Tropical Biomedicine 36(1): 81–93 (2019)

Prevalence and associated risk factors of Opisthorchis viverrini infections in rural communities along the Nam Kam River of Northeastern

Chuangchaiya, S.1*, Laoprom, N.2 and Idris, Z.M.3* 1Faculty of Public Health, , Chalermphrakiat Province Campus, 47000, Sakon Nakhon, Thailand 2Faculty of Science and Engineering, Kasetsart University, Chalermphrakiat Campus, 47000, Sakon Nakhon, Thailand 3Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000, Kuala Lumpur, Malaysia *Corresponding author e-mail: [email protected]; [email protected] Received 4 May 2018; received in revised form 26 November 2018; accepted 27 November 2018

Abstract. Opisthorchis viverrini infection is still one of the public health problems in Southeast Asia. In Thailand alone, more than three million people are estimated to be currently infected. Opisthorchiasis may cause severe biliary diseases, eventually leading to fatal cholangiocarcinoma. This study aimed to investigate the prevalence and risk factors of opisthorchiasis in two rural communities along the Nam Kam River in the northeastern region of Thailand. A cross-sectional study was conducted in of Sakon Nakhon Province and of Province in Thailand between March and June 2017. Faecal samples were collected and examined for the presence of O. viverrini by Kato-Katz technique. Socio-demographic variables and risk factors were collected by a standardised questionnaire. The overall prevalence of O. viverrini among 495 study participants was 15.4% (95% confidence interval [CI] 12.3 – 18.8). The parasite rate was higher in Nakhon Phanom than in Sakon Nakhon (P = 0.009) and differed significantly in adults <45 years old in both provinces (P <0.05). Multivariate regression analyses revealed being labourer (adjusted odd ratio [AOR] 4.7 [95% CI 1.08, 20.38], P = 0.039) and farmer (AOR 5.79 [95% CI 1.45, 31.91], P = 0.015) were positively associated with a higher risk of O. viverrini infection. This study highlights O. viverrini infection as a significant endemic disease with potential health problems among the water-based communities along the Nam Kam River. For country with high O. viverrini transmission, such information will be useful for designing efficient strategic interventions.

INTRODUCTION Thailand are the most effected with O. viverrini infections, where infection Opisthorchis viverrini, a fish-borne prevalence exceeding 50% at the village level trematode, is one of the neglected tropical is still commonly observed (Sithithaworn et diseases with considerable public health al., 2012; Sripa et al., 2015). The concentrated importance in Southeast Asia, particularly in distribution of O. viverrini in these regions Thailand (Keiser & Utzinger, 2009; Sripa et corresponds with the local presence of al., 2010; Utzinger et al., 2010). It has been natural intermediate hosts and the traditional estimated that more than 3.3 million people eating habits (Sriamporn et al., 2004; Sripa et in Thailand are infected with O. viverrini al., 2007). Apart from the northern areas, O. with varying distribution depending on the viverrini infection is also endemic in central endemic areas (Wongsaroj et al., 2014). Rural Thailand due to the migration of local people communities in north and northeastern from the northeastern provinces which

