Search

Infection with Strongyloides stercoralis is often asymptomatic but can be life-threatening in immunocompromised patients, which can be prevented by ivermectin (IVM) treatment. The efficacy of IVM has been reported to have lessened over time in some regions as a consequence of prolonged use and mass treatment campaigns. Ivermectin has been used in Thailand for more than a decade; therefore, we investigated the efficacy of a single dose (200 µg/kg) of IVM against in asymptomatic strongyloidiasis in northeastern Thailand. Fecal samples were collected before and 2 weeks after treatment and were analyzed for the presence of Strongyloides using a modified agar plate culture and the formalin-ethyl acetate concentration technique. Our results showed that single-dose IVM treatment successfully eliminated S. stercoralis infection in asymptomatic individuals in the endemic area with a 100% cure rate, indicating the high efficacy of IVM treatment in strongyloidiasis in northeast Thailand.

Strongyloidiasis, caused by Strongyloides stercoralis, is a neglected tropical disease with a global distribution. The infection can be fatal in immunocompromised individuals, and accurate diagnosis leading to timely treatment can save lives. Serodiagnosis is a sensitive method for diagnosis and is recommended for screening high-risk individuals. A point-of-care rapid test will facilitate the screening activities, especially in low-resource settings. This study aims to apply a new IgG4 immunochromatographic test using S. stercoralis recombinant antigen (SsRapid® cassette test) and to compare it with in-house IgG and IgG4 enzyme-linked immunosorbent assays (IgG- and IgG4-ELISAs) using native Strongyloides ratti antigen to investigate the epidemiology of strongyloidiasis in northeast Thailand. A total of 300 people participated, with 136 males and 164 females of a similar mean age. The reference tests were fecal examinations using the formalin-ethyl acetate concentration technique and an agar plate culture technique. The prevalence of S. stercoralis determined by SsRapid (81.7%) was significantly higher than that by fecal examinations (43.3%) or by antibody detection by IgG-ELISA (53.0%) or IgG4-ELISA (44.0%). The diagnostic sensitivities of SsRapid, IgG-ELISA, and IgG4-ELISA were found to be 93.9%, 77.7%, and 63.1%, respectively. The rate of positive tests by the SsRapid was significantly correlated to the levels of Strongyloides-specific IgG4 and IgG antibodies. By all diagnostic methods, male participants had a significantly higher prevalence of strongyloidiasis than females. Age was significantly associated with the concentration of specific serum IgG but not with the SsRapid grading score. In conclusion, SsRapid was shown to be a sensitive and valuable diagnostic test for the epidemiology study of strongyloidiasis.

Clonorchis sinensis, Opisthorchis viverrini and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts is important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes and hence contribute to reduction of CCA in endemic areas., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background and Aim: Captive animals are susceptible to parasitic diseases due to the stress and confinement they experience. In addition, they can serve as reservoirs of zoonotic parasites that have the potential to infect humans. To investigate this possibility, we estimated the prevalence of gastrointestinal (GI) parasites in captive mammals at Khon Kaen Zoo, Thailand., Materials and Methods: One hundred and forty-seven individual mammals (37 primates, 43 carnivores, 62 herbivores, and 5 rodents) were examined for parasitic infections by fecal examination daily for 3 consecutive days using the formalin-ethyl acetate concentration technique (FECT) and the agar plate culture method., Results: According to FECT, the overall prevalence of GI parasites was 62.6% (92/147). Within animal groups, the numbers were as follows: 67.6% (25/37) in primates, 23.3% (10/43) in carnivores, 85.5% (53/62) in herbivores, and 80.0% (4/5) in rodents. Using the agar plate culture method, 21.43% (27/126) were positive for Strongyloides spp. and hookworm infections. The GI parasites identified belonged to three categories: protozoa (including Entamoeba histolytica species complex, Entamoeba coli , Giardia spp., coccidia, and ciliated protozoa), trematodes (minute intestinal flukes and rumen flukes), and nematodes (strongyle/hookworm, Strongyloides spp., Ascarididae, and Trichuris spp.)., Conclusion: The findings of this study indicate the prevalence of several GI parasites in zoo animals with the potential for transmission to humans, given the animals' close proximity to both visitors and animal caretakers., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Sangpeng, et al.)

Background: Screening for opisthorchiasis, a parasitic worm infection affecting many millions of people in Southeast Asia, has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique (FECT) and Kato-Katz method. Although the urinary enzyme-linked immunosorbent assay (ELISA) has been used more recently, we developed a urinary antigen-based rapid diagnostic test (RDT) to simplify diagnosis and as a point-of-care testing (POCT) and field applications for surveillance and control of opisthorchiasis., Methods: A urinary Opisthorchis viverrini (OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV. The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA (n = 493). Cross-reactivities of urinary OV-RDT with other helminthiases coexisted with O. viverrini were determined (n = 96). A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis (n = 1629). The McNemar chi-square, Kruskal-Wallis and Cohen's kappa coefficient (κ-value) tests were used for statistical analyses., Results: Urinary OV-RDT had sensitivity of 94.2% and specificity of 93.2%, compared to faecal FECT. Urinary OV-RDT had high diagnostic agreement (Kappa = 0.842-0.874, P < 0.001) and quantitative correlation with urinary antigen ELISA (Kruskal-Wallis tests = 316.2, P < 0.0001) and faecal FECT (Kruskal-Wallis tests = 362.3, P < 0.0001). The positive rates by OV-RDT, ELISA and FECT were 48.9%, 52.5% and 49.3%, respectively. Cross-reactions of urinary OV-RDT with other helminthiases were few (2%). Field trials of urinary OV-RDT yielded comparable prevalence of O. viverrini between urinary OV-RDT (53.2%) and urinary antigen ELISA (54.0%). OV screening showed high diagnostic agreement (kappa > 0.8, P < 0.0001) between urinary OV-RDT and urinary antigen ELISA. The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT (86.6%) and urinary antigen ELISA (80.5%) were similar (P > 0.05)., Conclusions: The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis. The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening, control and elimination of opisthorchiasis, thereby contributing to a reduction in the disease burden in Southeast Asia., (© 2023. The Author(s).)

Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called " O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine . Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis . Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts.

Objective: Chronic Opisthorchis viverrini (OV) infection is the cause of advanced periductal fibrosis (APF), subsequently leading to cholangiocarcinoma (CCA). Natural killer (NK) cells can kill hepatic stellate cells (HSCs), the initiating cells for fibrosis formation, by using the interaction between the natural killer group 2 member D (NKG2D) receptor and its ligand on the HSCs. This can inhibit the fibrosis formation. Major histocompatibility complex class I chain-related A (MICA) is the ligand of the NKG2D receptor and has highly polymorphic characteristics that are involved in NKG2D binding and NK cell activation. This study aimed to investigate the polymorphism of MICA in OV-induced fibrosis., Method: MICA typing was performed by polymerase chain reaction- sequence specific primer (PCR-SSP) and sequencing in two groups: OV infection without fibrosis (N = 99) and with fibrosis (N = 290)., Result: Six alleles were identified and the MICA*010 allele had the highest frequency in both groups. The MICA*00201-02 allele was a protective factor for fibrosis (OR= 0.508, 95%CI= 0.34-0.76, Pc <0.05), while the MICA*019 allele was suggested to be a risk allele for fibrosis (OR=1.95, 95%CI=1.25-3.03, Pc<0.005). In addition, two motifs, glycine (G) at position 14 and glutamine (Q) at position 251, were negatively associated with fibrosis (G14: OR=0.508, 95%CI=0.34-0.76, Pc <0.05 and Q251: OR=0.586, 95%CI=0.41-0.84, Pc <0.05). Moreover, the distribution of the MICA-129 genotype also showed the protective genotype (Pc<0.05, OR=0.319, 95%CI= 0.12-0.54) for fibrosis. The MICA*00201-02 allele encoded all these motifs, and this suggested that it might lead to strong NK cell activation to kill HSCs, subsequently preventing fibrosis formation., Conclusion: This study described initial evidence suggesting that the polymorphism of the MICA gene might be a marker for OV-derived periductal fibrosis.

