Study of the population genetic structure of Opisthorchis-like eggs in northern Thailand using mitochondrial genes

Background Opisthorchis-like eggs are a public health problem in northern and northeastern Thailand. However, the genetic epidemiology and structure of these parasites in northern Thailand are unknown. Thus, this study investigated their population genetic structure using cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) nucleotide sequences. Methodology/Principal findings A study was conducted in the hill tribe regions of Chiang Mai Province, northern Thailand. Internal transcribed spacer 2 polymerase chain reaction and restriction fragment length polymorphism were used to distinguish 205 positive feces samples for Opisthorchis-like eggs. The results showed that the prevalence of O. viverrini and Haplorchis taichui was 10.5% and 38.2%, respectively, and the co-infection rate was 37.2%. To determine the genetic structure of O. viverrini and H. taichui using cox1 and nad1 genes, genetic analysis was performed using 30 randomly chosen fecal samples for Opisthorchis-like eggs. Pairwise FST analysis indicated that O. viverrini and H. taichui displayed nonsignificant genetic differentiation within Chiang Mai Province and between interpopulations from different geographic areas. Moreover, within the intrapopulation in Chiang Mai Province, cox1 presented higher gene flow than nad1 in O. viverrini, while nad1 demonstrated higher gene flow than cox1 in H. taichui. The neutrality tests based on Fu’s Fs indicated population expansion and selective sweep from bottleneck or hitchhiking in O. viverrini and H. taichui populations, supported by haplotype network patterns. Phylogenetic tree analysis based on cox1 and nad1 revealed the monophyly of O. viverrini and H. taichui and genetic relationships with other isolates collected from Thailand, Lao People’s Democratic Republic (PDR), and Vietnam. Conclusions/Significance This study investigated the molecular discrimination and genetic structure of Opisthorchis-like eggs in northern Thailand. The genetic information derived from this study could be associated with the background, molecular epidemiology, and disease severity of these parasites.


Introduction
In Thailand, food-borne trematode infections (especially human liver fluke [Opisthorchis viverrini] and minute intestinal flukes [MIFs] in the northern and northeastern regions) remain a public health problem [1,2].O. viverrini infections cause opisthorchiasis.Most clinical manifestations of opisthorchiasis are asymptomatic; however, chronic infection can lead to cholangiocarcinoma (CCA), which is diagnosed at a late stage, resulting in poor prognosis and difficult treatment [3][4][5][6][7][8].Thailand has the highest global prevalence of CCA, especially in the northeastern region (89 per 100,000 for females and 129 per 100,000 for males) [9].MIF infections are often asymptomatic, but heavy infections can cause intestinal irritation and inflammation, reducing nutrient and fluid absorption.Severe symptoms occur when parasite eggs pass through the intestinal wall into the bloodstream, damaging the internal organs [10].Currently, O. viverrini and MIF infections are diagnosed by detecting parasite eggs in feces under a light microscope using the Kato-Katz technique or formalin-ethyl acetate concentration technique (FECT) [11,12].However, O. viverrini eggs are difficult to distinguish from MIF eggs under a light microscope because they have similar size and shape; therefore, they are identified as Opisthorchis-like eggs [1,[13][14][15].The Chinese liver fluke (Clonorchis sinensis) was reported to have a prevalence of 23% among residents in central Thailand [16], and chronic infection with this parasite can lead to CCA.However, there has only been one report on the prevalence of C. sinensis in Thailand.Despite evidence showing the distribution of the freshwater snail Melanoides tuberculata, which may serve as the first intermediate host of C. sinensis in Thailand that could support its life cycle, comprehensive studies on the prevalence of this parasite in Thailand are lacking [17].Additionally, M. tuberculata is distributed in the northern, eastern, and central regions of Thailand [17], suggesting that the prevalence of this parasite should also be monitored in northern Thailand.Currently, molecular-based polymerase chain reaction (PCR) approaches are commonly used to differentiate Opisthorchis-like eggs.In these methods, internal transcribed spacers 1 and 2 (ITS2) [16,18,19] and mitochondrial genes (e.g., cytochrome c oxidase subunit 1 [cox1] [20,21], NADH dehydrogenase subunit 1 [nad1] [21,22], and NADH dehydrogenase subunit 2 [nad2] [23,24]) are used as molecular targets for discrimination.Moreover, multilocus enzyme electrophoresis [25,26], random amplified polymorphic DNA [27,28], microsatellite markers [29,30], and mitochondrial sequencing [15,22,31] have been used to examine the genetic structure and diversity of O. viverrini.Mitochondrial DNA sequences are widely used to analyze the genetic structures of parasites such as Fasciola hepatica [32,33], Paragonimus westermani [34,35], and Schistosoma spp.[36][37][38] because of their high mutation rates and maternal inheritance characteristics, allowing for a comprehensive understanding of their genetic variation and population structure.Thus, cox1 and nad1 could be used for investigating the genetic structure and diversity among O. viverrini populations in northern Thailand, where mitochondrial DNA sequence data are lacking.
In northern Thailand, the prevalence of MIF infections, especially Haplorchis taichui, is higher than that of O. viverrini infections.According to a previous study, the prevalence of H. taichui in Nan and Lampang Provinces was 70.8% and 69.0%, respectively, whereas O. viverrini was not found [39].In another study that performed stool examinations of residents in Chiang Mai Province, northern Thailand, the prevalence rates of O. viverrini and H. taichui were 10.5% and 38.2%, respectively, and the co-infection rate was 37.2% [2].Conversely, high prevalence of O. viverrini was not observed in several endemic locations of northern Thailand, which uncorrelated to CCA incidence, and MIFs infections do not produce CCA in this region [39].Since a high prevalence of Opisthorchis-like egg infections have been reported among hill tribe residents of Chiang Mai, northern Thailand, the Ministry of Public Health of Thailand and collaborating health agencies launched a national strategic plan to control liver fluke infections and CCA, with the goal of reducing the prevalence of these parasites and CCA in northern Thailand [2].However, the genetic structure of Opisthorchis-like eggs in these endemic areas is unknown.Therefore, the present study aimed to investigate the presence and genetic structure of Opisthorchis-like eggs collected from fecal samples of hill tribe residents in Chiang Mai Province, northern Thailand, using mitochondrial genes (cox1 and nad1).Using fecal samples containing Opisthorchis-like eggs can provide a representative sample of the parasite population within infected individuals and will reflect the overall genetic diversity present in the parasite population within the host population.Moreover, pooled parasite eggs from fecal samples of infected individuals enables more efficient resource utilization by reducing the number of individual samples collected and invasive processes required for each sample.The findings of this study are crucial for the development of disease prevention and long-term control strategies, surveillance efforts, and understanding the diversity and evolution of these parasites in northern Thailand.