81 maintained their traditional life style that also among communities has led to the includes raw food consumption (Rangsin et persistence of O. viverrini infections in al., 2009; Suwannahitatorn et al., 2013). many parts of these regions (Sripa et al., The life cycle of O. viverrini is complex, 2011; Sithithaworn et al., 2014). involving two intermediate hosts: snails of This study was conducted in two districts the genus Bithynia are the first intermediate along the Nam Kam River namely Phon Na host, whereas freshwater fishes i.e. Cyprinoid Kaeo and That Phanom, in Sakon Nakhon family serve as the second intermediate host Province and (Keiser & Utzinger, 2009). Human is infected respectively. Both provinces are situated in by O. viverrini through consumption of raw the northeastern region of Thailand or insufficiently cooked freshwater fish that approximately 500 km from the Thai capital harbours the infective stage of the parasites . Phon Na Kaeo District (352 km2) is called metacercariae. Consumption of located at the downstream of the Nam Kam undercooked fish dishes such as Koi pla is River, while That Phanom District (368 km2) deeply rooted in local culture in the affected is about 80 km upstream of Phon Na Kaeo. areas, where proper sanitation is minimal or The Nam Kam River is one of the absent (Utzinger et al., 2010). Transmission River’s tributaries which cover 3,440 km2 of is maintained by the frequent practice of open catchment area with a total length of 123 km. defecation that results in contamination of The river flows through two provinces water bodies. Although infection with O. starting from Nhong Harn swamp in Muang viverrini is often asymptomatic, chronic District of Sakon Nakhon and merges with infection may lead to a number of severe liver the great Mekong River in That Phanom and bile duct diseases (Mairiang & Mairiang, District in Nakhon Phanom. This river 2003; Keiser & Utzinger, 2009; Sripa et al., provides the water source for agriculture and 2010). The most severe sequelae of O. for various utilities in this region. Though viverrini infection is cholangiocarcinoma there are many developmental initiatives in (CCA), a bile duct cancer with extremely this region, poverty is still a major challenge. poor prognosis (Sripa et al., 2010; Sripa et Farming and fishing are the predominant al., 2011) and with high fatality rate economic activities for the river shore (Sithithaworn et al., 2014). In Thailand, communities. Farming is mostly subsistence- previous studies have reported a high based, particularly rice cultivation as well incidence rate of CCA in the northeastern as small-scale animal husbandry. Fishing is regions ranging from 93.8 to 317.6 per 100,000 done primarily by men in a variety of person-years (Sripa et al., 2007). freshwater bodies including rivers, swamps Today, preventive chemotherapy and paddy fields. Dietary habits are deeply combined with health education via vertical rooted in the local culture and include a programmes remain the main strategy for variety of meals and dishes putatively O. viverrini control in Thailand. It is containing metacercariae. implemented locally in target areas Previous epidemiological studies depending on problems and priorities in each conducted in seven Upper Northeastern community (Jongsuksuntigul & Imsomboon, provinces in Thailand showed that the 2003). Although most target areas are located prevalence of O. viverrini varied among in the northern and northeast regions which provinces: highest in Nakhon Phanom have been recognized as endemic area of (40.9%), followed by Sakon Nakhon (~28%) O. viverrini infection, many reports showed and lowest in Bueng Kan (~10%) that the prevalence was still high (Andrews (Thaewnongiew et al., 2014). Nevertheless, et al., 2008; Ong et al., 2016; Kim et al., 2017; according to the Province Statistic and Saenna et al., 2017). In addition, despite Demographic Office, the total prevalence of decades of control efforts, the high Opisthorchis infections in Nakhon Phanom availability of freshwater fish as major source in 2016 and Sakon Nakhon in 2015 were lower of protein (Grundy-Warr et al., 2012), and the than previously reported with 10.1% and continuous practice of raw fish consumption 11.2%, respectively. These contrasting figures

