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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. 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). 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. 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. [ABSTRACT FROM AUTHOR]

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. Halting the transmission of O. viverrini is crucial to preventing cases of cholangiocarcinoma, yet there is no consensus on the best strategies for large-scale parasite control. Quantitative modelling can provide insights and guide control programmes more rapidly and using fewer resources than clinical trials. To give reliable output, models depend on making accurate assumptions and using parameters inferred from relevant datasets. Mathematical models of O. viverrini dynamics have recently been published and provide a valuable starting point. However, many key epidemiological processes remain poorly understood, including the links between parasite exposure, anthelmintic treatment, and carcinogenesis. We highlight areas where new studies are required to fill knowledge gaps. Recent developments in statistical modelling and parasite genomics provide opportunities to make rapid gains. [ABSTRACT FROM AUTHOR]

Human infection with Opisthorchis viverrini , a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis. Global molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand. Multivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury. Several metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA. [ABSTRACT FROM AUTHOR]

The diagnosis of strongyloidiasis by coprological methods has a low sensitivity, underestimating the prevalence of Strongyloides stercoralis in endemic areas. Serodiagnostic tests for strongyloidiasis have shown robust diagnostic properties. However, these methods require a blood draw, an invasive and labor-intensive sample collection method, especially in the resource-limited settings where S. stercoralis is endemic. Our study examines a urine-based assay for strongyloidiasis and compares its diagnostic accuracy with coprological and serological methods. Receiver operating characteristic (ROC) curve analyses determined the diagnostic sensitivity (D-Sn) and specificity (D-Sp) of the urine ELISA, as well as estimates its positive predictive value and diagnostic risk. The likelihood ratios of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for each diagnostic positivity threshold. The urine ELISA assay correlated significantly with the serological ELISA assay for strongyloidiasis, with a D-Sn of 92.7% and a D-Sp of 40.7%, when compared to coprological methods. Moreover, the urine ELISA IgG test had a detection rate of 69%, which far exceeds the coprological method (28%). The likelihood of a positive diagnosis of strongyloidiasis by the urine ELISA IgG test increased significantly with increasing units of IgG detected in urine. The urine ELISA IgG assay for strongyloidiasis assay has a diagnostic accuracy comparable to serological assay, both of which are more sensitive than coprological methods. Since the collection of urine is easy and non-invasive, the urine ELISA IgG assay for strongyloidiasis could be used to screen populations at risk for strongyloidiasis in S. stercoralis endemic areas. [ABSTRACT FROM AUTHOR]

Infection with the liver fluke Opisthorchis viverrini sensu lato (s.l.), a group 1 carcinogen, is the most important risk factor for developing cholangiocarcinoma (CCA) in Southeast Asia. Cholangiocarcinoma is a fatal disease with the world’s highest incidence being found in northeast Thailand. Liver fluke infection occurs through eating raw or partially cooked cyprinid fish containing metacercariae and, therefore, the control of O. viverrini s.l. infection should lead to a reduction in CCA incidence. In this report, we review and analyze the age-prevalence profile data of O. viverrini to reveal temporal changes in patterns of prevalence pre- and post-control programs in Thailand. The profiles of O. viverrini prevalence have transformed from high prevalence in school children prior to 1983 to low prevalences after 1994. This pattern strongly suggests the influence of the health education program on the likelihood of school children becoming infected. In conjunction with current developments in health and socioeconomic conditions, we predict that the incidence of CCA will be reduced with time as the population cohorts that experienced the education programs reach the age at which CCA is most likely to develop, i.e. >50 years. The lessons learned in Thailand may be applicable to other areas endemic for human liver flukes. [ABSTRACT FROM AUTHOR]

The use of Strongyloides ratti as heterologous antigen for serodiagnosis of strongyloidiasis is preferable to Strongyloides from humans due to the ease and safety of antigen preparation. In Southeast Asia where Opisthorchis viverrini coexists with Strongyloides stercoralis, there has been no report in using S. ratti for serodiagnosis of S. stercoralis. In this study, performance of an enzyme-linked immunosorbent assay (ELISA) based on S. ratti was compared with that based on S. stercoralis for diagnosis of strongyloidiasis in areas where O. viverrini is co-endemic in Thailand. Of the 107 individuals, 50 (46.7 %) were positive for S. stercoralis by agar culture method and by ELISA; 82 (76.6 %) and 81 (75.7 %) were seropositive using S. ratti and S. stercoralis antigens, respectively. The levels of parasite-specific IgG to S. ratti and S. stercoralis antigen were significantly proportionally correlated ( P < 0.001). Mixed infections with O. viverrini have little effect on diagnosis of strongyloidiasis. Of 42 subjects who were infected with other parasites, there were no cross-reaction with Angiostrongylus cantonensis, Taenia spp., hookworms, Paragonimus spp., Clonorchis sinensis, Ascaris lumbricoides except for Fasciola spp. (1 of 5), and Opisthorchis viverrini (5 of 20). In spite of cross-reactivities, the results suggest that the S. ratti antigen provides an useful option for diagnosis of strongyloidiasis in an endemic area of opisthorchiasis with high sensitivity comparable to the S. stercoralis antigen and provide a basis for effective control strategies for strongyloidiasis. [ABSTRACT FROM AUTHOR]