Ethics statement
The research protocol for this study was reviewed and approved by the Institutional Review Board of the Faculty of Medicine Vajira Hospital.The study was granted an exemption in accordance with international guidelines for the protection of human research participants, including the Declaration of Helsinki, The Belmont Report, CIOMS Guidelines, the International Conference on Harmonization in Good Clinical Practice (ICH-GCP), and 45 CFR 46.101(b), under approval number COE: 19/2021X.As the fecal samples used in this study were collected as part of routine public health surveillance activities under the national strategic plan to control liver fluke infections and cholangiocarcinoma (CCA) in northern Thailand, implemented by the Ministry of Public Health in collaboration with other health agencies, these samples were anonymized and used exclusively for public health research purposes.Therefore, individual consent forms were not required.

Stool collection
A cross-sectional study was conducted on hill tribe communities located in Chaing Mai Province, northern Thailand.A total of 205 positive samples for Opisthorchis-like eggs were obtained from the stool bank of the Ministry of Public Health and collaborating health agencies.These fecal samples were collected from 94 infected individuals in Doi Tao District, 67 in Mae Tang District, and 47 in Chiang Dao District, as part of the national strategic plan to control liver fluke infections and CCA in 2020.However, these samples do not contain demographic data.The three districts are primarily rural in nature and surrounded by mountains and forests.Furthermore, the residents share a deep-rooted connection to the land and nature, with agriculture (especially rice cultivation) serving as a major economic activity.Furthermore, the residents include ethnic Thai individuals and various hill tribe communities such as Karen, Hmong, and Lahu.These people have a diverse culture; they speak their own languages and have preserved their ancient customs [40].

DNA extraction of Opisthorchis-like eggs from fecal samples
Each Opisthorchis-like egg-positive sample was sedimented using phosphate buffered saline (PBS) ethyl acetate concentration technique in accordance with Buathong's protocols [15].Briefly, 200 μL of each sample was thoroughly homogenized by mixing with 1.4 mL of ATL tissue lysis solution and breaking up the eggs through PBS incubation method and five freezethaw cycles using liquid nitrogen at 100˚C.Then, DNA extraction was performed using the QIAamp DNA Stool Mini Kit (Qiagen) in accordance with the manufacturer's instructions.Finally, the DNA was eluted to a volume of 50 μL with elution buffer and used as a template for PCR assays.

Discrimination of Opisthorchis-like eggs using PCR and restriction fragment length polymorphism (RFLP) assays
A total of 205 Opisthorchis-like egg-positive samples were subjected to PCR assay using RTFluke primers, which were designed by Traub et al. [16] and could amplify the ITS2 regions of human liver flukes and MIFs in the Heterophyidae family.PCR amplifications were performed in a total volume of 25 μL using a DNA template, 10 pmol of each RTFluke primer, and 1X KAPA2G Fast HotStart ReadyMix with dye (Roche).The PCR products were amplified using the Mastercycler personal (Bio-Rad).ITS2-PCR assay was performed in accordance with Traub et al.'s protocols [16].The amplicons of O. viverrini, C. sinensis, and H. taichui were 375, 381, and 526-bp, respectively.To differentiate between O. viverrini and C. sinensis, the 375 and 381-bp PCR products were subjected to discrimination analysis using the FauI restriction enzyme (New England Biolabs) based on RFLP.After digestion at 55˚C for 6 h, the PCR products for O. viverrini comprised 129 and 247-bp fragments, whereas C. sinensis yielded an undigested amplicon [15].The results of ITS2-PCR and RFLP assays for discriminating Opisthorchis-like eggs revealed only the presence of O. viverrini and H. taichui.Therefore, genetic analysis of cox1 and nad1 was performed for O. viverrini and H. taichui.