Background: Detection of parasite-specific IgG in urine is a sensitive method for diagnosis of strongyloidiasis and gives similar accuracy to serum IgG. However, there are no data concerning detection of IgG subclass in urine. To further explore the utility of diagnosis from urine samples, we evaluated the diagnostic performance of IgG4 in urine compared with parasitological and other immunological methods., Methods: The urine and sera included proven strongyloidiasis (group 1, n = 93), other parasitic infections (group 2, n = 40) and parasite negatives (group 3, n = 93). The performance of Strongyloides-specific IgG4 in urine for diagnosis of strongyloidiasis using fecal examinations as the reference standard was assessed., Results: With fecal examination as a gold standard, Strongyloides-specific IgG4 in urine had 91.4% sensitivity and 93.2% specificity while serum IgG4 had 93.6% sensitivity and 91.0% specificity. IgG4 in both urine and serum had almost perfect diagnostic agreements with fecal examination (Cohen's kappa coefficient was > 0.8). Cross-reactivity to Opisthorchis viverrini and Taenia spp. of IgG4 in urine were 7.5% and 12.5% in serum. Concurrent analyses of total IgG in urine and serum showed that the sensitivities (97.9-100%) and specificities (88.7-91.0%) were similar (P > 0.05). The sensitivity for parasitological examination by the formalin-ethyl acetate concentration technique (FECT) was 49.5% and that for agar plate culture technique (APC) it was 92.6%., Conclusion: Our findings showed that specific IgG4 detection in urine yielded similar diagnostic performance to the same biomarkers in serum. This suggests that accurate diagnosis of strongyloidiasis can be performed using urine samples and IgG4 is a valid choice of diagnostic marker. Further assessment is required to assess the utility of urine IgG4 for measuring the response treatment in strongyloidiasis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Human liver fluke infection through the consumption of raw freshwater fish is one of the foodborne parasitic infections of global concern. Despite decades of health campaign efforts, high prevalence of infection remains in different areas of the Lower Mekong Basin. This necessitates the consideration of the infection differences between places and the human-environment complexities of disease transmission. This paper used the socio-ecological model as a framework to unraveled the social science dimensions of liver fluke infection. We conducted questionnaire surveys in Northeast Thailand to gather participants' knowledge on liver fluke infection and reasons for raw fish consumption. We synthesized our findings with prior work to identify factors influencing liver fluke infection at four socio-ecological levels. At the individual level, gender and age differences in food consumption habits and personal hygiene of open defection presented the behavioral risks. At the interpersonal level, family tradition and social gathering affected the disease risk. At the community level, physical-social-economic environments of land use and modernization, community health infrastructure and health volunteer support accounted for the varying degree of infection. At the policy level, impacts of regional and national regulations on disease control, health system organization structure, and government development projects were of concerned. The findings provide insights into how infection risks are shaped by people's behavior, social connectedness, interactions with places, and the interplay of these multi-level socio-ecological influences. The framework therefore allows a more comprehensive understanding of liver fluke infection risks to inform a culturally sensitive and sustainable disease control program., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Streptococcus agalactiae (group B Streptococcus) sequence type 283 bacteremia, found almost exclusively in Southeast Asia, is associated with consuming raw freshwater fish, but some patients deny consumption. We detected fecal carriage in 5/184 (2.7%) persons in northeast Thailand. Human carriers might contribute to transmission or be the original source of this sequence type.

Detection of worm antigen in urine is a sensitive diagnostic method for opisthorchiasis, particularly for light-intensity infections; however, the presence of eggs in feces is essential for validating results from the antigen assay. To address the issue of low sensitivity of fecal examination, we modified the protocol for the formalin-ethyl acetate concentration technique (FECT) and compared it against urine antigen measurements for detection of the parasite Opisthorchis viverrini. First, we optimized the FECT protocol by increasing the number of drops for examinations from the standard two drops to a maximum of eight. We were able to detect additional cases after examination of ≥ 3 drops, and the prevalence of O. viverrini saturated after examination of ≥ 5 drops. We then compared the optimized FECT protocol (examining five drops of suspension) against urine antigen detection for the diagnosis of opisthorchiasis in field-collected samples. The optimized FECT protocol detected O. viverrini eggs in 25 of 82 individuals (30.5%) who had positive urine antigen tests but were fecal egg negative by the standard FECT protocol. The optimized protocol also retrieved O. viverrini eggs in 2 of 80 antigen-negative cases (2.5%). In comparison with the composite reference standard (combined FECT and urine antigen detection), the diagnostic sensitivity of examining two and five drops of FECT and the urine assay was 58.2, 67, and 98.8%, respectively. Our results show that multiple examinations of fecal sediment increase the diagnostic sensitivity of FECT and thus provide further support for the reliability and utility of the antigen assay for diagnosis and screening of opisthorchiasis.

Background: The direct reliance of humans on and their interactions with freshwater ecosystems in the Lower Mekong Basin have given rise to parasitic infections, which is particularly prevalent in Northeast Thailand where raw fish consumption is practiced. This study examined the interactions between environments, ecosystem (dis-)services, human raw fish consumption habits, and raw fish dish sharing on liver fluke infection risk., Method: Water fecal contents and the first intermediate snail host were sampled between June and September of 2019. One hundred twenty questionnaires were surveyed in two villages of different environmental surroundings, one next to a river and the other located inland, in Northeast Thailand. Multivariate regression analyses using linear mixed effect models assessed the influence of social, behavioral and perceptual factors on raw fish consumption frequency, willingness to avoid consumption and liver fluke infection status. Social network analysis compared the degree of raw fish dish sharing between the villages and assessed the probable influence of connections to fish procurement locations and sharing activities on liver fluke infection risk., Results: High abundance of the first intermediate snail host and presence of fecal contamination in water could endanger both villages to ecosystem disservices of parasitic transmission. The river-side village relied more on provisioning ecosystem services than the inland village (29.7% vs. 16.1% of villages) to consume raw fish as their main source of protein. Males in both villages (64.5 and 40.4 days/year for the respective villages) are also likely to consume koi pla and pla som, higher risk fish dishes, more frequently than females (4.1 and 4.3 days/year for the respective villages). The consumption habits of both villages were driven mostly by deriving cultural ecosystem services. Participation in raw fish dish sharing activities significantly reduced the odds of an individual being willing to avoid the consumption (Odds ratio = 0.19). Network analysis suggested that river-side villagers had a more direct raw fish dish sharing interaction and they procured fish from multiple locations; these characteristics might potentially account for more liver fluke infected households in the village., Conclusion: Villagers' raw fish consumption is driven by deriving cultural ecosystem services, and the geographic settings of the villages potentially affect villagers' fish procurement locations and infection risk. The findings underscore the linkages between villagers and their surrounding ecosystem environments as pertinent determinants for foodborne parasitic disease risk., (© 2023. The Author(s).)