Fish-borne zoonotic trematodes (FZT) including heterophyids and opisthorchiids are prevalent in native and aquaculture fish in Southeast Asia. FZT are reported highly prevalent in juvenile aquaculture fish that belonged to the family Heterophyidae, particularly Haplorchis taichui. Aquaculture fish are reported to have varying levels of natural infection with H. taichui, but data for their susceptibility as well as resistance to infection are not available. The aim of this study was to determine the susceptibility of five aquaculture fish to H. taichui. Experimental infections were performed by exposing fish individually to 0 (control), 50, 100, or 200 H. taichui cercariae for 12 h. Metacercarial burden was measured at 45 days postinfection. Three out of five fish species, silver barb ( Barbonymus gonionotus), common carp ( Cyprinus carpio), and mrigal ( Cirrhinus mrigala), were highly susceptible to H. taichui infection with percentage ranging from 93.33 to 100 %. The Nile and red tilapia were not susceptible to infection. Among the susceptible fish species, silver barb had higher intensity of H. taichui metacercariae than common carp and mrigal ( P < 0.001). Metacercarial burden significantly correlated with the dose of cercarial infection ( P < 0.001). Our findings that common aquaculture fish species have varying degrees of susceptibility to H. taichui infection provide important information to reduce parasite transmission in aquaculture fish. [ABSTRACT FROM AUTHOR]

Background: Snail intermediate hosts play a pivotal role in maintaining the life cycles of trematodes, including Opisthorchis viverrini. We investigated the emergence patterns of O. viverrini cercariae infecting Bithynia siamensis goniomphalos at foci in an endemic area in Vientiane Province, Lao PDR. Findings: Samples of B. s. goniomphalos collected during the hot-dry, rainy and cool-dry seasons were examined for O. viverrini infection by cercarial shedding. Emergence of cercariae from O. viverrini-positive snails was monitored daily from 06:00-18:00 h for seven consecutive days at 2 hourly intervals. Snail infections varied seasonally, being highest in the cool-dry season. Peak cercarial emergence was not consistent in different seasons, occurring between 08.00-10.00 h during the hot-dry season and between 12.00-14.00 h during the rainy and cool-dry seasons. The cercarial output was highest in the hot-dry season. The prevalence of infection and the emergence of cercariae were strongly dependent on snail size. Conclusions: This study shows that size of snails and environmental factors (such as season) may affect the emergence patterns of cercariae of O. viverrini in snails. These results have both fundamental and applied implications for opisthorchiasis epidemiology and control. [ABSTRACT FROM AUTHOR]

A previous population genetics study of Opisthorchis viverrini from a locality in an endemic area in Thailand found little genetic variation over time and second intermediate fish host species. Since a similar comparative analysis is not available for Lao PDR, we conducted a study of O. viverrini from different endemic foci in Vientiane Province, Lao PDR, based on spatial, temporal and fish host species. A total of 620 adult O. viverrini originating from the Nam Ngum River wetland were analysed at five previously defined polymorphic enzyme loci. Of these worms, 252 were from six different localities (spatial samples), 162 worms from different years (temporal samples) and 206 worms from four different cyprinid fish species. Significant heterozygote deficiency was found in most O. viverrini populations with levels of genetic differentiation ranging between F 0.0000 and 0.0197 suggesting that gene flow occurred at a variable rate. The role of temporal factors and fish host species had little influence on the level of genetic differentiation. As for O. viverrini from Thailand, these findings indicate that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. Unlike the results for O. viverrini from Thailand, spatial population substructuring may be the underlying population processes for O. viverrini in Lao PDR. These findings indicate that geographical variation may contribute to the transmission dynamics of the parasite with implications for parasite control. However, other host factors, such as snail intermediate hosts and mammal reservoir hosts, as well as human beings, may also play significant roles. [ABSTRACT FROM AUTHOR]