Evaluation of PCR assays to detect O. viverrini and H. taichui in fecal samples based on cox1 and nad1
A subset of 30 positive samples, confirmed through ITS2-PCR for O. viverrini and H. taichui, was randomly selected for further analysis using COX1-and NAD1-PCR assays and genetic analysis.The PCR-based assay targeting cox1 of O. viverrini was evaluated using primers designed by Bauthong et al. [21].To evaluate of nad1 of O. viverrini, the COX1-PCR assay of O. viverrini was performed in a total volume of 25 μL, comprising a DNA template, 10 pmol of each primer, and 1X KAPA2G Fast HotStart ReadyMix with dye (Roche).New primers with greater sensitivity were designed using Primer3web version 4.1.0(https://primer3.ut.ee/) and used in this study.The NAD1-PCR assay of O. viverrini was performed in a total volume of 25 μL, comprising a DNA template, 10 pmol of each primer (OVNADI-F, 5 0 -TCAG GTACG-CAGGTGGTT TG-3 0 ; OVNADI-R, 5 0 -CCTTCGCAAG GT TAACAGCC-3 0 ), and 1X KAPA2G Fast HotStart ReadyMix with dye (Roche).The PCR products of cox1 of O. viverrini were amplified in the Mastercycle personal (Bio-Rad).The PCR assay of OVNADI primers was conducted as follows: predenaturation at 95˚C for 5 min, followed by 30 cycles of denaturation at 95˚C for 45 s, annealing at 60˚C for 45 s, extension at 72˚C for 1 min, final extension at 72˚C for 10 min, and holding at 12˚C.
To investigate mitochondrial genes in H. taichui, the primers and optimal PCR assays of cox1 conducted by Thaenkham et al. were used [22].The COX1-PCR assay for H. taichui was conducted in a total volume of 25 μL, comprising a DNA template, 10 pmol of each primer, and 1X KAPA2G Fast HotStart ReadyMix with dye (Roche).The primers used for detecting nad1 of H. taichui were designed using Primer3web version 4.1.0(https:// primer3.ut.ee/).The primer sequences for the NAD1-PCR assay of H. taichui were as follows: HTNAD1-F, 5 0 GGTGGCTAGACACT CAGAGC-3 0 ; HTNAD1-R, 5 0 -AGCCCC-CAAAGCTAACATCC-3 0 .PCR assay was performed in a total volume of 25 μL, comprising a DNA template, 10 pmol of each primer, and 1X KAPA2G Fast HotStart ReadyMix with dye (Roche).The optimal reaction conditions for the NAD1-PCR assay of H. taichui were as follows: predenaturation at 95˚C for 5 min, followed by 30 cycles of denaturation at 95˚C for 45 s, annealing at 60˚C for 45 s, extension at 72˚C for 1 min, final extension at 72˚C for 10 min, and holding at 12˚C.
The PCR products were run on a 2% agarose gel and visualized using the Molecular Imager Gel Doc XR+ Imaging System (Bio-Rad).Thereafter, the PCR products of cox1 and nad1 from O. viverrini and H. taichui were individually excised from agarose gel and placed into 2 mL tubes for gel extraction and DNA purification using the QIAquick Gel Extraction Kit (Qiagen) in accordance with the manufacturer's protocols.Subsequently, the samples were sent to U2Bio (Thailand) for DNA sequencing.The DNA sequences obtained in this study were aligned using the ClustalW program incorporated within the MEGA-X software [41], which allows for precise alignment and comparison.After alignment, the sequences were compared with a comprehensive database of known nucleotide sequences using the Basic Local Alignment Search Tool to validate species identification and determine species origin.To obtain accession numbers of each haplotype, the nucleotide sequences of cox1 and nad1 from Opisthorchis-like eggs obtained in this study were submitted to the GenBank database.Subsequently, the accession numbers of cox1 and nad1 from Opisthorchis-like eggs were utilized for phylogenetic tree and haplotype network analysis.

