Molecular evidence of a high prevalence of Opisthorchis viverrini infection in an endemic community in Sakon Nakhon Province, northeastern Thailand

Abstract

Background

Opisthorchiasis caused by Opisthorchis viverrini infection is a major public health concern in Thailand. Despite decades of surveillance and control efforts, accurately estimating the true prevalence of O. viverrini remains a challenge. This study aimed to investigate the epidemiology of O. viverrini infection in an endemic community in northeastern Thailand using a combination of microscopic and molecular diagnostic methods.

Methods

A cross-sectional survey was conducted from May to July 2025 in Kusuman District, Sakon Nakhon Province. Demographic and behavioral data were collected using a standardized questionnaire. Stool samples were obtained from participants and examined using simple wet smear and modified Kato–Katz techniques to detect O. viverrini eggs and other intestinal parasites. In parallel, DNA was extracted from stool samples and polymerase chain reaction (PCR) targeting the internal transcribed spacer 2 (ITS2) gene was performed to confirm the presence of O. viverrini. Multiple logistic regression was used to investigate factors associated with infection.

Results

Among 246 participants, stool microscopy detected intestinal helminth infections in 10.2%, with O. viverrini being the most prevalent species (6.1%). Notably, all microscopy-positive O. viverrini cases were very light-intensity infections (< 500 eggs per gram [EPG] of stool). ITS2-specific PCR identified a higher prevalence of O. viverrini infection at 19.9%, detecting significantly more than three times as many cases as microscopy (P < 0.001). Multivariate regression analysis further showed that being male was associated with higher odds of O. viverrini infection (adjusted odds ratio [aOR] 5.55; 95% CI 2.41–12.83; P < 0.001), whereas participants aged 41–60 years had significantly lower odds compared to older individuals (aOR 0.08; 95% CI 0.03–0.19; P < 0.001).

Conclusions

This study demonstrates that O. viverrini infection remains highly prevalent in rural communities of northeast Thailand. Molecular diagnosis was more sensitive than conventional microscopy and key demographic risk factors were identified. These findings underscore the need for active surveillance and provide valuable data to guide and strengthen future prevention and control strategies for O. viverrini and other helminth infections in the region.

Supplementary Information

The online version contains supplementary material available at 10.1186/s41182-026-00929-3.

Background

Human liver fluke infection caused by Opisthorchis viverrini remains a significant public health problem in Southeast Asia. This burden is most evident across the Greater Mekong Subregion, particularly in Thailand, Lao People's Democratic Republic (Lao PDR), Cambodia, Vietnam and Myanmar, where riverine communities experience high endemicity [1, 2]. Together with Clonorchis sinensis and Opisthorchis felineus, O. viverrini is recognized by the World Health Organization (WHO) as a major foodborne trematode capable of causing substantial hepatobiliary disease in humans [3]. Infection with these flukes is strongly associated with cholangiocarcinoma (CCA), a malignancy classified as a Group 1 carcinogen, with the adult O. viverrini worm playing a central role in chronic inflammation and biliary pathology [4, 5]. The life cycle of these liver flukes involves Bithynia snails as the first intermediate host and cyprinid freshwater fish as the second, in which infectious metacercariae develop. Humans and other mammals acquire infection primarily through the consumption of raw or undercooked fish harboring metacercariae [6]. Following ingestion, the parasites migrate to the bile ducts, mature into adult flukes, and induce a range of clinical manifestations such as fever, anorexia, jaundice, and fatigue [7]. Chronic or heavy infections can lead to recurrent cholangitis, periductal fibrosis, cholecystitis, liver cirrhosis, cholelithiasis and ultimately CCA, underscoring the severe pathological burden associated with opisthorchiasis in endemic regions [7, 8].

In Thailand, more than 8 million people are infected with O. viverrini [2], with the northeastern region showing higher prevalence compared with other areas of the country [9]. Recent data report human infection rates of up to 18.2% in this region alone [10], highlighting the entrenched nature of transmission. Consequently, because chronic O. viverrini infection is strongly linked to severe hepatobiliary diseases, particularly CCA, the northeastern region of Thailand also records the highest incidence globally [11]. The national disease burden is substantial, with CCA attributable to liver fluke infection accounting for an estimated 70,745–138,221 disability-adjusted life years (DALYs) [12]. This persistent burden is driven by the parasite’s complex host range and environmental distribution, which sustain transmission despite long-term control efforts [13]. Even with public health education and other intervention programs, the prevalence of opisthorchiasis remains stubbornly high [5], emphasizing the urgent need for a more comprehensive understanding of its ecological and epidemiological dynamics.