Approximately 680 million people are at risk of infection with Opisthorchis viverrini (OV) and Clonorchis sinensis, with an estimated 10 million infected with OV in Southeast Asia alone. While opisthorchiasis is associated with hepatobiliary pathologies, such as advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), animal models of OV infection show that immune-complex glomerulonephritis is an important renal pathology that develops simultaneously with hepatobiliary pathologies. A cardinal sign of immune-complex glomerulonephritis is the urinary excretion of immunoglobulin G (IgG) (microproteinuria). In community-based studies in OV endemic areas along the Chi River in northeastern Thailand, we observed that over half of the participants had urine IgG against a crude OV antigen extract (OV antigen). We also observed that elevated levels of urine IgG to OV antigen were not associated with the intensity of OV infection, but were likely the result of immune-complex glomerulonephritis as seen in animal models of OV infection. Moreover, we observed that urine IgG to OV antigen was excreted at concentrations 21 times higher in individuals with APF and 158 times higher in individuals with CCA than controls. We also observed that elevated urine IgG to OV antigen could identify APF+ and CCA+ individuals from non-cases. Finally, individuals with urine IgG to OV antigen had a greater risk of APF as determined by Odds Ratios (OR = 6.69; 95%CI: 2.87, 15.58) and a greater risk of CCA (OR = 71.13; 95%CI: 15.13, 334.0) than individuals with no detectable level of urine IgG to OV antigen. Herein, we show for the first time the extensive burden of renal pathology in OV endemic areas and that a urine biomarker could serve to estimate risk for both renal and hepatobiliary pathologies during OV infection, i.e., serve as a "syndromic biomarker" of the advanced pathologies from opisthorchiasis. Author Summary: Approximately 680 million people risk infection with food-borne trematodes, including Opisthorchis viverrini (OV). Animal models show that significant kidney pathology results from OV infection as detected by antibodies in urine (microproteinuria). However, kidney pathology in humans infected with OV is often overlooked because it develops alongside more severe pathologies such as bile duct fibrosis and bile duct cancer. In Northeastern Thailand, the researchers observed that OV infected individuals had elevated levels of urine IgG against OV antigen that was not associated with the level of OV infection. The researchers observed that urine IgG to OV antigen was associated with bile duct fibrosis and bile duct cancer. Moreover, individuals with urine IgG to OV antigen also had elevated risk of bile duct fibrosis and bile duct cancer than individuals with no urine IgG to OV antigen. For the first time, OV infection has been shown to result in significant kidney disease in humans, which is also strongly associated with bile duct pathology. A urine-based assay that could indicate both renal and bile duct pathology from OV infection would be of profound benefit in Southeast Asia, especially in the resource-limited settings of the Mekong Basin region countries of Thailand, Laos and Cambodia. [ABSTRACT FROM AUTHOR]

Approximately 680 million people are at risk of infection with Opisthorchis viverrini (OV) and Clonorchis sinensis, with an estimated 10 million infected with OV in Southeast Asia alone. While opisthorchiasis is associated with hepatobiliary pathologies, such as advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), animal models of OV infection show that immune-complex glomerulonephritis is an important renal pathology that develops simultaneously with hepatobiliary pathologies. A cardinal sign of immune-complex glomerulonephritis is the urinary excretion of immunoglobulin G (IgG) (microproteinuria). In community-based studies in OV endemic areas along the Chi River in northeastern Thailand, we observed that over half of the participants had urine IgG against a crude OV antigen extract (OV antigen). We also observed that elevated levels of urine IgG to OV antigen were not associated with the intensity of OV infection, but were likely the result of immune-complex glomerulonephritis as seen in animal models of OV infection. Moreover, we observed that urine IgG to OV antigen was excreted at concentrations 21 times higher in individuals with APF and 158 times higher in individuals with CCA than controls. We also observed that elevated urine IgG to OV antigen could identify APF+ and CCA+ individuals from non-cases. Finally, individuals with urine IgG to OV antigen had a greater risk of APF as determined by Odds Ratios (OR = 6.69; 95%CI: 2.87, 15.58) and a greater risk of CCA (OR = 71.13; 95%CI: 15.13, 334.0) than individuals with no detectable level of urine IgG to OV antigen. Herein, we show for the first time the extensive burden of renal pathology in OV endemic areas and that a urine biomarker could serve to estimate risk for both renal and hepatobiliary pathologies during OV infection, i.e., serve as a “syndromic biomarker” of the advanced pathologies from opisthorchiasis. [ABSTRACT FROM AUTHOR]

Abstract: Bithynia snails are important hosts of the human liver fluke, Opisthorchis viverrini. Allozyme markers were used to examine genetic variation and the systematics of Bithynia snails from different geographical localities in Thailand and Lao PDR. Our results show that Bithynia siamensis goniomphalos contains two major evolutionary clades with at least nine morphologically similar but genetically distinct “cryptic” species (taxa). Clade A consists of snails from the Chi and the Mun River wetlands, Thailand and clade B consists of snails from the Nam Ngum River wetland, Lao PDR, as well as snails from some localities in the Songkram River wetland, Thailand. There is an association between O. viverrini cryptic taxa with B. s. goniomphalos cryptic taxa within the defined wetlands. Bithynia species are the critical amplifying component of O. viverrini and they control transmission from humans to fish second intermediate hosts. These findings are particularly important for understanding the transmission dynamics of O. viverrini. They are, in addition, a link in the life cycle of O. viverrini which can be used for developing and implementing programs to eliminate and control opisthorchiasis and its associated cholangiocarcinoma. [Copyright &y& Elsevier]

Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy–Weinberg equilibrium of 0.44–0.56. Assessment of population structure by pairwise FST analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini. Author Summary: Infection with the liver fluke (Opisthorchis viverrini) is a risk factor for cholangiocarcinoma (CCA), which is highly prevalent in Khon Kaen Province, Thailand. Within this Province, there is considerable variation in the epidemiology of opisthorchiasis among districts. Preliminary allozyme and mitochondrial DNA data indicate that genetic variation in O. viverrini occurs even over a small endemic area within the province. Here, we used microsatellite DNA analyses to examine the population genetic structure of O. viverrini from four geographically close localities. Analyses of adult worms based on 12 microsatellite loci revealed varying levels of genetic diversity and population substructuring between the four localities. Worms originating from one locality (Phu Wiang) had significantly higher genetic diversity than the other three localities. Data on the population genetic structure observed in these localities are similar to those found at a larger geographic scale. This provides background data to further investigate the biological and epidemiological significance of these genetic variants. [ABSTRACT FROM AUTHOR]

Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy–Weinberg equilibrium of 0.44–0.56. Assessment of population structure by pairwise FST analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini. [ABSTRACT FROM AUTHOR]

Abstract: Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host. [Copyright &y& Elsevier]

Abstract: Opisthorchiasis, together with its associated cholangiocarcinoma, is one of the most important human parasitic diseases on continental Southeast Asia. A great deal of epidemiological data from humans is available on this disease, particularly from the northeast of Thailand, however, only limited information is available on those aspects of the life cycle relating to its Bithynia (Gastropoda) and cyprinid fish intermediate hosts. Here we review the information which is available on the Bithynia hosts of Opisthorchis viverrini. Only one major ecological study has been carried out at one site on a single species of Bithynia. We show not only that detailed ecological studies are required to clarify the epidemiology of opisthorchiasis, but also that the taxonomic status of the Bithynia species transmitting O. viverrini requires clarification. [Copyright &y& Elsevier]

Abstract: This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the “96 Years of Opisthorchiasis. International Congress of Liver Flukes”. It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor. [Copyright &y& Elsevier]

Opisthorchis viverrini ( O. viverrini; known as human liver fluke) is a major health problem in the northeastern region of Thailand. Infection with O. viverrini is the cause of hepatobiliary disease and cholangiocarcinoma (CCA). Previous studies demonstrated specific antibodies to crude O. viverrini antigens in serum from O. viverrini-infected patients. However, no studies have measured specific antibodies to O. viverrini antigens in saliva from patients with opisthorchiasis and CCA. The objective of the study was to detect specific antibodies to crude O. viverrini antigens in saliva from patients with opisthorchiasis and CCA, and to evaluate their use for diagnosis of O. viverrini infection. Saliva samples from 23 control subjects, 30 opisthorchiasis patients, and 38 CCA patients were collected. ELISA was established for detection of salivary IgA and IgG to crude O. viverrini antigens. ANOVA was used to compare salivary IgA and IgG levels among groups. Salivary IgA to crude O. viverrini antigens in CCA patients was significantly higher than controls ( p = 0.007). Salivary IgG in CCA patients was significantly higher than opisthorchiasis patients and controls ( p = 0.010 and p < 0.001, respectively). The cut-off value from salivary IgG test demonstrated higher accuracy for positivity of O. viverrini infection than salivary IgA. In conclusion, specific antibodies to crude O. viverrini antigens were detected in saliva of patients with opisthorchiasis and CCA. Salivary antibodies reflect serum immune response to O. viverrini infection, and salivary IgG tends to be a good candidate for diagnosis of O. viverrini infection. [ABSTRACT FROM AUTHOR]

Abstract: Snails are the critical amplifying hosts of the liver fluke Opisthorchis viverrini, the causative agent of hepatobiliary disease and cholangiocarcinoma in the Mekong area of Southeast Asia. Bithynia funiculata, B. siamensis goniomphalos and B. s. siamensis are the first intermediate hosts of O. viverrini in Thailand. Morphological similarity between Bithynia species and subspecies creates problems for their taxonomic identification and an understanding of Bithynia systematics. In this study, multilocus enzyme electrophoresis (MEE) was applied to define genetic markers that could prove useful for investigating the systematics and population genetics of this genus in Thailand. Of the 34 enzymes examined, 20 encoding a presumptive 24 loci showed sufficient staining intensity and resolution for genetic interpretation. Of these, three loci were monomorphic and eight loci were diagnostic among the three Bithynia taxa. The remaining 13 loci were diagnostic between combinations of the three taxa. Fixed genetic differences were detected at 67–73% of loci among these taxa which in turn differed from a closely related species, Hydrobioides nassa, at 88% of loci. Seventy three percent fixed genetic differences were detected between B. funiculata and the two sub-species B. s. siamensis and B. s goniomphalos. Our data reveals similarly large genetic divergence, 67% fixed genetic differences, between B. s. siamensis and B. s. goniomphalos, which may well represent different species rather than subspecies as currently defined. The genetic markers detected will form the basis for subsequent comprehensive studies on the systematics and population genetics of Bithynia snails as well as for their role in the transmission of O. viverrini and opisthorchiasis. [Copyright &y& Elsevier]

Abstract: Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24h to 28days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis. [Copyright &y& Elsevier]