82 implied that either intensive control A witness observing this procedure also measures of Opisthorchis infections by the signed the consent form. For illiterate health authority might have significant participant, informed consent was read out impact in the reduction of the infections or and, after approval, the person signed with a the overestimation of the true burden of O. fingerprint. No children were involved in this viverrini in the study area. Furthermore, study. there is a dearth of information on the risk factors underlying each study area. The Study design and field procedure critical risk factors for O. viverrini infection A community-based survey was carried out are mainly associated with eating raw fish or between March and June, 2017. Two villages uncooked fish. Differences in education, namely Ban Phon Khae Noi in Sakon Nakhon economy, social practices and life style Province (Na Tong Watthana sub-district, amongst community has been found to Phon Na Kaew District) and Ban Fang Daeng influence the habit of consuming in Nakhon Phanom Province (Fang Daeng inadequately cooked fish (Sripa et al., 2017). sub-district, That Phanom District), were In response, we aimed to investigate the included in the study. These villages were current prevalence and associated risk selected based on, (i) their representa- factors of O. viverrrini infections using faecal tiveness of typical lowland villages samples from people living in Phon Na Kaeo characterised by the proximity of water- and That Phanom, in Sakon Nakhon Province bodies from which freshwater fish can be and Nakhon Phanom Province, respectively. caught; (ii) maintenance of the tradition of Results from this study provide valuable eating raw fish dishes, which is typical for information to direct improved health rural areas in the northeastern region of intervention efforts in order to identify and Thailand. The sample sizes for the different implement effective control measures, and study sites were calculated using the plan for the participation of the targeted following formula: N = z2 p (1 – p)/e2, where z communities in the area. is the confidence interval which is set at 95% (z-value of 1.96); p is the expected prevalence of O. viverrini infections of 11.2% MATERIALS AND METHODS for Sakon Nakhon and 10.1% for Nakhon Phanom (from available regional statistics) Ethics statement and e is the allowed error margin which is This study was approved by the Ministry of set to 4%. In addition, contingencies were Health of Thailand (reference no. 5/2560) and adjusted for by adding another 10% of the ethics committee of the Sawang Dan individuals, giving us a total of 503 Din Crown Prince Hospital (reference no. participants to be faecal sampled for the two SWDCPH 2017 – 009), which also approved study sites combined. the informed consent procedure. Permission Village leaders and household heads for the field work was obtained from the were informed about the study’s objectives Provincial Health Office and the District and procedures. A convenience sampling Health Office. Local health workers were strategy was used in this study, whereby briefed, using , on the residents were asked to come to selected participant information sheet and the need to survey point for study participation. Enrolled obtain written consent from the participants, participants were interviewed using and on how to administer the questionnaire standardised questionnaires to identify the as well as to obtain faecal samples. Village risk factors for O. viverrini infection. The meetings were held in the community hall questionnaires covered socio-demographic centers and inhabitants were given detailed aspects (i.e. age, gender, education level, explanations about the aims, procedures, marital status, occupation, income level, potential risks, and benefits of the study. alcohol consumption as well as present and Individual written informed consent was type of domestic animal) and behavioral obtained from all adult household members. aspects (i.e. raw fish consumption, previous

83 stool examination and positivity for helminth checked for errors. Data were processed infection, praziquantel intake and prior and analysed using Stata/SE version 13.1 for knowledge about O. viverrini infection). Windows (StataCorp, TX, USA). Differences With regards to the type of domestic animal, in proportions were tested using Chi-squared cats and dogs are the main animal reservoirs test or the Fisher’s exact test, while in the life cycle of O. viverrini, which they comparison for continues variables were are allowed to roam freely in the study areas. made using t-test. 95% confidence intervals All completed questionnaires were checked (95% CI) were estimated to provide for accuracy and completeness. uncertainty surrounding the point estimates. After proper instruction, a biohazard Univariate logistic regression was performed zipper bag containing stool container and to identify risk factors for the outcome of O. spatula, labeled with the subject’s name and viverrini infection as determined by Kato- identification number was distributed to each Katz thick smear. Odds ratios (OR) and 95% participant. The participants were visited by CI were also computed for the explanatory local health workers at home the following variables. All variables with a P <0.1 from a day for collection of the samples. All likelihood ratio test in univariate analyses collected samples were checked for correct were entered into a multivariate logistic labelling and quantity of sample, and regression model and stepwise backwards transported immediately in a cool box to the elimination was used to identify the main risk field laboratory. factors for infection. A P <0.05 was considered statistically significant. Parasitological data Study participants were invited to provide a single stool sample. Modified Kato-Katz thick RESULTS smears (Katz et al., 1972) were prepared after the stool collection using a commercially Characteristic of study population at available kit (Department of Helminthology, screening Mahidol University, Bangkok, Thailand). The A total of 495 participants, 245 from Sakon Kato-Katz technique is commonly employed Nakhon Province and 250 from Nakhon in epidemiological surveys of helminth due Phanom Province, were enrolled in the study. to the relative ease of operation and the The ages of the participants ranged from 15 possibility to quantify infection intensity, to 87 years with a median age of 52 years. which allows stratification into different Except gender, marital status, use of intensity classes based on cut-offs provided praziquantel and prior information of O. by the World Health Organization (WHO) viverrini infection, all other personal (2002). In this study, Kato-Katz thick smears characteristics of the study participants were allowed to clear for 30 minutes before varied significantly between the two examination under a light microscope (100x provinces (Table 1). Participants in Nakhon magnification). To eliminate bias, each faecal Phanom were slightly younger compared to sample was examined by three trained senior participants from Sakon Nakhon (mean 47.8 medical laboratory technologists who were vs 51.2 years). In Nakhon Phanom, a slight not informed about the health status and other majority had primary education (74.0% [95% details of the study participants. All samples confidence interval, 95% CI 68.1 – 79.3] vs were examined on the day of collection. The 72.2% [95% CI 66.1 – 77.8]) and work as farmer number of eggs per gram faeces (EPG) of (65.6% [95% CI 59.4 – 71.5] vs 62.0% [95% CI stool was calculated for each smear, and the 55.6 – 68.1]). Participants from Nakhon geometric mean of EPG was calculated for Phanom had lower alcohol intake but were infected individuals. more often living with domestic animals i.e. dogs. When considering factors more directly Statistical analysis related to helminth infections, more Survey data were double entered into participants in Nakhon Phanom were Microsoft Excel spreadsheet and cross- reported to have consumed raw fish in the