Despite decades of surveillance and control efforts, accurately estimating the true prevalence of O. viverrini remains a challenge. Previous molecular studies indicate that conventional diagnostic methods such as stool microscopy, may significantly underestimate infection rates due to low sensitivity and the presence of morphologically similar trematodes [14–17]. This discrepancy suggests that the actual burden of infection and consequently the risk of CCA, may be higher than previously reported, reinforcing the importance of adopting sensitive molecular diagnostic approaches [18]. To address this gap, the present study aimed to determine the prevalence of O. viverrini among residents in an endemic area of Sakon Nakhon Province in northeastern Thailand using a combination of the gold standard microscopic stool examination and a conventional molecular method targeting the internal transcribed spacer 2 (ITS2) gene of O. viverrini. Additionally, the study sought to identify potential demographic and behavioral risk factors associated with infection, providing evidence to better guide public health interventions and control strategies.

Methods

Ethical considerations

The study was approved by the Ethics Review Committee of the Sakon Nakhon Provincial Public Health Office (Reference number: SKN REC 2025-031). Informed consent was obtained from each patient before participation in the study.

Study area and sample collection

The study was conducted from June to July 2025 in a rural community of Kusuman District (17.36°N, 104.26°E), Sakon Nakhon Province, located 688 km east of Bangkok in northeastern Thailand (Fig. 1). This predominantly rural district is home to agricultural and fishing communities whose livelihoods are centered mainly on rice and rubber cultivation. It spans 454 km² and has a population of 10,330 according to the 2005 census (Department of Provincial Administration, Thailand). As in many other provinces in the region, O. viverrini is the only fish-borne trematode endemic to the area [19]. Accordingly, the provincial health authority adheres to three strategic guidelines of the Thai Ministry of Public Health for the prevention and control of O. viverrini infection: stool examination, health education, and treatment of infected cases with praziquantel [20].

Fig. 1.

Geographic location and environmental characteristics of the study area in Kusuman District, Sakon Nakhon Province, northeastern Thailand. The figure depicts the study site and representative freshwater habitats within the district, including wetlands and riverine environments that support the life cycle of O. viverrini and facilitate its transmission. Panels show: A map of Thailand indicating the location of Sakon Nakhon Province, B satellite view of Kusuman District highlighting major water bodies, namely C Ban Song Tao reservoir, D Nong Sa Eau reservoir and E nearby Nong Han Lake. The map was created with Google Maps and the photographs were taken by author PS

The required sample size was estimated using Cochran’s formula, N = z²p(1–p)/e², with a 95% confidence level (z = 1.96), where p is the expected prevalence of O. viverrini infections (7.1% from a previous study by modified Kato–Katz technique [21]), and e is the allowed error margin which set at 5%. This calculation provided a minimum reference samples size, to which an additional 25% was added to account for contingencies such as non-response and incomplete data, resulting in a minimum target of 126 participants. No further adjustment was applied despite the use of non-probabilistic sampling. Village leaders and heads of households were briefed on the study aims and procedures. Participants were recruited through convenience sampling, whereby adult residents of the selected community were invited to designated survey sites; therefore, the study population represents the surveyed community rather than with wider district or provincial population. Eligible individuals were interviewed using standardized, Thai-language questionnaire adapted from our previous studies in similar O. viverrini-endemic settings [22, 23]. The questionnaire was developed based on established instruments and underwent expert review for content validity, followed by pre-testing in a small subset for the target population to assess clarity, cultural appropriateness and feasibility. Minor revisions were made accordingly prior to field deployment. The questionnaire collected information on socio-demographic characteristics (i.e., age, gender, education level, marital status, occupation and income level) and behavioral factors associated with helminth infection (e.g., raw fish consumption and anti-helminthic drug intake). All completed questionnaires were carefully reviewed by the study team for missing, inconsistent or unclear responses. Participants were contacted when necessary to verify and complete the information.