Detection of anti-Strongyloides IgG in urine by enzyme-linked immunosorbent assay (ELISA) for diagnosis of strongyloidiasis reportedly has comparable performance to conventional serum assays. Initial comparisons of urine assays using commercial ELISA kits designated for serology have shown its diagnostic potential but sub-optimal accuracy. In the present study, we optimized urine ELISA protocols based on different antigen types and evaluated their accuracies in determining the epidemiology of strongyloidiasis in Northeast Thailand. Paired urine and fecal samples of 966 individuals from the study community were collected for three consecutive days and tested for strongyloidiasis. We compared three ELISA protocols using different antigens including crude S. stercoralis antigen (Ss-ELISA), crude S. ratti antigen (Sr-ELISA) and recombinant NIE antigen (NIE-ELISA) and fecal examination by agar plate-culture (APCT) technique and formalin-ethyl acetate concentration technique (FECT). The optimized ELISA protocols using three different antigen sources yielded significantly higher prevalence rates of strongyloidiasis (58.9-65.1%) than those by fecal examination methods (19.7%). The prevalence of strongyloidiasis determined by ELISA protocols significantly increased with age (p value < 0.0001) and males had higher prevalence than females (p value < 0.0001). Diagnostic agreements between ELISA protocols were moderate (κ = 0.461-0.586) and the agreement between each ELISA protocol and fecal examinations were slight (κ = 0.139-0.210). The results obtained by urine ELISA protocols using three different antigens showed comparable diagnostic performances, provided further supports for the utility of urine as an alternative clinical specimen for diagnosis of strongyloidiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Eamudomkarn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background/aims: Global liquid chromatography mass spectrometry (LC-MS) profiling in a Thai population has previously identified a urinary metabolic signature in Opisthorchis viverrini -induced cholangiocarcinoma (CCA), primarily characterised by disturbance in acylcarnitine, bile acid, steroid, and purine metabolism. However, the detection of thousands of analytes by LC-MS in a biological sample in a single experiment potentially introduces false discovery errors. To verify these observed metabolic perturbations, a second validation dataset from the same population was profiled in a similar fashion., Methods: Reverse-phase ultra-performance liquid-chromatography mass spectrometry was utilised to acquire the global spectral profile of 98 spot urine samples (from 46 healthy volunteers and 52 CCA patients) recruited from Khon Kaen, northeast Thailand (the highest incidence of CCA globally)., Results: Metabolites were differentially expressed in the urinary profiles from CCA patients. High urinary elimination of bile acids was affected by the presence of obstructive jaundice. The urine metabolome associated with non-jaundiced CCA patients showed a distinctive pattern, similar but not identical to published studies. A panel of 10 metabolites achieved a diagnostic accuracy of 93.4% and area under the curve value of 98.8% (CI = 96.3%-100%) for the presence of CCA., Conclusions: Global characterisation of the CCA urinary metabolome identified several metabolites of biological interest in this validation study. Analyses of the diagnostic utility of the discriminant metabolites showed excellent diagnostic potential. Further larger scale studies are required to confirm these findings internationally, particularly in comparison to sporadic CCA, not associated with liver fluke infestation., (© 2022 Indian National Association for Study of the Liver.)

Helminth transmission and morbidity are dependent on the number of mature parasites within a host; however, observing adult worms is impossible for many natural infections. An outstanding challenge is therefore relating routine diagnostics, such as faecal egg counts, to the underlying worm burden. This relationship is complicated by density-dependent fecundity (egg output per worm reduces due to crowding at high burdens) and the skewed distribution of parasites (majority of helminths aggregated in a small fraction of hosts). We address these questions for the carcinogenic liver fluke Opisthorchis viverrini , which infects approximately 10 million people across Southeast Asia, by analysing five epidemiological surveys ( n = 641) where adult flukes were recovered. Using a mechanistic model, we show that parasite fecundity varies between populations, with surveys from Thailand and Laos demonstrating distinct patterns of egg output and density-dependence. As the probability of observing faecal eggs increases with the number of mature parasites within a host, we quantify diagnostic sensitivity as a function of the worm burden and find that greater than 50% of cases are misdiagnosed as false negative in communities close to elimination. Finally, we demonstrate that the relationship between observed prevalence from routine diagnostics and true prevalence is nonlinear and strongly influenced by parasite aggregation.

It is known that Opisthorchis viverrini (OV) is the most significant risk factor for the development of cholangiocarcinoma (CCA); hence, it is also known as carcinogenic parasite. Effective control and elimination of OV infection should significantly reduce O. viverrini-related CCA. This chapter includes details of the three recently developed innovative tools, namely the Isan cohort database software, an OV-RDT for screening of O. viverrini, and an ultrasound telecommunication system. Past and current control programs, i.e., education, medication, and sanitation were discussed and stressed the need for a comprehensive control program which encompasses primary, secondary, and tertiary patient care programs for confirmation and management of suspected CCA cases. The approach of mathematical modeling for control of OV and CCA was also briefly described. Additionally, we highlighted the current progress toward control of OV and CCA in Thailand and potential for expansion into nearby countries in Southeast Asia., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Opisthorchis viverrini plays a key role as the carcinogenic liver fluke causing bile duct cancer in Southeast Asia. A comprehensive understanding of its life cycle, distribution, systematics, and population genetics is critically important as they underpin the effective development and establishment of future prevention and control programs that center on opisthorchiasis and cholangiocarcinoma. This chapter provides detailed information concerning the basic biology and updated information of O. viverrini related to its host life cycle, transmission route via raw, partially cooked or fermented freshwater cyprinid fish, endemic areas, and the discovery of new foci. Previous sequential studies over the last two decades on the phylogenetic and systematic relationships, genetic variation, and population genetics of O. viverrini as well as its snail intermediate host Bithynia spp. are presented and discussed, which have led to the currently known complex species level systematics and population genetics framework of this host-parasite system. Additionally, further directions for comprehensive research are suggested to provide a more complete understanding of liver fluke, O. viverrini-related cholangiocarcinoma., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Antigen detected in urine for the diagnosis of opisthorchiasis has a low daily variation; however, the longer term variability in antigen concentrations is unknown. In this study, we prospectively monitored Opisthorchis viverrini antigen concentrations for 30 consecutive days and at subsequent monthly intervals in a cohort of opisthorchiasis-positive individuals. On the basis of the monoclonal antibody-based ELISA, the profiles of antigen-positive rate and antigen concentration exhibited no significant change over 30 days with a mean proportion positive of 87.1% (range 73.7%-100%), and the average antigen concentration was 29.7 ± 2.2 ng/mL (mean ± SE). The urine antigen concentration at baseline was similar to the subsequent measurements at 2, 4, 6, and 10 months in the follow-up study (P > 0.05). The consistency and low daily and long-term fluctuation of O. viverrini antigen in urine demonstrates the reliability of urine assay for diagnosis of opisthorchiasis.