Abstract: Multilocus enzyme electrophoresis (MEE) and DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene were used to genetically compare four species of echinostomes of human health importance. Fixed genetic differences among adults of Echinostoma revolutum, Echinostoma malayanum, Echinoparyphium recurvatum and Hypoderaeum conoideum were detected at 51–75% of the enzyme loci examined, while interspecific differences in CO1 sequence were detected at 16–32 (8–16%) of the 205 alignment positions. The results of the MEE analyses also revealed fixed genetic differences between E. revolutum from Thailand and Lao PDR at five (19%) of 27 loci, which could either represent genetic variation between geographically separated populations of a single species, or the existence of a cryptic (i.e. genetically distinct but morphologically similar) species. However, there was no support for the existence of cryptic species within E. revolutum based on the CO1 sequence between the two geographical areas sampled. Genetic variation in CO1 sequence was also detected among E. malayanum from three different species of snail intermediate host. Separate phylogenetic analyses of the MEE and DNA sequence data revealed that the two species of Echinostoma (E. revolutum and E. malayanum) did not form a monophyletic clade. These results, together with the large number of morphologically similar species with inadequate descriptions, poor specific diagnoses and extensive synonymy, suggest that the morphological characters used for species taxonomy of echinostomes in South-East Asia should be reconsidered according to the concordance of biology, morphology and molecular classification. [Copyright &y& Elsevier]

Recent systematic studies of Opisthorchis viverrini based on multilocus enzyme electrophoresis (MEE) have shown that there are at least five genetic groups and possibly two cryptic species occurring in Thailand and Lao PDR each associated with a specific wetland system. A study based on mitochondrial DNA (mtDNA) gene analyses of an O. viverrini population from Savannakhet (SV, Lao PDR) clustered with several isolates from Thailand and Lao PDR although they originated from different river wetland systems. We used MEE to re-examine whether O. viverrini from SV was similar genetically to isolates from Thailand and Lao PDR. The allelic profiles of O. viverrini from SV and five different wetlands representing defined genetic groups of O. viverrini were recorded at 24 enzyme loci as opposed to only two loci of mtDNA. Contrary to previous studies, O. viverrini from SV was found to have fixed genetic differences at six to eight of the 24 loci examined (24.50-35.42%). Allelic data indicated that O. viverrini from SV differed from isolates in the Nam Ngum River wetland in Lao PDR (29.33% fixed genetic differences) and clustered with O. viverrini from Nakhon Phanom and Sakon Nakhon within the Songkram River wetland in Thailand but had fixed genetic differences from these at 24.5% of loci examined. Our data confirm the association between genetic groups of O. viverrini and specific wetland systems, and raise important questions regarding the significance of the genetic differences and relationships of O. viverrini from these wetlands. [ABSTRACT FROM AUTHOR]

Abstract: Opisthorchis viverrini is a carcinogenic foodborne trematode endemic in Southeast Asia especially in Thailand and the Lao People''s Democratic Republic. Opisthorchiasis causes hepatobiliary diseases and cholangiocarcinoma (bile duct cancer). Currently there is substantial evidence on genetic variation of O. viverrini but the information on population genetic structure is lacking. Because microsatellite DNA of this parasite is not available, we for the first time isolated and utilized microsatellite DNA as genetic markers to examine genetic diversity and the population structure of O. viverrini. Partial genomic DNA libraries were constructed by conventional and enrichment methods which yielded microsatellite-containing clones of 0.18–0.25% and 16.84%, respectively. Within 41 microsatellite loci isolated 36.59% were perfect, 60.98% were interrupted and 2.44% were compound microsatellites. The CA repetitions were the most frequent, followed by GT and CAT. Primers specific to the flanking regions of 12 microsatellite loci were developed to genotype 150 O. viverrini individuals from geographical localities in Thailand and Lao PDR. Allele numbers per locus ranged from 2 to 15, with the mean expected heterozygosity of 0.03–0.66. Analyses of O. viverrini from 5 localities revealed a high level of genetic diversity and had significant deviation from Hardy–Weinberg equilibrium. Significant heterozygote deficiency as well as heterozygote excess was detected across all localities indicating the possibility of selfing (inbreeding) as a predominant reproductive mode. Significant genetic differentiation (F ST) was also detected between worms from different localities with varying levels of genetic heterogeneity. We discuss our results in terms of what these novel microsatellite markers reveal about the epidemiology and transmission dynamics of this medically important parasite, both in terms of the current study and their potential for future comprehensive population genetic studies O. viverrini sensu lato in Southeast Asia. [Copyright &y& Elsevier]

Infection with Opisthorchis viverrini and its associated cholangiocarcinoma (CCA) is an underestimated problem in the Mekong region of Southeast Asia, despite the widespread use of praziquantel and health education measures for parasite control. Although data from Cambodia, Laos and Vietnam are rare, data from Thailand often show wide-ranging variability in epidemiological parameters, including human morbidity and the prevalence and incidence of CCA. The recent discovery of high levels of population genetic variability in O. viverrini in different wetlands in Thailand and Laos, which indicates the presence of sibling species, suggests that we have underestimated the complexity of this epidemiological situation. Future research should determine the relationship between the genetic variability of O. viverrini and patterns of opisthorchiasis-related disease. [Copyright &y& Elsevier]