Sample collection and parasitological examination

Study participants who met the inclusion criteria, namely individuals aged ≥ 21 years, residing in the study area for at least 6 months and willing to participate, were invited to provide a single stool sample for parasitological and molecular examination. The age cutoff reflects a higher likelihood of independent dietary habits, including raw or undercooked fish consumption, a key risk factor for O. viverrini infection. Individuals recently treated with praziquantel, severely ill or unable to provide a sample were excluded. After receiving standardized instructions, each participant was given a biohazard zipper bag containing a labeled stool container and spatula. Local health workers visited each household the following day to collect stool samples. A single stool sample of approximately 2–3 g (about the size of a golf ball) was obtained from each participant in a clean, leak-proof container, avoiding contamination with urine, water or soil. Upon receipt, all specimens were checked for proper labeling and sufficient volume, transported in a cool box maintained at 2–8 °C and processed within 2 h of collection at the field laboratory. Aliquots intended for molecular analysis were stored at −20 °C until DNA extraction.

The stool specimens were examined using the simple wet smear and modified Kato–Katz techniques, employing a commercial Kato–Katz kit supplied by the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (Lot number 11/67, manufactured in 2024), following established protocols [22, 23]. The kit comprises standard cardboard templates with a central 6 mm hole to ensure a standard amount of stool (approximately 41.7 mg) is used, nylon mesh, cellophane strips pre-soaked in glycerol-based clearing solution and applicator tools. The stool smears were allowed to clear for 30 min before microscopic evaluation, initially using a 10× objective and subsequently a higher-power objective to confirm suspected helminth eggs or larvae. To minimize observer bias, each sample was independently examined by three experienced senior medical laboratory technologists who were blinded to participants’ health information. Discrepancies between readers were resolved through joint review and discussion until a consensus was reached. Additionally, quality control was performed by re-examining a randomly selected 10% of slides by a senior parasitologist (SC) to ensure the accuracy of egg identification.

Duplicate Kato–Katz slides were prepared for each stool sample. Samples that were microscopy-negative but PCR-positive were re-examined to confirm the presence or absence of O. viverrini eggs. A fecal sample was considered positive for O. viverrini eggs when one or more eggs were observed in any of the two methods. The intensity of infection was assessed by counting O. viverrini eggs and converting the counts to eggs per gram (EPG) of stools using the Kato–Katz method. Infection intensity was categorized as none (0 EPG), light (< 1,000 EPG), moderate (1,000–10,000 EPG) or heavy (> 10,000 EPG) [24]. All examinations were completed on the day of collection and participants with confirmed helminth infections received appropriate anthelmintic treatment, including praziquantel and/or albendazole.

Molecular identification of O. viverrini

O. viverrini DNA was extracted from approximately 200 mg of stool specimens using the NucleoSpin DNA Stool Mini kit (Macherey–Nagel, Germany), which is suitable for isolating helminth egg DNA and removing PCR inhibitors from stool samples. DNA extraction was performed according to the manufacturer’s instructions, including an initial lysis step with vigorous vortexing to facilitate egg disruption, without additional mechanical disruption. The concentration and purity of the extracted DNA were assessed using a NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA), with an A260/A280 ratio of 1.7–2.0 considered indicative of high-quality DNA with minimal protein concentration. PCR amplification of the O. viverrini ITS2 of the ribosomal RNA (rRNA) gene was performed with minor modification to previously described protocols [17, 25]. All molecular analyses were conducted at the Genomic Laboratory, Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur. Briefly, PCR amplifications were carried out in a final volume of 50 μl, consisting of 5 μl DNA template; 5 μl each of 2.5 µM of RTFlukeFa (5'- CTTGAACGCACATTGCGGCC-3’) and RTFlukeRa (5'-CACGTTTGAGCCGAGGTCAG-3'); 25 μl of 2 × Taq Master Mix (Vazyme Biotech, China); and 10 μl ddH₂O. The PCR products were amplified using the Mastercycler Pro (Eppendorf, Germany). The PCR assay included an initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min. The expected ITS2 amplicon size for O. viverrini was 375 bp. PCR products were separated on a 1.5% agarose gel in 1× TBE buffer at 100 V for approximately 45 min at room temperature, and the bands were visualized using a Gel Doc EZ Imaging System (Bio-Rad, USA). Each PCR run included a positive control (genomic DNA from a stool microscopy- and PCR-confirmed specimen) and a negative control (nuclease-free water). All PCR assays were conducted by personnel (NWH and KS) blinded to the microscopy results.

Statistical analysis

Survey responses were independently double-entered into a Microsoft Excel database and subsequently cross-validated to minimize data entry errors. Statistical analyses were conducted using Stata/SE version 13.1 for Windows (StataCorp, USA). Differences in categorical variables were assessed using the Chi-squared test. Univariate logistic regression was used to examine associations between potential predictors and O. viverrini infection as determined by PCR, and odds ratios (OR) with corresponding 95% CI were generated. Variables with a likelihood ratio test P < 0.1 in the univariate analysis were entered into a multivariate logistic regression model, in which stepwise backward elimination was applied to identify independent risk factors. Statistical significance was defined as a P < 0.05.