Sequence analysis of cox1 and nad1 from O. viverrini and H. taichui to determine population genetic structure
The population genetic structure of O. viverrini and H. taichui was determined using a total positive sample from COX1-and NAD1-PCR assays.The cox1 and nad1 nucleotide sequences of O. viverrini and H. taichui were genetically analyzed for haplotype diversity (Hd) values, segregation sites across populations (S), and nucleotide diversity (Pi) using DnaSP version 5.10.1 [42].The genetic differentiation in O. viverrini and H. taichui populations was evaluated using pairwise F ST values estimated from DnaSP version 5.10.1 [42].F ST values between 0.0 and 0.05 imply small genetic differences between populations (or some gene flow), values between 0.05 and 0.15 indicate moderate genetic differences, and values greater than 0.15 suggest high genetic differentiation and low (or no) gene flow.The gene flow between O. viverrini and H. taichui populations was calculated using N e m = (1/F ST − 1)/4 [43].The genetic differentiation and gene flow of cox1 within the O. viverrini intrapopulation from Chiang Mai Province were analyzed and compared with the interpopulations from northeastern Thailand (MDM-5, BR-2, MD-5, SKPK18M-3, SKNH-5, H5, HH22-1, VTNK), eastern Thailand (SK), Lao PDR (VT, KM-1, SV-4, accession number JF739555), and Vietnam (BD1).Additionally, the genetic differentiation and gene flow of cox1 in O. viverrini from 36 isolates, comprising 15 recently identified haplotypes from cats isolated in northeastern Thailand (OM424139-OM424174), [44] were analyzed and compared with haplotypes obtained from Chiang Mai Province to determine genetic relationship and transmission dynamics.Furthermore, the genetic differentiation and gene flow of nad1 within the O. viverrini intrapopulation in Chiang Mai Province were evaluated and compared with the interpopulations from different geographic areas, including northeastern Thailand (KLp, KS, KBp, KPv, KK, DQ119551), eastern Thailand (SK/3), Vietnam (BD, OvL, DL3, QN, PY3, OvPY3), Lao PDR (VT/1, SV/2, KM/1, JF739555, CP/14), and Cambodia (KD/4).For H. taichui, the genetic differentiation and gene flow of cox1 were investigated within the intrapopulation from Chiang Mai Province and compared with the interpopulations across geographically distinct endemic regions, including northern Thailand (CR1, CR3, CR4, PY7), central Thailand (CH1), Vietnam (HG4, QT3, QT6, QT15, TH3, TH5, TH11, YB, KC404636), Lao PDR (BK1, BK3, SV1, CP1), and the Philippines (PHL1).Furthermore, the genetic differentiation and gene flow of nad1 in H. taichui were investigated and compared with the H. taichui interpopulations from other endemic locations, including Vietnam (HTAQT3, QT3) and Korea (NC_022433).Moreover, the neutrality tests of cox1 and nad1 of O. viverrini and H. taichui obtained in this study were analyzed for the historical dynamics of the population using Fu's Fs and Tajima's D tests from Arlequin version 3.1 [45].Fu's Fs test is a statistical method that examines the allele frequency distribution in a population, whereas Tajima's D test evaluates the difference between the number of segregating sites and the average number of pairwise differences in a DNA sequence alignment.A significantly negative value of

Phylogenetic tree and haplotype network analysis of O. viverrini and H. taichui using cox1 and nad1
The nucleotide sequences of cox1 and nad1 in O. viverrini and H. taichui were analyzed using the ClustalW program in the MEGA-X software.Maximum likelihood phylogenetic trees were constructed using the Tamura 3-parameter in the MEGA-X software with 1,000 replications of bootstrapping values based on cox1 and nad1 of O. viverrini and H. taichui.[46].The reference sequences used in the O. viverrini and H. taichui phylogenetic trees were obtained from the GenBank database.Haplotype networks were generated using the cox1 and nad1 nucleotide sequences of O. viverrini and H. taichui.The haplotype networks were computed using a median-joining network algorithm from Network 10, which could be accessed at the following URL: http://www.fluxus-engineering.com.Moreover, the reference isolates of O. viverrini and H. taichui were retrieved from the GenBank database.
The analysis of Opisthorchis-like egg infections by district revealed distinct prevalence rates.In Doi Tao District, the rate of single infection with O. viverrini was 5.3% (5/94), while no cases were found in Mae Tang and Chiang Dao Districts.Notably, single infections with H. taichui were higher in Doi Tao District, occurring in 50% (47/94) of the samples.Additionally, mixed infections involving both O. viverrini and H. taichui were observed in 36.2% (34/94) of the samples from this district.In Mae Tang District, single infections with H. taichui were prevalent at 56.7% (38/67), while mixed infections of H. taichui and O. viverrini occurred at 25.4% (17/67).In Chiang Dao District, the prevalence of single infections with H. taichui was 46.8% (22/47), whereas mixed infections with both O. viverrini and H. taichui were observed in 27.7% (13/47).Overall, the prevalence analysis by district revealed that single infections with H. taichui and mixed infections with both O. viverrini and H. taichui were common across all three districts.Furthermore, C. sinensis was not detected in this study.