84 85 past (P <0.001), undertaken previous stool only being a labourer (adjusted odd ratio examination (P = 0.024), and received [AOR] 4.7 [95% CI 1.08, 20.38], P = 0.039) and information regarding the infection (P = farmer (AOR 5.79 [95% CI 1.45, 31.91], P = 0.003), when compared to participants in 0.015) were significantly associated with a Sakon Nakhon. Finally, among the 381 higher risk of O. viverrini infections in the individuals reported of eating raw fish, the overall cohort, when compared to the proportion of raw fish consumption were unemployed group. highest in >55 age group (35.3% [95% CI 30.5 – 40.3]), attended primary education (74.3% [95% CI 69.6 – 78.6]) and worked as farmer DISCUSSION (67.5% [95% CI 62.5 – 72.1]). This study describes data on O. viverrini Prevalence and distribution of O. prevalence among 495 individuals living in viverrini two provinces in northeastern Thailand along The overall O. viverrini prevalence in the the Nam Kam River Basin. The present study study sample was 15.4% (76/495) and indicates significant local variation in O. significantly higher in Nakhon Phanom (19.6% viverrini prevalence between the two [95% CI 14.9 – 25.1]) than in Sakon Nakhon study areas; two northern regions with (11.0% [95% CI 7.3 – 15.6] (P = 0.009) (Fig. predominately rural inhabitants. The overall 1a). Nevertheless, the geometric mean EPG relationship between age and parasite (ranged from 24 – 1848 geometric mean EPG) prevalence was similar, albeit showed among O. viverrini positive individuals in variation in individuals below 45 years both areas were similar. No other intestinal between the study areas. The results also helminths were observed. In general, parasite show that the main risk factors associated rate in the overall cohort was not differed by with O. viverrini infection in the two age groups (P = 0.154), but the parasite rate communities were related to individual’s was significantly higher in the <35 group (P occupation as being labourers and farmers. = 0.025) and the 35 – 45 group (P = 0.003) in We examined the cross-sectional data to Nakhon Phanom than in Sakon Nakhon investigate the current prevalence of O. (Fig. 1b). Furthermore, in Nakhon Phanom, viverrini infection in settlements along the the prevalence of O. viverrini were Nam Kam River. Our study showed that the significantly higher than Sakon Nakhon for overall O. viverrini prevalence was high (i.e. females and farmers as well as having 15.4%) and local variation in prevalence was dogs as domestic animals (all P<0.05). also observed between the two communities Similarly, with regards to history of helminth living along the river’s course. Our study infections, the prevalence differed signifi- areas are situated in the northeastern region cantly in Nakhon Phanom than Sakon Nakhon of Thailand and was previously reported to (Table 2); high among those who had previous be the region with the highest frequency of stool examination (P = 0.038) and used of O. viverrini infections in the country praziquantel (P = 0.02), but low in those (Kaewpitoon et al., 2008). The habit and who previously tested positive for other frequency of eating raw or undercooked fish helminthes infections (P = 0.045). named Koi pla, a traditional dish that is commonly found in the north and the northeast Factors associated with opisthorchiasis of Thailand, therefore, contributed to the occurrence higher morbidity rate than other regions. The Comprehensive results for all significant differences in prevalence between the two co-variables associated with O. viverrini communities maybe due to their respective infection in univariate analysis (P <0.05) are distance to the waterbodies where villagers provided in Table 3, and these variables were procure fish for consumption. A recent study further used to build multivariate models with has shown that the distance to waterbody had stepwise forward selection. Final model in an impact on how villagers source the fish multivariate logistic regression showed that used in raw fish dishes (Ong et al., 2016).