Results

Characteristics of study population

A total of 246 participants were enrolled in this study (Table 1). Their ages ranged from 21 to 89 years, with a mean ± standard deviation (SD) of 56.2 ± 11.5 years. The majority of participants were female (61.4%), aged 41–60 years (54.9%), had completed secondary education (52.9%), were married (78.9%), worked as farmers (80.9%), and reported a monthly income of ≤ 8,000 Thai Baht (THB) (84.6%). Regarding variables directly related to health behaviors associated with helminth infections, the majority of participants reported a history of consuming raw cyprinid fish (77.6%), no prior stool examination (56.5%), no previous diagnosis of helminth infection (80.5%), and no prior use of anti-helminthic drugs (97.9%). Most participants also reported not consuming alcohol (63.4%), not practicing open defecation in water reservoirs (72.8%) and having previously received information about O. viverrini (98.4%).

Table 1.

Sociodemographic characteristics and health behaviors of study population in Sakon Nakhon Province, northeastern Thailand

Demographic characteristic	n (%)	95% (CI)
Gender
Male	95 (38.6)	32.5–45.1
Female	151 (61.4)	54.9–67.5
Age group
21–40	24 (9.7)	6.4–14.2
41–60	135 (54.9)	48.4–61.2
≥ 61	87 (35.4)	29.4–41.7
Education level
Primary	98 (39.8)	33.7–46.3
Secondary	130 (52.9)	46.4–59.2
Tertiary	18 (7.3)	4.4–11.3
Marital status
Single	18 (7.3)	4.4–11.3
Married	194 (78.9)	73.2–83.8
Divorced/widowed	34 (13.8)	9.8–18.8
Occupation
Farmer	199 (80.9)	75.4–85.6
Self-employed	20 (8.1)	5.1–12.3
Merchant	16 (6.5)	3.8–10.3
Government	11 (4.5)	2.3–7.9
Monthly income
THB ≤ 8000	208 (84.6)	79.4–88.8
THB > 8000	38 (15.4)	11.2–20.6
Health behavior
History of eating raw fish
Yes	191 (77.6)	71.9–82.7
No	55 (22.4)	17.3–28.1
History of stool examination
Yes	107 (43.5)	37.2–49.9
No	139 (56.5)	50.1–62.8
History of positive helminth
Yes	48 (19.5)	14.8–25.1
No	198 (80.5)	74.9–85.2
History of taking anti-helminths
Yes	5 (2.1)	0.7–4.7
No	241 (97.9)	95.3–99.3
Consume alcohol
Yes	90 (36.6)	30.6–42.9
No	156 (63.4)	57.1–69.4
Defecate on water
Yes	67 (27.2)	21.8–33.3
No	179 (72.8)	66.7–78.2
Received info on Opisthorchis
Yes	242 (98.4)	95.9–99.6
No	4 (1.6)	0.4–4.1

CI confidence interval, THB Thai Baht

Prevalence of helminth infections

The prevalence of intestinal helminth infections among the study participants is presented in Table 2. Based on stool microscopy, 25 of the 246 participants were found to have intestinal helminth infections, yielding an overall prevalence of 10.2%. Among the microscopy-positive cases, O. viverrini was the most frequently detected species (6.1%), followed by Strongyloides stercoralis (2.4%), Taenia spp. (0.8%), hookworm (0.4%) and Enterobius vermicularis (0.4%). Of the 15 samples positive for O. viverrini eggs, the stool EPG values ranged from 1 to 99 EPG (12 samples), 100 to 199 EPG (2 samples) and 200–499 EPG (1 sample), with all cases classified as very light-intensity infections. Using ITS2-PCR, 34 individuals who were negative by stool microscopy were found to be positive. Representative ITS2-PCR amplification results are shown in Fig. 2. Overall, ITS2-PCR detected a higher prevalence of O. viverrini infection at 19.9% (49/246), identifying more than three times the number of cases detected by stool microscopy and revealing a significantly greater proportion of infections (P < 0.001).

Table 2.