Abstract: The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples’ Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host–parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites. [Copyright &y& Elsevier]

Abstract: The liver fluke, Opisthorchis viverrini, is one of the major food borne trematodes in Southeast Asia, where infection causes hepatobiliary disease and subsequent development of cholangiocarcinoma. In Thailand, O. viverrini is most prevalent in the northeast where there is marked regional variation in the rate of infection in humans at provincial, district and village levels. To date, the roles of genetic variation of O. viverrini on this observed variability in infection, transmission and associated disease are not known. We have applied multilocus enzyme electrophoresis (MEE), specifically allozyme electrophoresis, to isolates of O. viverrini from Thailand and Laos to establish genetic markers to examine its systematics and population structure. Forty-six enzymes commonly found useful for genetic characterisation in parasitic helminths were screened, and of these, 33 enzymes gave sufficient staining and resolution to act as potential genetic markers. Sixteen enzymes were monomorphic and 17 enzymes were polymorphic in the pools of worms examined. Whether they are indicative of different enzyme loci, heterozygosity or unique genotypes within the pools of worms examined remains to be determined. Preliminary investigations examining five individual worms at enzyme loci where pools of worms showed multiple bands have confirmed the diagnostic value of the enzyme loci established as well as providing evidence of potential population sub structuring and heterozygosity. For the first time, we have established at least 17 enzymes that provide the basis to undertake comprehensive genetic analyses of the systematics and population structure of O. viverrini, a medically important food borne trematode in Southeast Asia. [Copyright &y& Elsevier]

Opisthorchis viverrini is a food borne trematode, important because of the sheer numbers of people infected and its serious morbidities such as hepatobiliary diseases and cholangiocarcinoma (CHCA). Although infections are identified throughout Southeast Asia, the epi-center is northeast Thailand, where high prevalence coexists with a high incidence of CHCA. In this review, we present the basic population features and the factors influencing transmission between the different hosts. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

One of the top ten foodborne parasitic infections of global concern is human infection of liver fluke through the consumption of raw or undercooked freshwater fish. This article examines the influences of human–environmental interactions of raw fish sharing and procurement connections on the geographic variation in liver fluke infection. We first discuss the networks embedded in the parasite life cycle that propagates disease transmission. Then, we collect 820 questionnaires from three provinces in northeast Thailand to visualize their fish-sharing and sharing-procurement networks, analyze how the connectivity differences in these networks might be linked to participants' consumption behaviors and environmental settings, and determine how all these together affect liver fluke infection risks. The results show that infection was positively associated with procuring fish from ponds, but negatively associated with procuring fish from rivers. Incorporating fish procurement sites substantially increases network connectivity, reinforcing the human connection to key fish sources as a potential risk factor for infection. The consideration of the connections between human, disease hosts, and the environment, compared to the previously human-centric approach of deworming, offers new insights for public health intervention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about a study that examined the likelihood of malaria reemergence in northeastern Thailand using data on changes in land use patterns.