86 Figure 1. Prevalence of Opisthorchis viverrini in Sakon Nakhon (SN; blue) and Nakhon Phanom (NP; red). (a) Area-specific prevalence and geometric mean of eggs per gram faeces (EPG). (b) Age-specific prevalence. (c) Gender-specific prevalence. (d) Prevalence by education level. (e) Prevalence by marital status. (f) Prevalence by type of occupation. (g) Prevalence by income level. (h) Prevalence by alcohol intake. (i) Prevalence by present of domestic animal. (j) Prevalence by type of domestic animal.

87 88 Differences in the infection levels in fish are social gathering where raw fish dishes are also dependent on the waterbody, thus can often available. Due to its location on the affect the risk of human exposure to the Thailand- international border, Nakhon infection (Ong et al., 2016). The strikingly Phanom is home to a large number of young high consumption of raw fish in Nakhon ethnic Laotians seeking job opportunities, Phanom (i.e. 99.6%) may also contribute to who may have different eating habits than the higher prevalence of O. viverrini in this the locals. Moreover, the consumption of raw area. Our direct observation revealed that fish dishes is part of one’s social obligation this environmental condition may contribute and integration among Laotian subcultures to the high O. viverrini infection rate (Xayaseng et al., 2013). Despite the little among the Nakhon Phanom population: the difference in prevalence among the age population lives along the Nam Kam River groups in our study, people at all ages are at and its streams with a large fish stock; the risk of being infected with O. viverrini. CCA abundance of freshwater fish leads to the is an Opisthorchis-associated cancer which regular consumption of raw fish. This can take 30 – 40 years to develop, people phenomenon has been explained by a infected with O. viverrini in their early age common process originating from the may only be diagnosed with this cancer unidirectional river flow that favours the during their most productive years of their displacement and downstream dispersion of life, with far reaching negative consequences fish (Blasco-Costa et al., 2013). Thus, the for their families and the entire communities unidirectional river flow supported the (Sriamporn et al., 2004; Andrews et al., 2008; existence of a longitudinal gradient in Steinmann et al., 2011). The interrelation of trematode abundance, which increases from O. viverrini infection and age becomes a downstream-to-upstream along the river. serious public health issue and requires Nevertheless, our study did not examine intergenerational (i.e. between children and possible environmental influences such as their parents) and transgenerational (i.e. proximity to different kinds of waterbodies across multiple generations) approaches in relation to the infection. Therefore, in designing health education campaigns, comparative multifactorial studies are such as delivering tailored health messages necessary to gain more insight into the and measures to each specific age group relationship between this environmental (Phongluxa et al., 2013). factor and the prevalence of O. viverrini In the overall cohort, there is clear infection in this region. evidence of infection prevalence being The prevalence of O. viverrini infection associated with low socio-economic status gradually increases in an age-dependent within the community. Our study has shown manner and reaching a plateau among the that the prevalence of O. viverrini were elderly. A similar age-specific pattern has significantly higher among farmers (Fig. 1f) been observed for O. viverrini infection in in Nakhon Phanom than in Sakon Nakhon and other parts of Thailand (Sithithaworn & working as a labourer or a farmer were both Haswell-Elkins, 2003; Kaewpitoon et al., independently associated with increased 2008; Chudthaisong et al., 2015), Laos odds of O. viverrini infections, thus (Sayasone et al., 2007; Sripa et al., 2011; highlighting the importance of both individual Forrer et al., 2012) and Cambodia (Yong et and community level poverties. This finding al., 2012). The overall prevalence of O. is consistent with the mounting evidence from viverrini in individuals less than 45 years other parts of the country that suggest people old was considerably lower than their older with lower socio-economic status have higher counterparts, furthermore, Nakhon Phanom risk to the infection (Ong et al., 2016; Kim et has a higher prevalence in all age groups than al., 2017; Prakobwong et al., 2017). In Sakon Nakhon. One reason for a higher previous studies, farmers were found to have infection prevalence among the older age higher infection prevalence than non-farmers groups in Nakhon Phanom is likely to be their (Forrer et al., 2012; Kaewpitoon et al., 2012), socializing behavior – actively involved in suggesting that the observed higher infection