Evaluation of COX1-and NAD1-PCR assays to detect O. viverrini and H. taichui using fecal samples
Thirty positive samples for O. viverrini were randomly selected from a total of 64 samples identified across three districts.These selected 30 samples for O. viverrini were then used to evaluate the COX1-and NAD1-PCR assays and for subsequent genetic analysis.The sensitivities of COX1-and NAD1-PCR assays of O. viverrini in 30 positive fecal samples were 86.7% (26/30) and 66.7% (20/30), respectively.After amplification, the PCR products of cox1 and nad1 in O. viverrini were 504 and 744-bp (Fig 1 ), respectively.Moreover, COX1-PCR assay showed higher sensitivity than NAD1-PCR assay for detecting O. viverrini in fecal samples.In addition, the OVNADI primers used in the NAD1-PCR assay for amplifying the nad1 gene in O. viverrini did not cross-react with other intestinal parasites in fecal samples, including Ascaris lumbricoides, hookworm, Taenia spp., Trichuris trichiura, H. taichui, Giardia intestinalis, Blastocystis sp., and Entamoeba coli.
Similarly, 30 positive samples of H. taichui were randomly selected from a total of 164 positive samples collected across three districts.These selected H. taichui positive samples were subsequently subjected to COX1-and NAD1-PCR assays for genetic analysis.The sensitivity of COX1-and NAD1-PCR assays for detecting H. taichui in fecal samples was 96.7% (29/30) and 83.3% (26/30), respectively.The PCR products of cox1 and nad1 in H. taichui were 375 and 1,040-bp (Fig 2 ), respectively.The HTNAD primers used in NAD1-PCR assay were first developed for detecting nad1 in H. taichui in fecal samples, and no cross-reactions with other intestinal parasites were noted, including O. viverrini.Furthermore, ITS2-PCR assay exhibited greater sensitivity than COX1-and NAD1-PCR assays for detecting O. viverrini and H. taichui in fecal samples.However, the difference was not statistically significant (p > 0.05; 95% confidence interval).

Population genetic structure analysis of cox1 and nad1 in O. viverrini and H. taichui
Genetic analysis was conducted on fecal samples of O. viverrini collected from Chiang Mai Province.The investigation of 26 cox1 and 20 nad1 nucleotide sequences revealed 9 cox1 haplotypes (CM1-CM9; accession numbers: OR514397-OR514405) and 10 nad1 haplotypes (CMNMU1-CMNMU10; accession numbers: OR528872-OR528881).Moreover, cox1 and nad1 in O. viverrini had 7 and 11 polymorphism sites (S) and presented haplotype diversity (Hd) values of 0.738 and 0.837, respectively.In addition, the nucleotide diversity (Pi) values of cox1 and nad1 in O. viverrini were 0.00244 and 0.00482, respectively (Table 2).From pairwise F ST analysis, O. viverrini within the intrapopulation in Chiang Mai Province revealed low genetic variation in cox1 (F ST = 0.03704) and high genetic variation in nad1 (F ST = 0.51020).However, the population of O. viverrini in this region showed no significant genetic difference (all p > 0.05).Gene flow estimation of O. viverrini showed that cox1 exhibited high gene flow (N e m = 5.75), whereas nad1 exhibited low gene flow (N e m = 0.396) (Table 3).[47].However, Tajima's D, Fu and Li's D, and Fu and Li's F tests did not yield statistically significant results (all p > 0.01) (Table 3).

Phylogenetic tree analysis of O. viverrini and H. taichui based on cox1 and nad1
The results of maximum likelihood phylogenetic tree analysis conducted on O. viverrini revealed that the tree topologies of cox1 and nad1 nucleotide sequences mostly formed a monophyletic group, consistent with reference isolates.The phylogenetic analysis of cox1 in O. viverrini collected from Chiang Mai (CM1-CM9) revealed that the majority of haplotypes formed monophyletic clusters and displayed a strong genetic relationship with reference isolates.Specifically, CM1, CM2, CM5, CM6, and CM9 haplotypes, obtained from Chiang Mai Province, were grouped together and shared genetic similarities with reference isolates originating from various regions, including northern Thailand (LP-1 and LP-5), northeastern Thailand (MDM-5, BR-2, MD-5, SKPK18M-3, SKNH-5, H5, HH22-1, VTNK, NP, SSK), Vietnam (BD1), and Lao PDR (JF739555).Moreover, two cat haplotypes (C4 and C24) were included in this clade, with C4 demonstrating a close genetic relationship with CM5 from Chiang Mai Province.Furthermore, CM4 and CM8 were closely related to haplotypes from northeastern Thailand (KK, NP, SSK) and Lao PDR (KM, SV-4).Additionally, CM8 obtained in this study exhibited close genetic similarity to cat haplotype C13-6, whereas haplotype CM7 was related to cat haplotypes C16 and C40-1.Moreover, haplotype CM3 displayed close genetic similarity to the reference isolate KPK18M-3 obtained from northeastern Thailand, with a bootstrap The phylogenetic tree of H. taichui using cox1 and nad1 nucleotide sequences from Chiang Mai Province also revealed a monophyletic group.In the phylogenetic tree of H. taichui based on cox1, nine haplotypes were grouped together with reference isolates from central Thailand (CH1) and Lao PDR (BK1, BK3, CP1, SV1).Moreover, haplotypes CMNU1 and CMNU4 were closely related to the reference isolate BK3, whereas haplotype CMNU1 was closely related to reference isolates BK1 and SV1 (Fig 4A).Based on nad1 nucleotide sequences from Chiang Mai Province, the phylogenetic tree of H. taichui showed that most haplotypes were within the same taxonomic group, being monophyletic and clustering with reference isolates from Vietnam (QT3 and HTAQT3).Furthermore, haplotypes CMHT10, CMHT11, and

