariae. The infection rate was 12.3% (79/640), predominantly in Cyclocheilichthys armatus, C. repasson, Puntioplites proctzysron, Hampala macrolepitota and Hampala dispar, respectively. The prevalence of O. viverrini metaceria was found covered 78.1% of areas, predominantly in Sida and KiaKham Thale So (Kaewpitoon et al., 2012a).
These figure indicate that O. viverrini infection is still a problem in this areas, a community-based approach to screen O. viverrini in highly risk areas are need required. Therefore, this study aimed to re-examine O. viverrini in 3 districts of Nakhon Ratchasima province using multi-stage sampling technique, between June and October 2015. This data is able useful for further therapy, curable, and planning of prevention and control.

Materials and Methods
A cross-sectional survey was a pilot project and conducted in 3 districts of Nakhon Ratchasima province, northeastern Thailand, between June and October 2015, included Bua Yai, Chum Phuang, and Mueang Yang district ( Figure 1). Bua Yai is a district in the northern part of Nakhon Ratchasima, and neighboring districts are (from the north clockwise) Waeng Noi district of Khon Kaen province, Bua Lai, Sida, Non Daeng, Khong, Ban Lueam, and Kaeng Sanam Nang of Nakhon Ratchasima Province. Bua Yai district is subdivided into 10 sub-districts, and 121 villages. This district is coverage areas 271.6 km 2 (104.9 sq mi), and has 84,133 populations. Chum Phuang is a district in the eastern part of Nakhon Ratchasima province, neighboring districts are (from the north clockwise) Prathai, Mueang Yang and Lam Thamenchai of Nakhon Ratchasima Province, Lam Plai Mat of Buriram province, and Huai Thalaeng and Phimai of Nakhon Ratchasima again. The district is subdivided into 9 sub-districts, and 130 villages. This district is coverage areas 540.6 km 2 (208.7 sq mi), and has 82,161 populations. Mueang Yang is a district in the northeastern part of Nakhon Ratchasima province, neighboring districts are (from the north clockwise) Ban Mai Chaiyapot, Phutthaisong and Kho Mueang of Buriram province, Lam Thamenchai, Chum Phuang and Prathai of Nakhon Ratchasima Province. The district is subdivided into 4 sub-districts, and 44 villages. This district is coverage areas 255.5 km 2 (98.6 sq mi), and has 28,359 populations. The main water resource is the Mun river.
Multi-stage sampling was used to select the participants in this studied. Briefly, total of 66,163 populations from 194,152 populations was selected with criteria of aged ≥30 years old. Populations at risk were screened by using miniverbal screening questionnaire contained the history with (1) opisthorchiasis; definitive diagnosed by medical doctor or related officers, (2) under-cooked fish consumption, (3) praziquantel used; given by medical doctor or related officers, (4) cholecystitis; definitive diagnosed by medical doctor or related officers, (5) relative family with  cholangiocarcinoma, (6) naïve northeastern people, (7) agriculture, and (8) alcohol consumption. Population at risk was identified following 1+2+3+4+5+6+7+8, who had a high score more than 5 points was selected, included 71, 121, and 163 participants from Mueang Yang, Bua Yai, and Chum Phuang district (Figure 2). Stools were collected and kept in labeled plastic bags and then transported in an icebox to the Clinical Pathological laboratory at the Suranaree University of technology Hospital, Suranaree University of Technology, Thailand, within a day after collection. Stool specimens were examined the O. viverrini and other known intestinal parasitic egg by the Modified Kato Katz thick smear procedures. Briefly, a small amount of stool material was placed on scrap paper and a piece of nylon sieve was pressed on top of it so that some of the stool sieved through the screen and accumulated on top of the 180 mesh nylon sieve. A spatula was scraped across the upper surface of the screen to collect the sieved stool. The spatula was used to deposit the stool in the orifice of the perforated plate on a microscope slide. The perpendicular 4x10x1 mm orifice was devised to contain exactly 0.04 g or 40 µL of stool, at one corner of the slide. The other side of the spatula was passed over a nylon sieve and over the perforated plate to remove excess stool. The plate was carefully removed by lifting, leaving behind a small square mould of sieved material. Opposite corner of other microscope slide was pressed on top of this, and a relatively thin smear was obtained by sliding over the slides in order to provide a thick smear in each slide (Meireles et al., 2008). All preparations were initially screened with a low-power (10x) objective lens. Suspected parasitic objects were subsequently examined under a high-power (40x) objective. The stool samples were preserved in 10%formalin for later confirmation, if needed. O. viverrini positive case was confirmed by 2 expert parasitologists before a definitive diagnosis was established. Patients who infected with other known parasitic were treated with anti-parasitic drugs and also attended the health education. The data was analyzed with descriptive statistics. Statistical data analysis was carried out using SPSS software version 12.0. The study protocol was approved by Suranaree University Ethical Review Committee, EC58-48.

Results
Total of 355 participant from 3 districts, was included in this study. The prevalence of O. viverrini infection was 2.25%. O. viverrini infection was slightly in female (3.17%) more than male (1.75%). The majorities of O. viverrini infection were found in age between 41-50 years old (4.49%), and followed by 61-70 years old (2.04%), 51-60 years old (1.54%), respectively. O. viverrini infection was found the highest in Mueang Yang district (2.82%), and followed by Bua Yai (2.48%), and Chum Phuang (1.84%), respectively. Baseline characteristics and infection are shown in Table 1. Other known parasitic infections were examined and found that two samples were infected Blastocystis hominis, and one sample was infected with Strongyloides stercolaris. Patients who infected with O. viverrini and other known intestinal parasitic were completed therapeutic treatment. O. viverrini infection in 3 districts between the surveyed data in year 2012 (Kaewpitoon et al., 2012c) Figure 3).

Discussion
Opithorchiasis is still a serious health problem in Nakhon Ratchasima province, Thailand.    (Harinasuta and Vajrasthira 1960;Thamavit et al., 1978;Harinasuta et al., 1984). In addition, O. viverrini has been classified as Type 1 carcinogens by the International Agency for Research on Cancer, World Health Organization (WHO) (IARC, 1994). Previously, the mortality rate of cholangiocarcinoma was reported and found that Nakhon Ratchasima province has 13.67-16.2 per 100,000 populations (Sripa and pairojkul, 2008). This figure indicates that Nakhon Ratchasima province should be intervened the health behavioral change particularly in these highly risk group. Furthermore, a provincial wide survey is need required, and screening of cholangiocarcnioma in infected participant and risk group is urgently concerned.