Exploring the second intermediate hosts and morphology of human- and cat-specific Opisthorchis viverrini-like populations.

Infection by the zoonotic fish-borne trematode, Opisthorchis viverrini, remains a crucial health issue in Thailand and neighboring countries. Recently, molecular analysis revealed two populations of putative O. viverrini: one found primarily in human hosts ("human-specific" population) and the other primarily in cats ("cat-specific" population). It is unclear how the infective stages (metacercariae) of these different populations circulate among definitive and reservoir hosts in nature. To gain an insight into this, mitochondrial cox1 and nad1 gene sequences of metacercariae from fish intermediate hosts were examined. None of 192 metacercariae from cyprinid fish in Lao PDR and Thailand had sequences typical of "cat-specific" O. viverrini, suggesting that cyprinid fish are not the main second intermediate hosts of this population. Interestingly, all 20 O. viverrini-like metacercariae from snakehead fish (Channa striata) shared 99.51% to 100% sequence identity with eggs from cats naturally infected in a previous study. Hence, we propose a modification of the known transmission dynamics of O. viverrini: consumption of metacercariae within snakehead fish provides another pathway for cats and (occasionally) humans to acquire infection. We also performed morphological comparisons of eggs, metacercariae, and adult flukes (raised in hamsters) of both Opisthorchis populations. The "cat-specific" population has eggs that are narrower and adults that are shorter and wider than in the human-specific population. The metacercaria of the "cat-specific" population is elliptical, while that of the "human-specific" population is oval, occasionally rounded. Our results confirmed that O. viverrini-like metacercariae from snakehead fish are the infective stages of the "cat-specific" fluke. This provides a new insight into the dissemination and transmission of each population in the second intermediate host. The identity of the cat-specific population is discussed.

Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Accuracy of a new rapid diagnostic test for urinary antigen detection and assessment of drug treatment in opisthorchiasis.

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.

Tetraspanins from the liver fluke Opisthorchis viverrini stimulate cholangiocyte migration and inflammatory cytokine production.

The liver fluke Opisthorchis viverrini (Poirier, 1886) (Digenea) secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalised by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (rLEL-Ov-TSP-2) and tetraspanin-3 (rLEL-Ov-TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adult O. viverrini (Ov-ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells (P < 0.05), whereas rLEL-Ov-TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hours (P < 0.05) and 48 hours (P < 0.01) time points. In like fashion, H69 cholangiocytes co-cultured with both Ov-ES and rLEL-Ov-TSP-3 underwent significantly elevated Il-6 and Il-8 gene expression for at least one of the time points assessed. Finally, both rLEL-Ov-TSP-2 and rLEL-Ov-TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated that O. viverrini CD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.

Tetraspanins from Opisthorchis viverrini stimulate cholangiocyte migration and inflammatory cytokine production.

The liver fluke Opsithorchis viverrini secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalized by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (rLEL- Ov -TSP-2) and tetraspanin-3 (rLEL- Ov -TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adult O. viverrini ( Ov- ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells ( P <0.05), whereas rLEL- Ov -TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hr ( P <0.05) and 48 hr ( P <0.01) time points. In like fashion, H69 cholangiocytes co-cultured with both Ov -ES and rLEL- Ov -TSP-3 underwent significantly elevated Il-6 and Il-8 gene expression for at least one of the time points assessed. Finally, both rLEL- Ov -TSP-and rLEL- Ov -TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated that O. viverrini CD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.

Small extracellular vesicles but not microvesicles from Opisthorchis viverrini promote cell proliferation in human cholangiocytes.

Chronic infection with O. viverrini has been linked to the development of cholangiocarcinoma (CCA), which is a major public health burden in the Lower Mekong River Basin countries, including Thailand, Lao PDR, Vietnam and Cambodia. Despite its importance, the exact mechanisms by which O. viverrini promotes CCA are largely unknown. In this study, we characterized different extracellular vesicle populations released by O. viverrini (OvEVs) using proteomic and transcriptomic analyses and investigated their potential role in host-parasite interactions. While 120k OvEVs promoted cell proliferation in H69 cells at different concentrations, 15k OvEVs did not produce any effect compared to controls. The proteomic analysis of both populations showed differences in their composition that could contribute to this differential effect. Furthermore, the miRNAs present in 120k EVs were analysed and their potential interactions with human host genes was explored by computational target prediction. Different pathways involved in inflammation, immune response and apoptosis were identified as potentially targeted by the miRNAs present in this population of EVs. This is the first study showing specific roles for different EV populations in the pathogenesis of a parasitic helminth, and more importantly, an important advance towards deciphering the mechanisms used in establishment of opisthorchiasis and liver fluke infection-associated malignancy.