Background: Glycoprotein sialylation changes are associated with severe development of various cancers. We previously discovered the sialylation of serotransferrin (TF) in cholangiocarcinoma (CCA) using glycoproteomics approach. However, a simple and reliable method for validating sialylation of a specific glycobiomarker is urgently needed., Methods: We identified the altered glycosylation in CCA tissues by glycoproteomics approach using mass spectrometry. An enzyme-linked lectin assay (ELLA) was developed for determining the serum levels of sialylated TF in CCA, hepatocellular carcinoma (HCC) and healthy controls in training and validation cohorts., Results: The nine highly sialylated glycoforms of TF were markedly abundant in CCA tumor tissues than in control. Serum SNA-TF and MAL1-TF were significantly higher in CCA patients. Under receiver operating characteristic curve, serum SNA-TF concentrations significantly differentiated CCA from healthy control. Higher SNA-TF were significantly correlated with severe tumor stages and lymph node metastasis. The combined SNA-TF, MAL1-TF, and CA19-9 as a novel glycobiomarkers panel demonstrated the highest specificity (96.2%) for distinguishing CCA from HCC patients. In CCA patients with low CA19-9 levels, SNA-TF in combination with CA19-9 achieved in 97% diagnostic accuracy., Conclusions: Sialylated serotransferrin glycoforms could be used as a novel glycobiomarker for diagnosis and prediction of clinical severity in CCA patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) is a crucial biomarker for oxidative DNA damage and carcinogenesis. Current strategies for 8-oxo-dG detection often require sophisticated instruments and qualified personnel. In this study, cysteamine-stabilised gold nanoparticles (cyst-AuNPs) were synthesised and used for colorimetric detection of 8-oxo-dG in urine. Sensing of 8-oxo-dG is based on the anti-aggregation of cyst-AuNPs, mediated by the specific recognition of 8-oxo-dG and its aptamer. In the absence of 8-oxo-dG, the aptamer was adsorbed onto the surface of cyst-AuNPs, resulting in aggregation and the development of a purple colour solution. Upon addition of the target molecule 8-oxo-dG, the aptamer specifically bound to it and could not induce the aggregation of cyst-AuNPs, leading to the dispersion of cyst-AuNPs in the solution. Simple visual examination could be used to monitor the purple-to-red colour change that started at 12 nM, a threshold concentration for visual analysis. The absorbance at 525 nm increased in direct relation to the number of the target molecule 8-oxo-dG. This aptamer/cyst-AuNPs system showed excellent sensing ability for the 8-oxo-dG concentration in the range of 15-100 nM, with a detection limit as low as 10.3 nM and a detection time of 30 min. Interference experiments showed that the developed colorimetric strategy had a good sensitivity. This simple and rapid colorimetric method has successfully been applied to inspect 8-oxo-dG concentration in real urine samples and provided recoveries between 93.6 and 94.1%, with a limit of quantification (LOQ) of 34.3 nM, which was comparable with an enzyme-linked immunosorbent-based detection of 8-oxo-dG. This new, easy-to-use, and rapid method could be used as an alternative and initiative strategy for the development of an on-site analysis of 8-oxo-dG in urine., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Antigen detection in urine using an enzyme-linked immunosorbent assay (ELISA) is more sensitive than fecal examination for diagnosis of opisthorchiasis and for assessment of the effects of drug treatment. It is not known whether day-to-day variation of urine composition, including levels of Opisthorchis viverrini antigen, influences the urine assay. We investigated this topic with the cooperation of participants from two localities in Northeast Thailand. Project participants were screened for parasite infections for three consecutive days using the quantitative formalin-ethyl acetate concentration technique (FECT) to detect O. viverrini eggs and the urine ELISA for detection of O. viverrini antigen. A subset of participants (n = 801) with matched fecal and urine samples were analyzed for comparison of inter-day prevalence estimates and the performance of the urine assay compared against FECT for diagnosis of opisthorchiasis. The daily prevalence measured by the urine assay ranged between 29.0%-30.2% while those by FECT ranged between 11.9%-20.2%. The cumulative three-day prevalence estimate determined by the urine antigen assay was 30.3%, which was significantly higher than that by FECT (20.2%, p < 0.05). A significant positive correlation was found between the concentration of antigen in urine and fecal egg counts (p < 0.001). Overall, the urine assay had better diagnostic performance for opisthorchiasis than fecal examination by FECT. The high sensitivity plus negligible daily variation of O. viverrini antigen in urine indicates the utility of the urine assay for diagnosis, as well as population screening, of opisthorchiasis., Competing Interests: The authors have declared that no competing interests exist.

Background: Control and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep ® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections., Methods: A cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases., Results: The initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05)., Conclusions: As the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis., (© 2022. The Author(s).)

Infection by Opisthorchis viverrini causes significant health problems, including cholangiocarcinoma (CCA); thus control and elimination of this trematode is an important strategy for the reduction of CCA. Currently, urine and copro antigen detection is more sensitive than parasitological examination of the feces for the diagnosis of opisthorchiasis. Given limitations in human studies, we used an animal model to quantify the parasite antigen profiles in urine and feces in O. viverrine-infected hamsters, and postdrug treatment. The positive detections of O. viverrini antigen began from week 1 in urine and week 2 in feces after infection until week 28 of the study. The recoveries of O. viverrini worms were detected starting from week 1 and eggs of O. viverrini were detected in feces from week 3 after infection and remained detectable throughout the study period. There was a significant positive correlation of urine and copro antigen levels with the number of fecal egg counts (P < 0.01) and worm recovery (P < 0.01). In the drug-treatment experiment, treatment of infected hamsters with praziquantel significantly reduced worm burden, fecal egg output, and antigen in urine and feces compared with the untreated controls (P < 0.001). At 4 weeks posttreatment, the egg and worm reduction rates were 100% and 95.5%, respectively. The positive antigen detections in urine and feces corresponded with partial worm clearance from praziquantel treatment. This study demonstrated a direct link of urine and copro antigen tests with worms infecting the liver thereby reaffirming the reliability of urine and copro antigen assay in opisthorchiasis diagnosis.

Several trematodes including Opisthorchis viverrini utilize Bithynia siamensis goniomphalos as a snail intermediate host in their life cycles. In order to capture a comprehensive range of host–parasite interactions and their transmission dynamic patterns, B. s. goniomphalos were sampled monthly over 4 consecutive years in an irrigated paddy-field habitat in northeast Thailand. Using a standard cercarial shedding method, a high diversity of trematodes (17 types) was recovered. Virgulate xiphidiocercariae were the most prevalent (7.84%) followed by O. viverrini (0.71%). In addition to seasonal and environmental factors, the quantity of irrigation water for rice cultivation correlated with transmission dynamics of trematodes in B. s. goniomphalos. The peak prevalence of all trematode infections combined in the snails shifted from the cool-dry season in 2010–2012 to the hot-dry season in 2013 associated with an increasing quantity of water irrigation. A low frequency of mixed trematode infections was found, indicating that the emergence of virgulate cercariae, but not of O. viverrini, was negatively impacted by the presence of other trematodes in the same snail. Taken together, the observed results suggest that interactions between host and parasite, and hence transmission dynamics, depend on specific characteristics of the parasite and environmental factors including irrigated water for rice cultivation.

Detection of IgG in urine is an efficient method comparable to that in serum for diagnosis of strongyloidiasis, but the effects of daily variation in urine dilution on diagnostic accuracy are not clearly known. This study evaluated the effects of urine concentration on the detection of parasite-specific IgG by urine enzyme-linked immunosorbent assay (ELISA), particularly in individuals with borderline results or false-negative diagnosis. Optimal concentration conditions were established by comparing Strongyloides -specific IgG antibody levels between unconcentrated and concentrated urine in participants with different infection intensities, namely, healthy control (HC), low-negative (LN), high-negative (HN), and low-positive (LP) groups. The optimal condition was selected and validated in a field trial study. The final urine concentration protocol required centrifugation at 4,000 × g at 4°C for 10 mins using the Amicon concentrator tube. This protocol was validated in groups of participants with various diagnoses according to urine ELISA and fecal examination ( n  = 148). The concentrated-urine ELISA increased the proportion of positive results in the LN group by 68.2% and by 100% in the HN group. Significantly elevated IgG antibody levels were seen in the LP group. In the group that was false negative by urine ELISA but positive by fecal examination ( n  = 28), concentrated-urine ELISA yielded 100% positive results. Overall, the frequency estimates of Strongyloides stercoralis were 23.6% by fecal culture, 27% by standard urine ELISA, and 90.5% by concentrated-urine ELISA. The concentration of urine samples prior to analysis by ELISA improved the sensitivity for diagnosis and is potentially useful in the diagnosis of strongyloidiasis in immunocompromised individuals or in low-prevalence areas.

Infection with the foodborne trematode, Opisthorchis viverrini, is a major public health issue in southeast Asia. The freshwater snail, Bithynia siamensis goniomphalos, is an intermediate host of O. viverrini and other trematode species. Understanding the effects of environmental conditions and infecting trematodes on B.s. goniomphalos snails is thus crucial for the potential influences on trematode transmission. This study measured environmental variables of water and soil properties, and analyzed B.s. goniomphalos snails for their trematode infection, snail shell length, and inorganic elemental concentration, from 30 localities in northeast Thailand. The results showed that prevalence of trematode infection in B.s. goniomphalos was 3.82%. Nine types of trematode cercariae were identified, with virgulate type 1 as the most common (1.23%). Opisthorchis viverrini-infected snails were mostly found in low-humic gley soils in Sakon Nakhon Province, and were associated with water dissolved oxygen and soil pH. Compared with uninfected snails, larger sizes were observed in virgulate type 1 and pleurolophocerca-infected snails, whereas hypercalcification was noticed in virgulate type 1, virgulate type 3, and pleurolophocerca-infected snails. Infected snails were more sensitive toward environmental conditions, possibly because of the dynamic parasitic processes between trematodes and hosts. Among the environmental factors, soil texture (i.e., sand, silt, and clay compositions) exhibited more significant correlations with B.s. goniomphalos shell characteristics regardless of the trematode infection types. The findings of this study underscore the need to consider the effects of environmental conditions and trematode species-specific pathogenic processes for a more effective and sustainable parasitic control and prevention effort.