Prevalence of intestinal helminth infections among study population based on stool microscopy and ITS2-PCR in Sakon Nakhon Province, northeastern Thailand

Intestinal helminth infections	n (%)	95% (CI)
Overall prevalence by stool microscopy	25 (10.2)	6.7–14.6
Overall prevalence by ITS2-PCR*	49 (19.9)	15.1–25.5
Specific prevalence by stool microscopy
O. viverrini**	15 (6.1)	3.5–9.9
Strongyloides stercoralis	6 (2.4)	0.9–5.2
Taenia spp.	2 (0.8)	0.1–2.9
Hookworm	1 (0.4)	0.0–2.2
Enterobius vermicularis	1 (0.4)	0.0–2.2

CI confidence interval, N number of study population, n number of positive individuals

^*A total of 34 individuals were negative for O. viverrini (0 EPG) by stool microscopy, but positive by ITS2-PCR

^**Of 15 positive O. viverrini eggs samples, EPG of stool examined are as follows: 1–99 EPG (12 samples), 100–199 EPG (two samples), and 200–499 EPG (one sample), for which all positive samples were considered light intensity

Fig. 2.

Representative 1.5% agarose gel electrophoresis showing specific 375 bp PCR amplicons targeting the internal transcribed spacer 2 (ITS2) region. Lane M: 100 bp DNA markers; lane N: negative control (nuclease-free water); lane P: positive control (genomic DNA from a stool microscopy- and PCR-confirmed specimen). Lanes 2, 10, 12 and 14 show positive bands, whereas lanes 1, 3–9 and 11 show no detectable amplification

Factor associated with opisthorchiasis occurrence

Univariate and multivariate analyses of risk factors for acquiring O. viverrini infection based on ITS2-PCR are summarized in Table 3. ITS2-PCR was used for this analysis because it detected a higher prevalence of O. viverrini infection compared to stool microscopy, providing a more sensitive measure of true infection status. All covariates that were significant in the univariate analysis (P < 0.05) were subsequently included in the multivariate model using stepwise forward selection. In the final multivariate logistic regression model, being male was significantly associated with higher odds of O. viverrini infection (adjusted odds ratio [aOR] 5.55; 95% CI 2.41–12.83; P < 0.001). Conversely, participants aged 41–60 years had significantly lower odds of infection (aOR 0.08; 95% CI 0.03–0.19; P < 0.001) compared to those in the older age group.

Table 3.

Univariate and multivariate analysis of risk factors for acquiring O. viverrini infections based on ITS2-PCR in Sakon Nakhon Province, northeastern Thailand

Demographic characteristic	Cases		Prevalence (%) (95% CI)	Unadjusted OR (95% CI)	P-value	Adjusted OR (95% CI)	P-value
	n	N
Gender
Male	30	95	31.6 (22.4–41.9)	3.21 (1.68–6.12)	< 0.001	5.55 (2.41–12.83)	< 0.001
Female	19	151	12.6 (7.7–18.9)	1.00		1.00
Age group
21–40	0	24	0	–		–
41–60	12	135	8.9 (4.7–15.1)	0.13 (0.06–0.27)	< 0.001	0.08 (0.03–0.19)	< 0.001
≥ 61	37	87	42.5 (31.9–53.6)	1.00		1.00
Education level
Primary	20	98	20.4 (12.9–29.7)	1.28 (0.34–4.86)	0.715	1.34 (0.26–7.06)	0.728
Secondary	26	130	20 (13.5–27.9)	1.25 (0.34–4.64)	0.739	1.42 (0.28–7.41)	0.671
Tertiary	3	18	16.7 (3.6–41.4)	1.00		1.00
Marital status
Single	4	18	22.2 (6.4–47.6)	0.69 (0.18–2.61)	0.579	0.62 (0.11–3.42)	0.579
Married	35	194	18 (12.9–24.2)	0.53 (0.23–1.21)	0.129	0.53 (0.18–1.55)	0.246
Divorced/widowed	10	34	29.4 (15.1–47.5)	1.00		1.00
Occupation
Farmer	39	199	19.6 (14.3–25.8)	2.44 (0.31–19.61)	0.402	0.46 (0.03–6.91)	0.574
Self-employed	6	20	30 (11.9–54.3)	4.29 (0.44–41.37)	0.208	6.02 (0.32–114.17)	0.232
Merchant	3	16	18.8 (4.1–45.6)	2.31 (0.21–25.66)	0.496	1.96 (0.12–32.79)	0.641
Government	1	11	9.1 (0.2–41.3)	1.00		1.00
Monthly income
THB ≤ 8000	44	208	21.2 (15.8–27.3)	1.77 (0.65–4.81)	0.262	2.75 (0.57–13.99)	0.206
THB > 8000	5	38	13.2 (4.4–28.1)	1.00		1.00
History of eating raw fish
Yes	39	191	20.4 (14.9–26.8)	1.15 (0.53–2.49)	0.715	1.43 (0.53–3.85)	0.482
No	10	55	18.2 (9.1–30.9)	1.00		1.00
History of stool examination
Yes	26	107	24.3 (16.5–33.5)	1.62 (0.86–3.04)	0.133	1.51 (0.67–3.41)	0.321
No	23	139	16.6 (10.8–23.8)	1.00		1.00
History of positive helminth
Yes	13	48	27.1 (15.3–41.8)	1.67 (0.81–3.47)	0.169	1.58 (0.63–3.98)	0.332
No	36	198	18.2 (13.1–24.3)	1.00		1.00
History of taking anti-helminths
Yes	2	5	40 (5.3–85.3)	2.75 (0.45–16.93)	0.275	2.09 (0.23–18.61)	0.511
No	47	241	19.5 (14.7–25.1)	1.00		1.00
Consume alcohol
Yes	20	90	22.2 (14.1–32.2)	1.25 (0.66–2.37)	0.493	1.69 (0.72–3.99)	0.231
No	29	156	18.6 (12.8–25.6)	1.00		1.00
Defecate on water
Yes	13	67	19.4 (10.8–30.9)	0.96 (0.47–1.94)	0.901	0.81 (0.31–2.16)	0.669
No	36	179	20.1 (14.5–26.7)	1.00		1.00
Received info on Opisthorchis
Yes	48	242	19.8 (15–25.4)	0.74 (0.08–7.29)	0.798	0.51 (0.01–38.35)	0.757
No	1	4	25 (0.6–80.6)	1.00		1.00