89 rate in people who are farmers is not permit georeferencing of individual occupationally associated. Furthermore, household locations and the complete higher percentage of participants who were climate data from each study areas. farmers (67.5%) claimed to have consumed Therefore, it was not possible to use raw fish than participants with other spatiotemporal analysis to investigate occupations in the study areas. This is patterns within individual sites. A recent because farmers in our study areas often spatiotemporal study in the same region has harvest fish from Nam Kam River or from their highlighted the highly dynamic nature of O. rice fields and then prepare and consume the viverrini transmission, together with catches on the spot. Agricultural labourers intensified ecological process modulated by may also consume raw fish in our study areas human and environmental factors (Kim et al., from the popular dish of Koi pla due to its 2016). As such, investigation on the spatial ease of preparation, wide distribution and and temporal variations in our study area, relatively cheap price. In addition, most of along with social and ecological under- the labourers in the study areas come from standings of the parasite transmission the neighboring country, and the finding that dynamics will be needed to provide parasitic infections are prevalent among additional information for future interventions these labourers demonstrate the need for planning. targeted control measures in foreign In conclusion, this study provides new labourers working in Thailand. Nevertheless, insight into the distribution of O. viverinni no clear pattern was observed between infection in Sakon Nakhon and Nakhon infection rates and the disclosure of income. Phanom provinces. It highlights O. viverrini Thus, we believe that the reasons for the infection as a significant endemic disease higher risk of O. viverrini infections in socio- with potential health problems in the water- economically deprived groups (i.e. farmer based communities along the Nam Kam and labourer) in the study areas may be River. The high prevalence of O. viverrini centered around the lifestyle compounded infection in these areas, particularly in with poverty in this region. Nakhon Phanom Province, is alarming and This study has several limitations. First, warrants urgent public health intervention. while the convenience sampling approach In Nakhon Phanom Province, raw fish used in this study was efficient and cost- consumption is common and O. viverrini effective, it has an inherent selection bias. prevalence is higher than in Sakon Nakhon. Our survey was conducted on a house-to- Further, socio-economically deprived house basis during the weekdays, meaning groups, namely labourers and farmers, are that only older adults (i.e. above 46 years) associated with opisthorchiasis occurrence. were disproportionally represented (Table 1). This implies a potential benefit to direct Over-representation of this age group among additional O. viverrini control activities to our samples may likely overestimate the true labourers and farmers, such as improving parasite prevalence in the study areas. access to diagnosis and treatment. Overall, Second, due to the imperfect sensitivity of this study calls for the local authorities and the Kato-Katz technique, the true O. viverrini communities in the northeastern provinces prevalence maybe considerably higher than of Thailand to integrate their actions and the rate reported here (Johansen et al., 2010). address O. viverrini infection by building Third, our study has undersampled younger awareness and strengthening knowledge and population that could have led to an health practices on the infection. underestimation of the overall parasite prevalence. Many adolescents in both study Acknowledgments. We would like to extend areas are in school or refused to be sampled our gratitude to the communities and when our survey was held. Opisthorchis community leaders for their support and infection in this younger age group is not well participation in the survey. We wish to characterised and warrants further sincerely thanks all members in the field investigation. Fourth, the survey design did team. We are grateful to the director of Phon

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