Introduction: Opisthorchis viverrini (OV) is a major cause of infection in Southeast Asia. Previous studies in mouse models have shown that OV infection can contribute to immune-complex glomerulonephritis (GN). However, OV infection in human kidney tissue has never been demonstrated. Herein, we evaluated the association of OV infection with biopsy-proven glomerular disease., Methods: This study was performed in adult patients who underwent kidney biopsy between July 2016 and February 2017. All kidney tissue samples were processed using the standard techniques for renal pathological diagnoses and immunohistochemistry techniques to detect OV antigen. Pre-implanted donor kidney tissue samples were used as controls. The participants were also assessed for OV infection by serum OV immunoglobulin G antibody (Ab) levels and/or presence of OV eggs in stool., Results: Forty-three renal tissue samples from glomerular disease patients and 50 from transplant donors were included in the study. Mean age in the GN group was 41.7 ± 15.9 years, estimated glomerular filtration rate (eGFR) was 70.65 ± 36.61 mL/min/1.73 m2, and median proteinuria was 3.17 (1.70-4.95) g/day. Lupus nephritis (LN) was the most common diagnosis (32.6%), followed by IgA nephropathy (23.3%), IgM nephropathy (18.6%), and primary membranous nephropathy (MN; 7%). The OV antigen was observed in kidney tissue from patients with IgA nephropathy, LN, primary MN, focal segmental glomerulosclerosis, and IgM nephropathy. By contrast, no OV antigen was detected in tissue samples from the control group. The presence of OV antigens was observed in glomerular endothelial cells, mesangial cells, tubular cells, and peritubular capillaries. The odds ratio of positive serum OV Ab to predict the presence of OV antigen in kidney tissues was 4.47 (p = 0.057), and there was a negative correlation between levels of serum OV Ab and eGFR (r = -0.31, p = 0.04)., Discussion/conclusion: This is the first study to demonstrate the presence of OV antigen in human kidney tissue, which indicates that OV infection may be associated with biopsy-proven glomerular diseases., (© 2022 S. Karger AG, Basel.)

The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and subsequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini, and distributed throughout endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and population genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations commenced some 20 years ago., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Haemonchus contortus is one of the most economically important parasitic nematodes affecting small ruminant livestock worldwide. This study was conducted to elucidate the genetic diversity and population structure of this nematode in Thailand based on mitochondrial DNA markers, the nicotinamide dehydrogenase subunit 4 (nad4) and the cytochrome c oxidase subunit 1 (cox1) genes. One hundred and thirty-six adult worms were obtained from 86 abomasa of slaughtered goats from 13 different localities in 5 regions of Thailand. Identification to the genus Haemonchus was done using morphology. DNA sequences of the nuclear ribosomal second internal transcribed spacer (ITS2) identified each specimen to species: three fixed nucleotide (SNP) differences distinguished H. contortus from H. placei. Genetic analysis defined 118 and 122 unique haplotypes in partial sequences of nad4 (alignment length 723 bp) and cox1 (645 bp) genes, respectively. Nucleotide diversities were 0.031 and 0.043 for nad4 and cox1 genes, respectively. Low genetic differentiation was observed among H. contortus samples from various provinces in Thailand. This is the first study on the genetic diversity and population structure of H. contortus of goats in Thailand. This study has provided insights into the transmission dynamics of this parasitic nematode, information which is essential for farm management and parasite control., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Phenotypic diversity in urinary metabolomes of different geographical populations has been recognized recently. In this study, urinary metabolic signatures from Western (United Kingdom) and South-East Asian (Thai) cholangiocarcinoma patients were characterized to understand spectral variability due to host carcinogenic processes and/or exogenous differences (nutritional, environmental and pharmaceutical). Urinary liquid chromatography mass spectroscopy (LC-MS) spectral profiles from Thai (healthy = 20 and cholangiocarcinoma = 14) and UK cohorts (healthy = 22 and cholangiocarcinoma = 10) were obtained and modelled using chemometric data analysis. Healthy metabolome disparities between the two distinct populations were primarily related to differences in dietary practices and body composition. Metabolites excreted due to drug treatment were dominant in urine specimens from cholangiocarcinoma patients, particularly in Western individuals. Urine from participants with sporadic (UK) cholangiocarcinoma contained greater levels of a nucleotide metabolite (uridine/pseudouridine). Higher relative concentrations of 7-methylguanine were observed in urine specimens from Thai cholangiocarcinoma patients. The urinary excretion of hippurate and methyladenine (gut microbial-host co-metabolites) showed a similar pattern of lower levels in patients with malignant biliary tumours from both countries. Intrinsic (body weight and body composition) and extrinsic (xenobiotic metabolism) factors were the main causes of disparities between the two populations. Regardless of the underlying aetiology, biological perturbations associated with cholangiocarcinoma urine metabolome signatures appeared to be influenced by gut microbial community metabolism. Dysregulation in nucleotide metabolism was associated with sporadic cholangiocarcinoma, possibly indicating differences in mitochondrial energy production pathways between cholangiocarcinoma tumour subtypes. Mapping population-specific metabolic disparities may aid in interpretation of disease processes and identification of candidate biomarkers., (© 2021. The Author(s).)

Human strongyloidiasis is one of the neglected tropical diseases caused by infection with soil-transmitted helminth Strongyloides stercoralis. Conventional stool examination, a method commonly used for diagnosis of S. stercoralis, has low sensitivity, especially in the case of light infections. Herein, we developed the droplet digital polymerase chain reaction (ddPCR) assay to detect S. stercoralis larvae in stool and compared its performance with real-time PCR and stool examination techniques (formalin ethyl-acetate concentration technique [FECT] and agar plate culture [APC]). The ddPCR results showed 98% sensitivity and 90% specificity, and real-time PCR showed 82% sensitivity and 76.7% specificity when compared with the microscopic methods. Moreover, ddPCR could detect a single S. stercoralis larva in feces, and cross-reactions with other parasites were not observed. In conclusion, a novel ddPCR method exhibited high sensitivity and specificity for detection of S. stercoralis in stool samples. This technique may help to improve diagnosis, particularly in cases with light infection. In addition, ddPCR technique might be useful for screening patients before starting immunosuppressive drug therapy, and follow-up after treatment of strongyloidiasis.