CI confidence interval, n number of individuals positive for O. viverrini, N number of individuals tested for O. viverrini, OR odds ratio, THB Thai Baht

Discussion

Infection with O. viverrini continues to be a significant public health issue and a primary risk factor for cholangiocarcinoma (CCA) throughout the Greater Mekong subregion, particularly in northern and northeastern Thailand [5, 8]. A recent large-scale survey across 21 provinces in northern, northeastern, and eastern Thailand using quantitative fecal examination found that the overall prevalence of O. viverrini remained high at 12.2%, reaching up to 30.2% in Kalasin Province [26]. The same study also reported that Sakon Nakhon Province, a neighboring province of Kalasin, had a similarly high prevalence of nearly 30%, highlighting localized hotspots of intense transmission and the need for more targeted and intensified surveillance in these areas [26]. To better understand the current endemicity of O. viverrini, this study applied a conventional molecular method targeting the ITS2 gene, alongside microscopic stool examination in a small rural community in Sakon Nakhon Province. This study found that stool microscopy detected O. viverrini in only 6.1% of participants, with all positive cases classified as very light-intensity infections (< 500 EPG). Whereas, ITS2-PCR identified a much higher prevalence of 19.9%, demonstrating the superior sensitivity of molecular diagnosis. Additionally, male participants were at higher risk of infection, while adults aged 41–60 years were less likely to be infected compared to older individuals.

The prevalence of O. viverrini infection detected by stool microscopy in the present study setting in Sakon Nakhon Province was 6%. This estimate is comparable to the 7% reported in a previous study conducted in 2020 [27], but lower than the 11% and 16% reported in two studies from 2017 [22, 24]. However, it is markedly lower than the prevalence observed in a recent large-scale survey reporting nearly 30% infection in 2025 [26]. Differences in prevalence across studies may be attributed to variations in study design, sampling approaches and the characteristics of the populations examined. Temporal shifts in which have not been assessed in the present study such as community behaviors, environmental conditions and the impact of ongoing control initiatives may also contribute to the heterogeneity of infection rates reported over time. In Thailand, stool microscopy, particularly egg-based methods such as the Kato–Katz technique, remains the most widely used diagnostic approach for O. viverrini infection [23, 28]. The WHO recommends Kato–Katz as the primary diagnostic tool for detecting and monitoring Opisthorchis eggs in stool specimens, considering it the “gold standard” due to its low cost, standardized methodology, and suitability for use in resource-limited field settings [28]. Although Kato–Katz is quantitative and allows estimation of infection intensity based on egg counts, its sensitivity decreases in low-prevalence settings, making it less reliable for monitoring control programs as transmission declines [29]. Furthermore, in the current transmission setting, as in the study area, stool-based diagnosis for O. viverrini may be less accurate because very light infections produce very few eggs [30]. In addition, Opisthorchis eggs can easily be confused with those of minute intestinal flukes, leading to misclassification by an untrained microscopist [31]. Thus, relying solely on stool microscopy may be insufficient to inform national public health strategies for helminth control and elimination, particularly for O. viverrini. Therefore, incorporating more sensitive diagnostic approaches is needed to accurately assess the true burden and intensity of these parasitic infections in endemic areas of Thailand.