Epidemiology of strongyloidiasis determined by parasite-specific IgG detections by enzyme-linked immunosorbent assay on urine samples using Strongyloides stercoralis, S. ratti and recombinant protein (NIE) as antigens in Northeast Thailand.

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.

Peptide derived from progranulin of the carcinogenic liver fluke, Opisthorchis viverrini stimulates cell hyperproliferation and proinflammatory cytokine production.

Purpose: Progranulin (PGRN) is a secreted glycoprotein growth factor with roles in wound healing, inflammation, angiogenesis and malignancy. An orthologue of the gene encoding human PGRN was identified in the carcinogenic liver fluke Opisthorchis viverrini. Methods: Sequence structure, general characteristics and possible function of O. viverrini PGRN was analyzed using bioinformatics. Expression profiles were investigated with quantitative RT-PCR, western blot and immunolocalization. A specific peptide of Ov-PGRN was used to investigate a role for this molecule in pathogenesis. Results: The structure of the gene coding for O. viverrini PGRN was 36,463 bp in length, and comprised of 13 exons, 12 introns, and a promoter sequence. The Ov-pgrn mRNA is 2,768 bp in length and encodes an 846 amino acids with a predicted molecular mass of 91.61 kDa. Ov-PGRN exhibited one half and seven complete granulin domains. Phylogenetic analysis revealed that Ov-PGRN formed its closest relationship with PGRN of liver flukes in the Opisthorchiidae. Transcripts of Ov-pgrn were detected in several developmental stages, with highest expression in the metacercaria, indicating that Ov-PGRN may participate as a growth factor in the early development of O. viverrini. Western blot analysis revealed the presence of detected Ov-PGRN in both soluble somatic or excretory/secretory products, and immunolocalization indicated high levels of expression in the tegument and parenchyma of the adult fluke. Co-culture of a human cholangiocyte cell line and a peptide fragment of Ov-PGRN stimulated proliferation of cholangiocytes and upregulation of expression of the cytokines IL6 and IL8. Conclusion: Ov-PGRN is expressed throughout the life cycle of liver fluke, and likely plays a key role in development and growth.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

Silencing of Opisthorchis viverrini Tetraspanin Gene Expression Results in Reduced Secretion of Extracellular Vesicles.

Inter-phylum transfer of molecular information is exquisitely exemplified in the uptake of parasite extracellular vesicles (EVs) by their target mammalian host tissues. The oriental liver fluke, Opisthorchis viverrini is the major cause of bile duct cancer in people in Southeast Asia. A major mechanism by which O. viverrini promotes cancer is through the secretion of excretory/secretory products which contain extracellular vesicles (OvEVs). OvEVs contain microRNAs that are predicted to impact various mammalian cell proliferation pathways, and are internalized by cholangiocytes that line the bile ducts. Upon uptake, OvEVs drive relentless proliferation of cholangiocytes and promote a tumorigenic environment, but the underlying mechanisms of this process are unknown. Moreover, purification and characterization methods for helminth EVs in general are ill defined. We therefore compared different purification methods for OvEVs and characterized the sub-vesicular compartment proteomes. Two CD63-like tetraspanins (Ov-TSP-2 and TSP-3) are abundant on the surface of OvEVs, and could serve as biomarkers for these parasite vesicles. Anti-TSP-2 and -TSP-3 IgG, as well as different endocytosis pathway inhibitors significantly reduced OvEV uptake and subsequent proliferation of cholangiocytes in vitro. Silencing of Ov-tsp-2 and tsp-3 gene expression in adult flukes using RNA interference resulted in substantial reductions in OvEV secretion, and those vesicles that were secreted were deficient in their respective TSP proteins. Our findings shed light on the importance of tetraspanins in fluke EV biogenesis and/or stability, and provide a conceivable mechanism for the efficacy of anti-tetraspanin subunit vaccines against a range of parasitic helminth infections.

Helicobacter pylori GroEL Seropositivity Is Associated with an Increased Risk of Opisthorchis viverrini-Associated Hepatobiliary Abnormalities and Cholangiocarcinoma.

Despite the synergistic effect of Opisthorchis viverrini and Helicobacter pylori co-infection on pathogenesis of severe hepatobiliary abnormalities (HBA) including advanced periductal fibrosis and replace with cholangiocarcinoma (CCA) have been established, the immune response to H. pylori in O. viverrini infected population has never been explored. Hence, this study aimed to investigate the antibody responses to 2 immunogenic H. pylori proteins in O. viverrini-infected patients with HBA and CCA. The risk analysis by multinomial logistic regression revealed that GroEL seropositivity was associated with higher risks of hepatobiliary abnormalities and CCA with adjusted odds ratios (95% confidence intervals) of 2.11 (95% CI=1.20-3.71, P=0.008) and 2.13 (95% CI=1.21-3.75, P=0.009), respectively. These findings indicate that GroEL seropositivity might be a biomarker for early detection of O. viverrini associated HBA and CCA.