Haplotype network of O. viverrini and H. taichui based on cox1 and nad1 nucleotide sequences
The haplotype networks of O. viverrini based on cox1 and nad1 collected from Chiang Mai Province and reference isolates were constructed and showed star-like patterns, indicating a rapidly expanding population.The haplotype network of O. viverrini based on cox1 revealed two distinct groups: human and cat haplotypes.Haplotype A emerged as the predominant and possibly ancestral haplotype.In addition, singletons (CM5, CM7, and CM9) retrieved from Chiang Mai Province exhibited genetic differences from other haplotypes (Fig 5A).Moreover, five cat haplotypes (i.e., C4, C13-6, C16, C40-1, and C24) were grouped within the cluster of human haplotypes.Haplotypes CM1, CM2, and CM6 obtained from Chiang Mai Province showed a close genetic relationship with cat haplotype C4, whereas haplotype CM8 formed a cluster with cat haplotype C13-6.Furthermore, haplotype SK was distinct from human haplotypes and categorized with cat haplotype C51.Among the cat haplotypes, haplotype CAT1 was the predominant one, comprising 14 isolates (Fig 5A ).
A haplotype network of O. viverrini using nad1 also presented population expansion.Haplotype CMNMU1 was dominant and exhibited high frequency, whereas haplotypes CMNMU6, CMNMU7, CMNMU8, CMNMU9, and CMNMU10 displayed obvious distinct genetic differences from another haplotype (Fig 5B ).
The cox1 haplotype network of H. taichui showed that haplotype CMNMU5 had a high frequency and was predominant.Haplotypes A and B and other haplotypes retrieved from Chiang Mai Province were genetically closely related.Moreover, haplotypes CMNMU2, CMNMU3, CMNMU4, CMNMU6, CMNMU7, and CMNMU8 were all singletons and demonstrated close genetic relationship to haplotypes A and B (Fig 6A).The nad1 haplotype network of H. taichui revealed that haplotype A was dominant and possibly the ancestral haplotype.In addition, several haplotypes from Chiang Mai Province, including CMHT2, CMHT3, CMHT4, CMHT6, CMHT7, CMHT8, CMHT9, CMHT10, CMHT11, CMHT13, and CMHT14, were observed as singletons, suggesting rapid population growth.Furthermore, haplotypes CMHT11, CMHT10, CMHT15, CCMHT13, and CMHT14 showed distinct divergence patterns with increasing distance from other haplotypes, particularly CMHT14 (Fig 6B).