Notably, when ITS2-PCR was used alongside stool microscopy in the current study, the detection rate increased substantially to 19.9%, identifying more than threefold the number of cases found by microscopy alone. This enhanced detection capacity is consistent with previous molecular studies demonstrating the higher sensitivity of ITS2-PCR in different endemic settings. For example, ITS2-PCR detected O. viverrini infection in 7% of individuals in Kratie Province, northeastern Cambodia [18], and identified a prevalence of 10% in a rural community in Chachoengsao Province, central Thailand [17]. In contrast, a much higher prevalence of 31% was reported using the same molecular assay among communities in Chiang Mai Province in northern Thailand [32]. In the present study, all microscopy-positive cases exhibited very light-intensity infections (< 500 EPG). While ITS2-PCR detected a substantially larger number of infections, earlier studies have reported variable molecular detection rates at low egg burdens, including reduced sensitivity at < 200 EPG in Khon Kaen Province [14] and detection rates of 79–83% in low-intensity settings in Lao PDR and northern Thailand [16, 32]. Differences in sample processing, DNA extraction efficiency, PCR optimization and the use of fresh versus preserved stool samples may partly explain these discrepancies. However, it should be noted that, in the absence of sequencing confirmation, the molecular prevalence reported here may include undetected co-infections or cross-reactivity with morphologically similar minute intestinal flukes, representing an important limitation. Nonetheless, the moderate agreement observed between the composite reference standard and ITS2-PCR underscores the diagnostic challenge posed by low-intensity infections and highlights the complementary value of molecular methods in detecting infections that may be missed by conventional stool examinations in lightly endemic settings.

The results of the analysis of risk factors for opisthorchiasis revealed the traditional role of demographic data such as the male gender, which had a significantly higher risk of O. viverrini infection compared to females, a pattern that has been consistently observed in previous studies across northeastern Thailand [22, 23, 26, 33–37]. This gender disparity appears to be driven by a complex interplay of behavioral, cultural and social factors. Traditional dietary practices play a major role in the higher prevalence of infection among men. Consumption of raw or undercooked freshwater fish, which is a primary source of O. viverrini infection, is more prevalent among males, often due to cultural habits, convenience and social norms [38]. Limited cooking facilities and preparation methods in rural households further reinforce the consumption of these high-risk foods. Socialization patterns also contribute; such as communal meals or gatherings often involve shared dishes of raw or lightly prepared fish, increasing exposure risk among men who are more likely to participate in such events [22, 23]. Although females in this community may also consume raw fish, the frequency, quantity and context of consumption are generally lower, which helps explain the gender disparity in infection rates [39]. This finding highlights that male-specific behaviors are a critical determinant of O. viverrini infection in endemic areas. Addressing this risk through targeted interventions including health education, promotion of proper cooking and food-handling practices and behavioral modifications, is essential for reducing infection and controlling transmission in rural communities.

This study also showed a clear age-dependent increase in O. viverrini prevalence. Adults aged ≥ 61 years had more than four times the prevalence of those aged 40–60 years, indicating significantly lower odds of infection in the latter age group. This age-specific pattern has likewise been documented elsewhere in northeastern Thailand [22, 23, 35]. The higher prevalence in older adults likely reflects long-term, persistent infections acquired decades earlier rather than repeated re-infections, consistent with the fact that 97.7% of participants reported no prior use of anti-helminthic drugs. Historical cohort effects may also contribute, as older generations were raised with stronger traditions of consuming chopped raw fish salad (Koi pla) or extensively fermented fish (Pla ra) and had less access to health education or anthelmintic programs, whereas middle-aged adults may have adopted reduced-risk behaviors due to past control campaigns and changing dietary norms [40]. Additionally, diagnostic and detection biases can influence observed prevalence. Older adults often carry higher-intensity infections that are easier to detect by microscopy, while lighter infections in treated or less-exposed middle-aged adults may go undetected unless more sensitive methods are used [18, 32]. In Sakon Nakhon Province, targeted health education and behavior-change interventions should focus on older adults, particularly addressing the continued practice of consuming raw or undercooked freshwater fish. Regular community-based screening using more sensitive diagnostic methods, combined with periodic praziquantel treatment, is also recommended to reduce the long-term infection burden in this high-risk age group.