Simple Summary: The zoonotic liver flukes Opisthorchis viverrini and Clonorchis sinensis infect small mammals, such as cats, dogs, pigs, rodents, and rabbits, as well as humans. Human infection subsequently develops into bile duct malignancy, also referred to as cholangiocarcinoma (CCA). Understanding the molecular systematics and population genetics of these liver flukes has an important role in prevention and control, and is important in comprehending their roles in zoonotic transmission. Different molecular markers have varying evolution rates and contain different genetic information. Polymorphic genetic markers are necessary and more suitable for such investigations. Therefore, we screened seven intron regions of the taurocyamine kinase gene (TK) to determine their potential as genetic markers for population genetic investigations of the liver flukes O. viverrini and C. sinensis which were collected from a range of geographical isolates and animal hosts. We identified a suitable intron region of TK, i.e., intron 5 of domain 1 (TkD1Int5) as having the most potential as a polymorphic marker. Results showed that TkD1Int5 is effective in examining the genetic variation and heterozygosity of O. viverrini and C. sinensis, but further studies are required to better understand the role of different species of animals as reservoir hosts of these zoonotic liver flukes. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Helminth infections have proven recalcitrant to control by chemotherapy in many parts of Southeast Asia and indeed farther afield. This study isolates and examines the influence of different aspects of the physical and social environment, and uneven intervention effort contributing to the pathogenic landscape of human Opisthorchis viverrini infections. Methodology: A cross-sectional survey, involving 632 participants, was conducted in four villages in northeast Thailand to examine the impact on prevalence and parasite burden of the reservoir dam environment, socio-economic, demographic, and behavioral factors, and health center intervention efforts. Formalin-ether concentration technique was used for diagnoses, and multivariate models were used for analyses. Principal Findings: The importance attributed to O. viverrini infections varied among health centers in the four study villages. Villages where O. viverrini infections were not prioritized by the health centers as the healthcare focus were at a higher risk of infection (prevalence) with odds ratio (risk factor) of 5.73 (3.32–10.27) and p-value < 0.01. Priority of healthcare focus, however, did not appear to influence behavior, as the consumption of raw fish, the main source of O. viverrini infections in the study area, was 11.4% higher in villages that prioritized O. viverrini infections than those that did not (p-value = 0.01). Landscape variation, notably proximity to reservoir, affects vulnerability of local population to infection. Infection intensity was higher in population located closer to the reservoir with risk ratio of 2.09 (1.12–4.02) and p-value < 0.01. Patterns of infection intensities among humans were found to match fish infection intensity, where higher infection intensities were associated with fish obtained from the reservoir waterbody type (p-value = 0.023). Conclusions/Significance: This study demonstrated the importance of environmental influence and healthcare focus as risk factors of infections in addition to the socio-economic, demographic, and behavioral factors commonly explored in existing studies. The reservoir was identified as a crucial source to target for opisthorchiasis intervention efforts and the need to consider infection intensity in disease control efforts was highlighted. The holistic approach in this study, which underscores the close relationship between the environment, animals, and humans in development of human infections or diseases, is an important contribution to the framework of One Health approach, where consideration of helminth diseases has largely been overlooked. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Glycoproteomics showed increased sialylation of serotransferrin in CCA tissues. • Reverse lectin-ELISA was developed to detect sialylated-serotransferrin in serum. • SNA-TF and MAL1-TF significantly higher in liver fluke-related CCA patients. • There is a strong correlation of serum SNA-TF levels with clinical severity of CCA. • Serotransferrin glycoform could be used as diagnostic and predictive marker for CCA. 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. 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. 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. Sialylated serotransferrin glycoforms could be used as a novel glycobiomarker for diagnosis and prediction of clinical severity in CCA patients. [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini sensu lato is a food-borne trematode which is classified as a class 1 carcinogen, with infection potentially leading to cholangiocarcinoma. Snails of the genus Bithynia act as the first intermediate hosts and an amplifying point in the parasite life cycle. In order to investigate seasonal effect on transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos, cercarial emergence and output profiles were monitored at different season. A total of 4533 snails originating from Sakon Nakhon Province, Thailand, collected during the three main seasons, were analyzed for O. viverrini s.l. infection. Emergence of O. viverrini s.l. cercariae from snails was monitored daily from 06:00 to 18:00 h for seven consecutive days. The prevalence of infection in the snails was highest in the hot-dry season and declined in the rainy and cool-dry seasons. Peak cercarial emergence occurred between 08:00 and 10:00 h during the rainy and cool-dry seasons and between 10:00 and 12:00 h during the hot-dry season. The cercarial output was highest in the hot-dry season, similar to a previous study from Lao People's Democratic Republic (PDR). Average cercarial output/snail in Thailand was higher than in Lao PDR. The number of cercariae emerging from the snails was strongly related to snail size, but the relationship between prevalence of infection and snail size differed between seasons. Observed discrepancies in the emergence patterns and per capita cercarial release may reflect differences in environmental, snail, and/or parasite factors particularly biological characteristics between the cryptic species of O. viverrini s.l. and B. s. goniomphalos from Thailand and Lao PDR. [ABSTRACT FROM AUTHOR]

Infectious diseases associated with dams in many parts of the world suggest a strong possibility of similar occurrence in Southeast Asia, but little is known about the influences of dams on disease occurrences in Southeast Asia, where a wide range of water-related diseases are present. An important public health issue in Southeast Asia is opisthorchiasis, the infection from liver fluke spread by ingesting raw or undercooked freshwater fish. This study investigated the effects of the Ubolratana reservoir in Thailand onOpisthorchis viverriniinfection through the analyses of fish species assemblage and fish host infection in the reservoir and rivers, human fish consumption behavior, and their interactions. Multivariate analyses for community ecology and surveys of human practices were used to examine human–environment interactions involved inO. viverrinitransmission. The results showed that the reservoir and the rivers harbored different fish species of variedO. viverriniinfection densities, with the reservoir having higher overall infection rates than the rivers. Although the preferred species for raw fish dishes was found with low infection, several high-infection species were commonly consumed. The reservoir might have implications for opisthorchiasis risk through the potential change of fish species assemblage and the supply of highO. viverrini–infected fish to most of the villages around it. This study underscored the need to consider human–environment interactions for understanding the risks of disease transmission. [ABSTRACT FROM AUTHOR]