Discussion
This study first investigated the genetic structure of Opisthorchis-like eggs in northern Thailand using mitochondrial genes, namely, cox1 and nad1.The results of ITS2-PCR assay of 205 fecal samples collected from Chiang Mai Province showed the high prevalence of H. taichui (79.5% overall) and O. viverrini (31.2% overall).This finding indicates that O. viverrini plays a major role in causing CCA in northern Thailand.Because of their similar endemic areas and second intermediate host, a high prevalence of co-infections (59.0%) between O. viverrini and H. taichui was also observed, which were commonly found in northern Thailand.In addition, the findings of this study show that using a light microscope for diagnosis could lead to misdiagnosis because O. viverrini and H. taichui have similar egg morphologies.
The genetic structures of O. viverrini and H. taichui were analyzed using the cox1 and nad1 genes.COX1-PCR assay of O. viverrini and H. taichui showed higher sensitivity than NAD1-PCR assay.Thus, COX1-PCR assay is more suitable for studying the prevalence of Opisthorchis-like eggs because the sensitivity for detecting cox1 is greater than that for nad1.Genetic structure analysis of O. viverrini isolated from Chiang Mai Province, northern Thailand, using mitochondrial genes demonstrated that nad1 in O. viverrini displayed higher genetic variation than cox1, as supported by haplotype diversity, segregation sites, pairwise F ST analysis, and gene flow estimation.Moreover, the genetic difference of O. viverrini using nad1 within the intrapopulation in Chiang Mai Province was higher than that between interpopulations from different geographical endemic areas.A previous study suggested that the genetic differentiation of nad1 in O. viverrini reduced genetic variation within interpopulations while increasing it between intrapopulation [22].Therefore, nad1 could be more suitable for investigating genetic variation and structure than cox1 in O. viverrini.The genetic structure of O. viverrini in Ching Mai Province based on cox1 revealed low genetic differentiation and high gene flow within the intrapopulation.This finding suggests that equivalent allele frequencies exist, which could prevent genetic drift despite having different geographically endemic areas.However, the genetic structures of cox1 and nad1 in O. viverrini were similar since genetic differentiation did not show significant differences within or between different geographical areas.The population of O. viverrini in Chiang Mai Province was expanding, and selective sweep and haplotype network patterns also showed rapid population growth.However, the results of Tajima's D test were not significant, and many samples are required for further investigation.The maximum likelihood phylogenetic trees based on cox1 and nad1 in O. viverrini isolated from humans showed genetic monophyly and shared a genetic relationship with reference haplotypes from different geographically endemic areas.Moreover, the phylogenetic tree and haplotype network of cox1 in O. viverrini revealed that five haplotypes (C4, C13-6, C16, C24, C40-1) isolated from cats were clustered with human haplotypes, which was similar to that in a previous study [44].Furthermore, haplotype SK was found in the same cluster with cat haplotypes, indicating that O. viverrini infections are not strictly host-specific.Additionally, haplotypes CM1, CM2, CM6, and CM8 obtained from Chiang Mai Province could be transmitted between residents in this province and cats, which serve as reservoir hosts.Additionally, haplotypes BD, MDM-5, BR-2, MD-5, LP-5, H5, LP-1, JF739555, SKNH-5, CM1, CM2, CM6, and C4 may have carried an older and dominant allele compared with singletons.Likewise, nad1 of O. viverrini collected from Chiang Mai Province displayed strong genetic relation to haplotypes from Vietnam (OvPY3 and PY3), implying that they might be ancient ancestral haplotypes.Recently, O. viverrini populations in Lower Mekong Basin were found to show genetic monophyly and grow without geographical barriers [22].Gene flow plays a crucial role in the genetic structure of O. viverrini populations and is frequently influenced by the host's movement patterns, particularly in regions with complex transmission dynamics.For example, the movement of the second intermediate host, cyprinoid fish, which typically inhabit freshwater bodies such as rivers, lakes, and ponds, is affected by factors including water currents, migration for spawning purposes, and environmental changes.Similarly, human movement, including travel and migration, can impact the spread of parasites in endemic areas, as can the movements of reservoir hosts (e.g., domestic cats and dogs), which may interact with infected cyprinoid fish or contaminated water sources.Additionally, Bithynia spp., the first intermediate host, usually inhabit freshwater habitats, including ponds, marshes, and slow-moving streams, and their movements are primarily confined to their aquatic environment and influenced by factors such as water flow, temperature, and habitat availability [22].Therefore, the host's movement, especially definitive hosts including humans and cyprinoid fish, could facilitate the spread of parasite genotypes across wide geographic areas, potentially contributing to genetic variation among O. viverrini populations.
The genetic structure of H. taichui based on cox1 and nad1 revealed high variation but no significant population differences.The intrapopulation differences in Chiang Mai Province are lower than the interpopulation differences in different geographically endemic areas.The genetic differences within H. taichui intrapopulation in Chiang Mai Province did not show a significant difference probably because they had similar genetic structure.Gene flow (i.e., the movement of specific alleles from geographically isolated populations) can support local adaptation.Consequently, the population of H. taichui in Chiang Mai Province, northern Thailand, showed low gene flow levels, resulting in high genetic variation within intrapopulation and low variation between interpopulations.In 2019, a study reported a significant prevalence of Opisthorchis-like egg infections within the hill tribe population of Chiang Mai Province, northern Thailand.The findings revealed a particularly noteworthy prevalence rate, with H. taichui and O. viverrini accounting for 75.4% and 37.2% of infections, respectively [2].Moreover, the prevalence of O. viverrini in this province was higher than that (7.2%) reported in a previous study conducted in the lower northern region of Thailand [48].Therefore, the genetic structure of O. viverrini in this area could be representative of O. viverrini in northern Thailand.Furthermore, the Opisthorchis-like eggs in this study were derived from the hill tribe population.Hence, understanding the population's genetic structure and gene flow is important for providing epidemiological data and identifying genetic diversity and differentiation patterns across geographical areas or ecological niches.However, a limitation of this study is the use of pooled parasite eggs from infected individuals, which may lead to the presence of multiple genotypes within hosts.This contrasts with conventional population genetic studies that often utilize individual parasite stages (e.g., egg, miracidium, cercaria, metacercariae, or adult worms) for genetic analysis.Nevertheless, pooled parasite eggs from infected individuals can allow us