This study has several limitations. Its cross-sectional design limits the ability to establish causality and prevents assessment of temporal changes in community behaviors, environmental conditions and the effects of ongoing control initiatives, all of which may influence infection rates. Participants were also recruited through convenience sampling from a single rural community, which may introduce selection bias, as volunteers may differ from non-participants in health status or risk behaviors. Furthermore, the sample size was not adjusted for the non-probabilistic sampling design. Therefore, the estimated prevalence should be interpreted with caution as community-specific and not as representative of the broader population. The reliance on a single stool sample per participant may have reduced the sensitivity of stool microscopy because helminth egg excretion is intermittent, particularly in light-intensity infections. This methodological constraint likely contributed to the low microscopy detection rate observed and partially explains the wider performance gap between microscopy and PCR. In addition, although molecular methods were employed, the study focused primarily on the current prevalence of O. viverrini and did not analyze its genetic diversity or differentiate it from other human liver flukes or common minute intestinal flukes, as previously implemented [17, 24]. It is particularly important to determine the true prevalence of O. viverrini, as it is more endemic in this region and is strongly associated with CCA. Nonetheless, the actual molecular prevalence of other fluke species in the study area remains unknown. Furthermore, all participants were drawn from a single community in northeastern Thailand, limiting the generalizability of the findings to other districts or regions in Thailand. The study also excluded individuals younger than 20 years due to infection risk, leaving a gap in understanding transmission dynamics in younger populations. Future research should include broader geographic coverage, younger age groups and more comprehensive molecular analyses to better capture the epidemiology of liver fluke infections in Thailand.

Conclusions

In conclusion, this study confirms that O. viverrini infection remains prevalent in the surveyed endemic community in Sakon Nakhon Province of northeastern Thailand. While stool examinations remain essential for routine diagnosis and assessment of infection intensity, ITS2-PCR was able to detect parasite DNA in very light infections that may not be detectable by microscopy. Molecular testing should therefore be considered a complementary surveillance tool, particularly in low-endemicity or near-elimination settings, rather than a replacement for standard parasitological methods. Key demographic risk factors associated with infection, including male gender and older age group, were identified, highlighting specific vulnerable populations who are at greater risk of acquiring O. viverrini infection and may benefit most from targeted interventions. The continued persistence of opisthorchiasis in these areas underscores the need for sustained and enhanced public health measures. Overall, these findings emphasize the importance of continuous surveillance, the incorporation of sensitive diagnostic methods and targeted screening programs to reduce the infection burden and improve health outcomes in endemic communities.

Abbreviations

CCA

Cholangiocarcinoma

DALY

Disability-adjusted life years

EPG

Egg per gram

FETC

Formalin-ether concentration technique

ITS2

Internal transcribed spacer 2

OR

Odds ratio

PCR

Polymerase chain reaction

WHO

World Health Organization

Author contributions

S.C. and Z.M.I designed the study. P.S., W.P., and S.C. collected specimens and performed field laboratory investigations. P.S., N.W.H., K.S., and Z.M.I performed molecular laboratory analysis. P.S., S.C., N.W.H., K.S., and Z.M.I analyzed the data. P.S. drafted the initial manuscript which was then critically reviewed by S.C. and Z.M.I. All authors reviewed and approved the final version of the manuscript for publication.

Funding

This work was partially supported by the Geran Ganjaran Penerbitan (Grant No. GP-K019336) and the Geran Fundamental Fakulti Perubatan (Grant No. FF-2025-202) from Universiti Kebangsaan Malaysia (UKM).

Data availability

The datasets generated and analyzed during the current study are not publicly available due to the inclusion of personally provided information. However, they are available from the corresponding author upon reasonable request with appropriate justification.

Declarations

Ethics approval and consent to participate

This study was conducted according to the guidelines stipulated in the Declaration of Helsinki. All procedures involving participants in this survey were approved by the Ethics Review Committee of the Sakon Nakhon Provincial Public Health Office (Reference number: SKN REC 2025-031). Informed consent was obtained from each patient before participation in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.