Reaching the goal of control, elimination and eradication of the Neglected Tropical Disease in a foreseeable future provides significant challenges at the ground level especially regarding helminthiasis. Helminths are still mainly diagnoses by egg identification in stool, methods with low sensitivity and for most species low specificity. Cross-sectoral collaboration with regard to zoonoses is almost non-existing and cross-validation by inter-laboratory evaluation of diagnostic tests is not a common practice. The aim of this review was to elucidate the dilemma of helminth diagnosis using zoonotic trematodes as examples. Much progress has been made improving the diagnostic sensitivity of Opisthorchis and Clonorchis using DNA-based techniques but the specificity of these tests is still a challenge due to the many most common but neglected intestinal trematodes. The burden of these diseases and ways to control them remains to be elucidated. Although efficacious drugs are available, the effectiveness of mass drug administration remains to be assessed. The importance of animal reservoirs and ways to control the diseases in animals are yet unknown. Diagnostic challenges regarding Schistosoma japonicum and Schistosoma mekongi include the many light infections and the persisting influx from the animal reservoirs. The sensitivity of the faecal based techniques suited morbidity control but will be insufficient for elimination of the helminths. More accurate diagnostic tools are required and new algorithms for detection and progression of helminth elimination will be needed. Standardized inter-laboratory test validation, inter-sectoral collaboration and establishment of an international One Health diagnostic platform, sharing best practices on diagnosis of helminth zoonoses, could all significantly contribute to control and elimination of these diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Genetic differentiation between two synonymous echinostomes species, Artyfechinostomum malayanum and Artyfechinostomum sufrartyfex was determined by using the first and second internal transcribed spacers (ITS1 and ITS2), the non-coding region of rDNA as genetic makers. Of the 699 bp of combined ITS1 and ITS2 sequences examined, 18 variable nucleotide positions (2.58 %) were observed. Of these, 17 positions could be used as diagnostic position between these two sibling species, whereas the other one variation was intraspecific variation of A. malayanum. A clade of A. malayanum was closely aligned with A. sufrartyfex and clearly distance from the cluster of other echinostomes. Our results may sufficiently suggest that the current synonymy of these species is not valid. [ABSTRACT FROM AUTHOR]

Abstract: Opisthorchis viverrini is one of the most common and medically important food-borne parasites in the Lower Mekong area of Southeast Asia. As we learn more about its ecology, pathology and epidemiology we see the need to consider more deeply the socio-cultural dynamics with which food-borne species complexes are associated. This paper argues that the Mekong region is characterized by strong livelihoods and life-style associations within wetland ecosystems, which are inseparable from human eating habits (“raw attitudes”). Within the fish-rice economies of the region there are many long-cherished food cultures based on eating raw, semi-cooked and fermented fish dishes, which are known to lead to opisthorchiasis, and potentially cholangiocarcinoma. This paper examines evidence from northeast Thailand showing that dedicated health outreach campaigns do help to reduce prevalence of opisthorchiasis over time. For disease prevention and health education approaches to be most effective, they must be sensitive to culture, livelihood economics, gender, and age. Further integrative, inter-disciplinary and international research must incorporate the complex dynamics of parasite ecology, human behavior, socio-economics, and public health awareness. [Copyright &y& Elsevier]

Abstract: A total of 314 individual Echinostoma revolutum were collected at different locations and times from domestic ducks from Khon Kaen Province, Thailand and Vientiane Province, the Lao People''s Democratic Republic (PDR). Genetic variation of these parasites was analyzed using multilocus enzyme electrophoresis at three polymorphic loci namely, glucose-6-phosphate dehydrogenase (G6pd), malic enzyme (Me) and peptidase valine-leucine (PepA). High levels of genetic variability were found within and between populations. Significant heterozygote deficiencies compared with the predictions under Hardy–Weinberg equilibrium were detected in populations from Thailand and the Lao PDR for all loci except G6pd-1. Significant genetic differentiation was observed between spatially separated populations from Thailand and the Lao PDR. This as also true for some samples collected at different times in Thailand. The variability found may be consistent with a Wahlund effect, genetic drift and/or other factors such as the population structure of snail hosts. Our data provide further insight into the process of genetic divergence within and among geographically and temporally isolated populations of E. revolutum, and potentially other medically important echinostomes in Southeast Asia. [Copyright &y& Elsevier]

Echinostomatidae are common, widely distributed intestinal parasites causing significant disease in both animals and humans worldwide. In spite of their importance, the taxonomy of these echinostomes is still controversial. The taxonomic status of two species, Echinostoma revolutum and Echinoparyphium recurvatum, which commonly infect poultry and other birds, as well as human, is problematical. Previous phylogenetic analyses of Southeast Asian strains indicate that these species cluster as sister taxa. In the present study, the first internal transcribed spacer (ITS1) sequence was used for genetic characterization and to examine the phylogenetic relationships between an isolate from Thailand with other isolates available from GenBank database. Interspecies differences in ITS1 sequence between E. revolutum and E. recurvatum were detected at 6 (3%) of the 203 alignment positions. Of these, nucleotide deletion at positions 25, 26, and 27, pyrimidine transition at 50, 189, and pyrimidine transversion at 118 were observed. Phylogenetic analysis revealed that E. recurvatum from Thailand clustered as a sister taxa with E. revolutum and not with other members of the genus Echinoparyphium. Interestingly, this result confirms a previous report based on allozyme electrophoresis and mitochondrial DNA that E. revolutum and E. recurvatum in Southeast Asia are sister species. Hence, the taxonomic status of E. recurvatum in Thailand, as well as in Southeast Asian countries needs to be confirmed and revised using more comprehensive analyses based on morphology and other molecular techniques. [ABSTRACT FROM AUTHOR]