to capture a broad parasite population in infected individuals within a geographic area or host population within the same local endemic areas.Moreover, fecal samples with pooled parasite eggs increase the likelihood of detecting rare alleles or low-frequency genetic variants within the parasite population.In this study, the number of parasite eggs recovered from each sample was not quantified, which directly impacts the amount of DNA available for genetic analysis.Insufficient DNA due to a low number of eggs can result in unsuccessful amplifications or unreliable sequencing outcomes, leading to inaccurate genetic analysis and compromising the interpretation of genetic information.Previous studies demonstrated successful PCR amplification of samples with light infections containing Opisthorchis-like eggs.The successful amplification can be attributed to meticulous DNA extraction protocols, optimized PCR assays, and primers with high sensitivity, which facilitate nucleotide sequencing and subsequent genetic analysis [15,49].Additionally, samples yielding PCR-negative results may potentially limit the comprehensiveness of genetic analysis in these parasite populations.In addition, analyzing the genetic structures of parasites in fecal samples allows us to investigate intraspecific interactions and their consequences for parasite transmission and evolution in their definitive host.In this study, a subset of 30 positive samples that were randomly selected from ITS2-PCR was utilized for genetic analysis to serve as a subpopulation and represent the broader population.Subsequently, the genetic information derived from this subpopulation was analyzed to infer the genetic traits of the broader population, providing insights into their diversity, relationships, and potential for further investigation.This process involved examining patterns of genetic variation, elucidating evolutionary connections, and evaluating the effects of factors such as genetic drift and natural selection.This process involved examining genetic variation patterns, elucidating evolutionary connections, and evaluating the effects of factors such as genetic drift or natural selection.Additionally, this preliminary assessment of the genetic structure of Opisthorchis-like eggs in the northern region of Thailand informs decisions on whether to expand the study further or refine research methods.However, genetic analysis conducted on a subpopulation may not be entirely generalizable to the entire population.If the subpopulation does not accurately reflect the broader population, there is a risk of either overestimating or underestimating certain genetic characteristics.The genetic variation of Opisthorchis-like eggs in Chiang Mai Province, northern Thailand, may be influenced by several factors such as parasite lifespan, spatial distribution, random mating size, distribution of intermediate snail host population, and seasonal infective dynamics rather than geographic distance.For example, the genetic structure and expansion of O. viverrini and H. taichui in Chiang Mai Province, northern Thailand, may depend on seasonal factors.O. viverrini infections are limited to the hot-dry season, whereas H. taichui infections peak during the hot-dry season followed by a gradual decrease throughout the rainy and cool-dry season [50].Furthermore, the lifespan of O. viverrini and H. taichui significantly influences their genetic structures as H. taichui has a shorter lifespan, which could reduce gene flow across populations.Additionally, their life cycles require intermediate hosts, leading to a population bottleneck during dry seasons.The gene flow between O. viverrini and H. taichui populations may be correlated with local adaptations and environmental changes, rather than geographic distance.However, due to the limited number of samples and localities available for investigating the genetic structure of Opisthorchis-like eggs in northern Thailand, further research is required.Expanding the number of localities and increasing the sample size will be crucial to accurately clarify the genetic differentiation in this region.In addition, next-generation sequencing (NGS) technologies should be utilized to conduct a more comprehensive genetic analysis and detect rare and low-frequency alleles, thereby providing a more detailed and indepth genetic landscape of the parasite population in this endemic area.Moreover, another genetic marker, such as microsatellite loci, should be applied to reveal the population genetic structure of these parasite in this endemic area.In addition, molecular epidemiology data of intermediate hosts between seasons and reservoir hosts remain insufficient and should be examined to understand their genetic structure.The genetic structure of Opisthorchis-like egg parasites may also be related to clinical manifestations and pathology, particularly CCA.Thus, the information obtained in this research will contribute to control and treatment programs that may impact individuals residing in endemic areas.
In conclusion, this study is the first to examine the genetic structures of Opisthorchis-like eggs from hill tribe population in northern Thailand.Genetic discrimination of Opisthorchislike eggs from Chaing Mai Province, northern Thailand, revealed the high prevalence of H. taichui and O. viverrini and co-infection of both parasites.This finding indicates that O. viverrini may be related to the incidence of CCA in northern Thailand.Moreover, genetic analysis of O. viverrini and H. taichui showed no significant genetic differentiation either within or between populations, suggesting that these populations are monophyletic.In addition, H. taichui and O. viverrini populations revealed population expansion and selective sweep in the study area, and both parasites displayed close genetic relationships with isolates obtained from other geographic regions, such as northern Thailand, Lao PDR, and Vietnam.
Fu's Fs or Tajima's D test suggests an excess of rare alleles, which may indicate recent population expansion, selective sweeps, or background selection.Conversely, a significantly positive value may indicate population reduction, balanced selection, or population subdivision.Furthermore, extensions of Fu's Fs and Tajima's D tests, known as Fu and Li's F and Fu and Li's D tests, were performed using DnaSP version 5.10.1 [42].These tests are useful for detecting population expansions or bottlenecks.A significantly negative value of Fu and Li's F or Fu and Li's D test suggests an excess of singleton mutations, indicating population expansion or positive selection.By contrast, a significantly positive value suggests a deficit of singleton mutations, which may indicate population reduction or purifying selection.

Fig 1 .
Fig 1. PCR products of nad1 in Opisthorchis viverrini after amplification with OVNADI primers.The PCR products of NAD1-PCR assay of O. viverrini were 744-bp.M is the 100-bp DNA ladder, P is the positive control, S1-S3 show the NAD1-PCR products of O. viverrini from fecal samples, and N is the negative control.https://doi.org/10.1371/journal.pntd.0012445.g001

Fig 2 .
Fig 2. PCR products of nad1 in Haplorchis taichui after amplification with HTNAD1 primers.The PCR products of NAD1-PCR assay of H. taichui were 1,040-bp.M is the 100-bp DNA ladder, P is the positive control, S1-S4 show the NAD1-PCR products of H. taichui from fecal samples, and N is the negative control.https://doi.org/10.1371/journal.pntd.0012445.g002