Liver fluke infection through the consumption of raw or undercooked freshwater fish is a major public health problem in the Mekong Region. Despite the extensive efforts of liver fluke health campaigns, Northeast Thailand still reports high human infection prevalence as consumption of raw fish dishes has diminished but not ceased. This study examines the roles of social-cultural factors, particularly the influences of masculinity and misinformation, on liver fluke infection risk. Participant observation, questionnaire surveys and semi-structured interviews were conducted in four villages in Kalasin Province, Thailand, to scrutinize reasons for raw fish consumption, gender differences in raw fish culture, processes of liver fluke information dissemination, and the extent of information mismatch. Our results show that one of the key reasons (76.9%) underlying continued raw fish consumption are deeply embedded cultural practices associated with ways of rural life. About 30% of the participants indicated that they would not avoid eating raw fish, regardless of knowing the health consequences. Gender difference is evident, with 75.6% of males consuming raw fish salad (koi pla), compared to 42.7% of females. Some male participants associate raw meat consumption with virility and strength. Such beliefs underscore the cultural linkage of koi pla consumption with masculinity. Misconceptions of liver fluke life cycle and risk of infection remain, as only 15.3% of the participants correctly selected raw fish as the food source for liver fluke infection while 84.2% misunderstood that other raw foods could lead to infection. The multi-layered and hierarchical structure of public health information dissemination from medical professionals to health officers and village health volunteers to villagers has contributed to information mismatch between different layers. Our study builds on others which call for multi-pronged scientific and social strategies, as well as culturally attuned approaches to public health messaging. The study raises masculinity and misinformation as relevant considerations in disease prevention. Incorporating grounded research and gendered perspectives are part of appreciating the cultural roots of raw fish consumption. Realizing the significant role of village health volunteers in information dissemination and in supplying coherent public health messages is vital for effective health campaigns., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The freshwater snails, Bithynia are the first intermediate hosts of the liver fluke, Opisthorchis viverrini, the causative agent of cholangiocarcinoma (CCA) in Southeast Asia. In Thailand, there are three traditionally recognized taxa of Bithynia: Bithynia funiculata; B. siamensis siamensis; B. s. goniomphalos. This study examines the geographical distribution and genetic structure of Bithynia species from five previously reported water catchments and six new catchments in Thailand. Of these, three new catchments Kok, Wang, and Nan are from the north and the remaining three new catchments are Phetchaburi, Prachuap Khiri Khan Coast, Mae Klong from the west of Thailand. We sampled 291 Bithynia snails from 52 localities in 11 catchment systems in the northern, western and central regions of Thailand. Mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S ribosomal DNA (16S rDNA) sequences were used to examine genetic diversity of Bithynia snails which revealed 200 and 27 haplotypes of COI and 16S rDNA, respectively. However, as 16S rDNA is a conserved gene, it is not suitable to distinguish Bithynia at the species and sub-species levels in our study. The phylogenetic tree and haplotype network analyses included sequences of COI from GenBank. B. funiculata was found only in the north of Thailand and the genetic structure did not differ among populations. Genetic differentiation (Φ ST ) analyses showed that B. s. goniomphalos contained three distinct lineages. Lineage I contained B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR. Lineage II contained all B. s. goniomphalos from the Prachin Buri and Bang Pakong catchment systems in eastern and central Thailand, including samples from all catchment systems in Cambodia. While lineage III contained B. s. goniomphalos from the Songkram and Nam Kam catchment systems in Thailand and the Nam Ngum and Huai Som Pak catchment systems in Lao PDR. Furthermore, results showed that all samples of B. s. siamensis were classified into one lineage and placed phylogenetically between B. s. goniomphalos lineages I and II. Thus, the taxonomic status of B. s. goniomphalos and B. s. siamensis requires reassessment, and they should be reclassified as belonging to the species complex "Bithynia siamensis sensu lato"., (Copyright © 2021. Published by Elsevier B.V.)

Ivermectin (IVM) is a widely used anthelmintic. However, with widespread use comes the risk of the emergence of IVM resistance, particularly in strongyloidiasis. Adenosine triphosphate (ATP)-binding cassette (ABC) transporter genes play an important role in the IVM-resistance mechanism. Here, we aimed to establish an animal experimental model of IVM resistance by frequent treatment of Strongyloides ratti with subtherapeutic doses of IVM, resistance being evaluated by the expression levels of ABC transporter genes. Rats infected with S. ratti were placed in experimental groups as follows: 1) untreated control (control); 2) treated with the mutagen ethyl methanesulfonate (EMS); 3) injected with 100 µg/kg body weight of IVM (IVM); 4) treated with a combination of EMS and IVM (IVM+EMS). Parasites were evaluated after four generations. Extent of IVM resistance was assessed using IVM sensitivity, larval development, and expression of ABC genes. By the F4 generation, S. ratti in the IVM group exhibited significantly higher levels of IVM resistance than did other groups according to in vitro drug-sensitivity tests and inhibition of larval development (IC50 = 36.60 ng/mL; 95% CI: 31.6, 42.01). Expression levels of ABC isoform genes (ABCA, ABCF, and ABCG) were statistically significantly higher in the IVM-resistant line compared with the susceptible line. In conclusion, IVM subtherapeutic doses induced IVM resistance in S. ratti by the F4 generation with corresponding upregulation of some ABC isoform genes. The study provides a model for inducing and assessing drug resistance in Strongyloides.

Strongyloides stercoralis affects more than half a billion people worldwide, and hyperinfection in immunocompromised patients can be fatal. Elimination of this neglected tropical disease requires field-applicable diagnostic tools. We conducted a laboratory evaluation of a lateral flow rapid dipstick test (SsRapid™) using sera samples from a Strongyloides-endemic area in northeast Thailand. Group 1 was S. stercoralis-positive and larvae- and/or antibody-positive (according to the IgG ELISA) (N = 100). Group 2 had negative fecal examination and IgG ELISA results (N = 25). Group 3 had other parasitic infections and negative IgG ELISA results (N = 25). The results showed good diagnostic sensitivity (82%) and excellent specificity (96%). Suggested improvements in the SsRapid™ test include increased diagnostic sensitivity and conversion to the more robust cassette format. Field studies should be performed as well.

The colorimetric AuNPs-LISA is a new, powerful technique for the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, the diagnostic sensitivity of the colorimetric AuNPs-LISA is powerless to screen ultralow concentrations of OvAg in urine samples in cases of early stage liver fluke infection. This work, we aimed to improve the diagnostic sensitivity of the colorimetric AuNPs-LISA by developing a new fluorescence AuNPs-LISA. O-phenylenediamine (OPD) was used as the chromogenic substrate instead of the tetramethylbenzidine (TMB) of the colorimetric AuNPs-LISA. Interestingly, the fluorescence of the OPD oxidation product by the peroxidase-like activity of labelled AuNPs can be extremely enhanced by a non-ionic surfactant, especially the Triton X-100. The proposed assay exhibited a dynamic linear detection of OvAg concentration in the range of 34.18 ng mL -1 to 273.44 ng mL -1 with the limit of detection at 36.97 ng mL -1 which the detection sensitivity enhancement around 1200-fold when comparing with the colorimetric AuNPs-LISA. This model exhibits high diagnosis sensitivity, specificity and accuracy, 91.28%, 91.75%, and 91.59%, respectively, compared to the traditional ELISA. The fluorescence AuNPs-LISA showed excellent potential for the diagnosis of OvAg in urine samples from endemic areas. This will provide an effective tool for the detection, control and elimination of human opisthorchiasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA) has a high immune response with chronic inflammation and oxidative stress. CD44 and Nestin, two cancer stem cell (CSC) markers, play major roles in cancer cell survival. Effects of immune response and expression CSC markers on survival of patients with CCA remain unclear., Objective: To investigate the effects of level of OV IgG together with CSC marker expression and also the combination of these markers on survival of CCA patients after curative resection., Methods: All serum specimens from CCA patients who underwent curative surgery from 2005 to 2015 were examined for IgG for OV antigen by ELISA. Tissue specimens were studied for CD44 and Nestin expression. Survival analysis by Cox proportional hazard model was used for estimating hazard ratio (HR) with a 95% confidence interval (CI)., Results: In this study, 122 (69.3%) of 176 were positive for OV IgG, and 35 (19.9%) were considered to have high-positive OV IgG. CD44s positive expression was found in 54 (40%), CD44v6 high expression in 96 (69.6%), CD44v8-10 high expression in 87 (63.5%) and Nestin high expression in 21 (16.1%). Multivariate survival analysis found that high-positive OV IgG and late stage tumor were independent prognostic factors with the adjusted HR of 2.24 (95% CI 1.27-3.93) and 2.78 (95% CI 1.46-5.29), respectively. Subgroup analysis in early and late stage CCA showed that a combined positive OV IgG and CD44s expression with the high expression of CD44v8-10 had the significantly poorest prognosis with HR of 3.75 (95% CI 1.61-8.72) and HR of 1.76 (95% CI 1.02-3.03), respectively., Conclusion: A high level of OV IgG as well as a high level of CSC markers resulted in an aggressive CCA. OV IgG level together with CSC markers can be used as the prognostic markers for CCA patients' survival. The study of the CD44 pathway is promising for adjuvant treatment., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Titapun et al.)

Recent work has found urine analysis to be as sensitive as serology for diagnosis of strongyloidiasis. Here, we examined the daily variation of Strongyloides-specific IgG in urine by qualitative and quantitative ELISA and its effects on diagnostic accuracy and reliability. In the first part of the study, matched urine and fecal samples were collected from project participants in northeast Thailand for three consecutive days. Urine samples were analyzed for Strongyloides-specific IgG by ELISA using Strongyloides ratti as the antigen source. Performance of urine ELISA was compared with parasitological diagnosis by agar plate culture technique (APCT) and formalin-ethyl acetate concentration technique (FECT). In the second part of the study, urine IgG levels were compared daily for thirty consecutive days. The prevalence of Strongyloides infection, as measured by urine ELISA for three consecutive days, was significantly higher than that found using parasitological methods (63.1% vs. 22%). There was slight daily variation in prevalence estimates according to urine ELISA while there were significant variations according to parasitological examination methods over three consecutive days. For the 3-day experiment, urine ELISA had 83-86% diagnostic sensitivity when compared with the fecal examination method or with a composite standard (combined results from fecal examination methods (APCT or FECT) and/or urine ELISA). The levels of parasite-specific IgG in urine were stable throughout both the 3-day and the 30-day studies. In conclusion, diagnosis of strongyloidiasis by urine ELISA is more sensitive than by fecal methods, with minimal daily variation for qualitative and quantitative diagnosis. Urine ELISA has potential for clinical diagnosis and population screening of strongyloidiasis., (Copyright © 2021. Published by Elsevier B.V.)

Transmission of the carcinogenic liver fluke Opisthorchis viverrini is ongoing across Southeast Asia. Endemic countries within the region are in different stages of achieving control. However, evidence on which interventions are the most effective for reducing parasite transmission, and the resulting liver cancer, is currently lacking. Quantitative modelling can be used to evaluate different control measures against O. viverrini and assist the design of clinical trials. In this article we evaluate the epidemiological parameters that underpin models of O. viverrini and the data necessary for their estimation, with the aim of developing evidence-based strategies for parasite control at a national or regional level., Competing Interests: Declaration of Interests No interests are declared., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues., Competing Interests: The authors have declared that no competing interests exist.

Several foodborne zoonotic trematodes of the family Opisthorchiidae have been reported to infect people, specifically two genera, Clonorchis and Opisthorchis. Three species Clonorchis sinensis, Opisthorchis felineus and Opisthorchis viverrini are the most extensivley studied of the Opisthorchiidae. At least 680 million people worldwide are at risk of infection of these liver flukes through the consumption of raw or partially cooked freshwater cyprinid fish. An estimated 45 million people in Europe and Asia are currently infected with these liver flukes. Of these, the 35 million are infected with C. sinensis mainly in China, 10 million with O. viverrini in Southeast Asia, and 1.2 million with O. felineus in Eastern Europe and Russia. These liver flukes have been proven to be causative agents of bile duct cancer or cholangiocarcinoma (CCA). A multidisciplinary program should be implemented involving comprehensive research on molecular genetics, diagnosis, treatment, prevention, as well as educational and control programs should progressively be introduced and applied in endemic regions of O. viverrini, O. felineus and C. sinensis throughout their ranges, hence, opisthorchiasis and clonorchiasis free communities can be realised globally., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Infection by the small liver fluke, Opisthorchis viverrini, causes serious public health problems, including cholangiocarcinoma, in Thailand and southeastern Asian countries. Previous studies have reported that O. viverrini represents a species complex with varying levels of genetic differentiation in Thailand and Lao PDR. In this study, we re-examined population genetic structure and genetic diversity of O. viverrini using extensive samples of the parasite collected over 15 years from 12 geographical localities in Thailand and eight localities in Lao PDR. Parasite life-cycle stages of 721 individuals of O. viverrini (91 cercariae, 230 metacercariae and 400 adult worms) were genotyped using 12 microsatellite loci. Metacercariae exhibited genetic diversity comparable with that of experimentally raised adults: metacercariae can therefore be used to represent O. viverrini populations without the need for laboratory definitive hosts. Data obtained from larval as well as adult worms identified two distinct genetic clusters of O. viverrini. Sequences of a portion of the mitochondrial cox1 gene strongly supported the existence of these two clusters. One, the widespread cluster, was found at all sampled sites. The second cluster occurred only in Phang Khon District, Sakon Nakhon Province (SPk), within the Songkram River wetland in Thailand. A striking feature of our data relates to the temporal dynamics of the SPk cluster, which was largely replaced by representatives of the widespread cluster over time. If the SPk cluster is excluded, no marked genetic differences were seen among O. viverrini populations from Thailand and Lao PDR. The underlying causes of the observed population structure and population dynamics of O. viverrini are not known., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Background: Serum antibody for Opisthorchis viverrini (OV) is strong evidence for a history of OV infection in people. Currently, no studies have examined whether varying cholangiocarcinoma (CCA) prevalence levels are linked to previous OV infection nor have they provided comprehensive assessment and characterization of OV-associated CCA., Objective: Our study examined the prevalence of serum IgG antibodies for OV-positive CCA cases and determined whether there were correlations of IgG antibodies with histopathologic features, HER2, PD-L1, and FGFR2 expression, as well as their roles on the patients' survival., Methods: The study involved 221 CCA surgical patients at Khon Kaen University Hospital, Thailand, from 2005 to 2017. Serum specimens were tested for OV IgG by ELISA. CCA tissue microarrays were used to examined for HER2, PD-L1, and FGFR2 expression. Logistic regression was used to investigate an association between factors and IgG. Cox regression was used to determine factors that affected CCA patient survival., Results: IgG for OV were positive in 162 cases, and the prevalence was 73.3% (95% CI=68.0-78.7). About three quarters (78.3%) had large duct type tumors and concomitant intraductal papillary neoplasm of bile ducts (IPNB) occurred in 92 (50%) cases. HER2 expression was positive in 94 (61.4%) cases. Positive PD-L1 and FGFR2 expression occurred in 125 (83.9%) and 100 (67.1%) cases. IgG for OV had no significant correlation to any histological feature but had significant correlation with HER2 overexpression with adjusted OR=2.32 (95% CI=1.09-4.96, P =0.03). Cases of CCA with OV IgG positive had a significantly poor prognosis with adjusted HR=1.66 (95% CI=1.13-2.43, P =0.01)., Conclusion: We found a high prevalence of serum IgG for OV-positive CCA patients and a correlation with overexpression of HER2. Moreover, IgG for OV and HER2 expression indicated poor survival of CCA. Therefore, future clinical studies for anti-HER2 treatments should focus on OV-associated CCA., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Titapun et al.)

Background: A urine antigen assay was applied to evaluate chemotherapeutic outcomes and reinfection patterns of opisthorchiasis in Thailand., Methods: We used a prospective study design by following opisthorchiasis subjects at baseline and post-treatment using a urine antigen assay and faecal examination by the formalin-ethyl acetate concentration technique (FECT)., Results: The antigen of Opisthorchis viverrini in urine diminished within 4 weeks after praziquantel treatment. Concurrent faecal examinations by FECT showed that faecal eggs were negative at 4 weeks after treatment. In a subsequent study, reinfection rates and intensity patterns of O. viverrini were evaluated at 48 weeks after praziquantel treatment. Within a group of subjects with curative treatment (n=137), 16.8% became reinfected according to FECT and 27.7% according to the urine antigen assay (p<0.05). There were significant correlations in intensity of infection between pretreatment and at 48 weeks post-treatment in both faecal egg counts and antigen levels in urine., Conclusions: The results suggested that in addition to screening, the urine antigen assay is an efficient tool for monitoring outcomes of drug treatment and reinfection in opisthorchiasis. Due to the ease of urine sample collection and handling, the urine assay becomes an alternative method to faecal examination for diagnosis and monitoring of opisthorchiasis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

The freshwater snail Bithynia siamensis goniomphalos serves as the first intermediate host of liver fluke Opisthorchis viverrini, a foodborne parasite, of which human infection has persisted in Southeast Asia for decades. The snail Filopaludina martensi martensi has been proposed as a biological control agent against B.s. goniomphalos, but knowledge on the snail ecology and population dynamics between the two species remains rudimentary. This study investigated selected abiotic and biotic factors influencing the distribution and abundance of B.s. goniomphalos and F.m. martensi. Water quality, soils, and snails were collected from 34 localities in Northeast Thailand. Soil properties and snail soft tissue elemental concentrations were analyzed. Experiments were performed to examine interspecific competition. Statistical analysis was conducted to explore the associations between water and soil properties and soft tissue elemental concentrations. The results showed that B.s. goniomphalos had the highest mean dominance in streams and red-yellow podzolic soils, while F.m. martensi snails preferred ponds and latosol soils. Negative correlation in species abundances was found between the two species. Interspecific competition was detected, with B.s. goniomphalos growth rates hampered by the presence of F.m. martensi. Despite the possibility of using F.m. martensi to control B.s. goniomphalos, B.s. goniomphalos exhibited a greater adaptability to different water and soil properties, suggesting that the species could colonize a wide range of environmental conditions. This study provides further insights into the ecology of the two snail species, underscoring the importance of considering abiotic factors when assessing the possible biological control agent to control O. viverrini transmission.

Echinostomes are a diverse group of digenetic trematodes that are difficult to classify by predominantly traditional techniques and contain many cryptic species. Application of contemporary genetic/molecular markers can provide an alternative choice for comprehensive classification or systematic analysis. In this study, we successfully characterized the intron 5 of domain 1 of the taurocyamine kinase gene (TkD1Int5) of Artyfechinostomum malayanum and the other two species of the 37 collar-spined group, Echinostoma revolutum and Echinostoma miyagawai, whereas TkD1Int5 of Hypoderaeum conoideum cannot be amplified. High levels of nucleotide polymorphism were detected in TkD1Int5 within E. revolutum and E. miyagawai, but not in A. malayanum. Thus, TkD1Int5 can be potentially used as genetic marker for genetic investigation of E. miyagawai and E. revolutum. We therefore used TkD1Int5 to explore genetic variation within and genetic differentiation between 58 samples of E. miyagawai and five samples of E. revolutum. Heterozygosity was observed in 17 and two samples with 16 and three insertion/deletion (indel) patterns in E. miyagawai and E. revolutum, respectively. Heterozygous samples were then cloned and nucleotide sequence was performed revealing the combined haplotypes in a particular sample. Based on nucleotide variable sites (excluding indels), the 72 E. miyagawai and seven E. revolutum haplotypes were subsequently classified. The haplotype network revealed clear genetic differentiation between E. miyagawai and E. revolutum haplogroups, but no genetic structure correlated with geographical localities was detected. High polymorphism and heterogeneity of the TkD1Int5 sequence found in our study suggest that it can be used in subsequent studies as an alternate independent potential genetic marker to investigate the population genetics, genetic structure, and possible hybridization of the other echinostomes, especially the 37 collar-spined group distributed worldwide.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), an oxidized form of guanosine residues, is a critical biomarker for various cancers. Herein, a sensitive citrate-capped gold nanoparticle-based aptasensor device has been developed for the detection of 8-oxo-dG in urine. We previously designed a 38-nt anti-8-oxo-dG-aptamer by a computer simulation and the experimental validation has been performed in the present work. The analytical performance of the 38-nt aptamer from the in silico design was compared with the parent 66-nt aptamer. This assay is based on the principle of salt-induced aggregation of citrate-capped gold nanoparticles. Based on this sensing mechanism, the difference between the absorbance in the presence and absence of 8-oxo-dG at λ = 525 nm (ΔA525) increased linearly as a function of 8-oxo-dG concentrations in the ranges of 10-100 and 15-100 nM for 38-nt and 66-nt aptasensors, respectively. This method can provide detection limits of 6.4 nM for 8-oxo-dG in the 38-nt aptasensor and 13.2 nM in the 66-nt aptasensor. Similar to the 66-nt aptamer, the shortened aptamer, 38-nt long, can provide high sensitivity and selectivity with rapid detection time. In addition, using the 38-nt aptamer as a recognition component in the developed portable low-cost device showed high sensitivity in the detection range of 15-100 nM with a detection limit of 12.9 nM, which is much lower than the threshold value (280 nM) for normal human urine. This easy-to-use device could effectively and economically be utilized for monitoring 8-oxo-dG in real urine samples and potentially serve as a prototype for a commercial device., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Opisthorchis viverrini is a fish-borne zoonotic trematode that causes significant public health problems in Southeast Asia. Its life cycle requires Bithynia snails as the first intermediate hosts, fish, and human and/or carnivore hosts. This study assessed impacts of land use practice for rice cultivation and seasonality on the transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos in rice paddy field habitats. The transmission of O. viverrini cercariae in B. s. goniomphalos was monitored at monthly intervals over a 4-year period from January 2010 to December 2013. From a total of 59,727 snails examined by standard cercarial shedding, the prevalence of O. viverrini was 0.7% (range, 0.0-4.1%). The prevalence of O. viverrini infection in B. s. goniomphalos varied with the amount of rainfall, with peaks of infection occurring in the cool-dry season, that is, after each rainy season. A shift of peak prevalence from cool-dry to hot-dry season observed in 2013 was associated with the increase in preceding water irrigation to support the production of second annual rice crop. Significant positive correlations were found between the prevalence and intensity of cercarial infection and the size of snails. Our results revealed substantial variation between years so that to have a clear understanding of the population dynamics of this complex system, studies should be conducted over an extended period (> 1 year). Results from this study highlight that water irrigation schemes in rice paddy cultivation and seasonality have a significant effect on the prevalence of O. viverrini in B. s. goniomphalos .

Infection with the liver fluke Opisthorchis viverrini is the major causative factor inducing cholangiocarcinoma in the Mekong region of Southeast Asia. Northeast Thailand has the highest incidence of this cancer worldwide leading to about 20,000 deaths every year. Infection with the liver fluke comes from eating raw or undercooked fish, a tradition in this area that can potentially be countered by education programs at school level. Here we develop a school-based health education model, based on protection motivation theory (PMT), including module design, learning materials, student activities, and capacity building amongst teachers. This education program was applied and tested in primary school to pupils (9-13 years) in Khon Kaen province, northeast Thailand. Using a randomized control trial, four schools served as intervention groups ( n = 118 pupils) and another four acted as controls ( n = 113 pupils). Based on PMT constructs, we found that the pupils in the intervention group had significantly greater knowledge and perceived the severity, vulnerability, response efficacy, and self-efficacy parameters concerning the dangers of eating raw fish and of developing cholangiocarcinoma than those in the control schools ( p < 0.05). All of the PMT constructs measured were significantly intercorrelated with each other ( p < 0.001). At the same time, some background knowledge, from community-based education programs, was present in the control schools. The result from this initial study suggests that PMT can be used to predict protective attitude as well as behavior changes in evaluating the consequence of school health intervention programs.

The enzyme-linked immunosorbent assay (ELISA) is currently a powerful technique for the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, its sensitivity and analysis time need to be improved. In the present study, we aimed to improve the signal enhancing system of traditional ELISA by using gold nanoparticles (AuNPs) with peroxidase-like activity on its surface instead of the horseradish peroxidase (HRP) system. The catalytic activity of the AuNPs probe can be boosted by the gold enhancing solution and the addition of ATP. The catalytic ability of the AuNPs probe depended on the probe and the H 2 O 2 concentration. The proposed approach can reduce the number of the traditional ELISA steps with better detection sensitivity. Interestingly, the limit of detection (LOD) of the test was 23.4 ng mL -1 , substantially lower than the 93.8 ng mL -1 for the traditional ELISA. The AuNPs-LISA assay showed higher sensitivity and specificity, 93.81% and 91.34%, respectively, compared to the traditional ELISA. The proposed assay was successfully applied for the detection of OvAg in urine samples. This will provide an effective tool for the detection, control and elimination of human opisthorchiasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)