Monoclonal Antibodies Targeting an Opisthorchis viverrini Extracellular Vesicle Tetraspanin Protect Hamsters against Challenge Infection.

Opisthorchis viverrini causes severe pathology in the bile ducts of infected human hosts, and chronic infection can culminate in bile duct cancer. The prevention of infection by vaccination would decrease opisthorchiasis-induced morbidity and mortality. The tetraspanin protein, Ov-TSP-2, is located on the membrane of secreted extracellular vesicles (EVs), and is a candidate antigen for inclusion in a subunit vaccine. To address the role of anti-Ov-TSP-2 antibodies in protection, we assessed the protective capacity of anti-Ov-TSP-2 monoclonal antibodies (mAbs) against opisthorchiasis. Two anti-TSP-2 IgM mAbs, 1D6 and 3F5, and an isotype control were passively transferred to hamsters, followed by parasite challenge one day later. Hamsters that received 3F5 had 74.5% fewer adult flukes and 67.4% fewer eggs per gram of feces compared to hamsters that received the control IgM. Both 1D6 and 3F5 (but not the control IgM) blocked the uptake of fluke EVs by human bile duct epithelial cells in vitro. This is the first report of passive immunization against human liver fluke infection, and the findings portend the feasibility of antibody-directed therapies for liver fluke infection, bolstering the selection of TSPs as components of a subunit vaccine for opisthorchiasis and fluke infections generally.

Repeated Ivermectin Treatment Induces Ivermectin Resistance in Strongyloides ratti by Upregulating the Expression of ATP-Binding Cassette Transporter Genes.

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.

Phylogeography and demographic history of Thai Pediculus humanus capitis (Phthiraptera: Pediculidae) revealed by mitochondrial DNA sequences.

Pediculus humanus (human louse) is a hematophagous insect that feeds on human blood. It is distributed worldwide. Understanding phylogeography and population-genetic structure of the human louse will illuminate the evolution of this insect and the dynamics of how resistance alleles might spread in the landscape. In this work, we used mitochondrial (cox1 and cytb genes) sequences of the human louse to investigate genetic diversity, population-genetic structure and demographic history of the louse in Thailand. Human lice in Thailand belonged to mitochondrial clades A and C. Most genetic variation was attributed to intra-region 65.71% within provinces for clade A and 68.92% for clade C, while inter-region level was 34.40% among provinces within regions for clade A and 20.09% for clade C. Neutrality and other indices suggested that louse populations from clades A and C in Thailand have experienced a population expansion. But head lice from Khon Kaen Province in clade C demonstrated a significant recent population bottleneck or natural selective pressure with constant population size. Head lice in Thailand showed varying degrees of low to high genetic differentiation at the level of province with many populations being genetically distinct from each other among regions and within the same region. Knowledge of the clades present in Thailand and that gene flow occurs between regions will assist in developing appropriate strategies for management of head lice at the local level in the country.

Analysis of Daily Variation for 3 and for 30 Days of Parasite-Specific IgG in Urine for Diagnosis of Strongyloidiasis by Enzyme-Linked Immunosorbent Assay.

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.

Hepatobiliary morbidities detected by ultrasonography in Opisthorchis viverrini-infected patients before and after praziquantel treatment: a five-year follow up study.

Infection of the liver fluke, Opisthorchis viverrini (OV) is an important public health problem in northeast Thailand and adjacent countries, where people have a habit of eating raw or undercooked fish. A community case-control study was carried out with 8,936 participants from 89 villages, in Khon Kaen province, Thailand. There were 3,359 OV-infected participants all of whom underwent ultrasonography of upper abdomen for the evaluation of hepatobiliary morbidity. The participants with advanced periductal fibrosis (APF) by ultrasound (n = 785) were invited to undergo annual follow-up ultrasonography for five years after praziquantel treatment. The sonographer was blinded with respect to status of OV infection at each visit. The study findings revealed variability in the study population profile of the hepatobiliary morbidities before and after praziquantel treatment over the follow up interval. At the end of the study, 32 (30.8%) out of 104 participants showed no relapse of APF whereas, by contrast, 39 (37.5%) participants showed relapse or persistent APF since the outset of the study (≥ two consecutive visits). The APF in most follow-up visits was significantly associated with male sex, with intrahepatic duct stones, with the width of the gallbladder "pre" minus "post" fatty meal, and with the ratio of left lobe of the liver to aorta. Five cases of suspected cholangiocarcinoma were observed over the five years of follow-up. This long-term ultrasound follow-up study demonstrates a significant incidence of persistent APF in over one-third of opisthorchiasis cases after praziquantel treatment, findings that support the prospect of ongoing cholangiocarcinogenesis long after successful elimination of liver fluke infection among the population.

Immunomics-guided discovery of serum and urine antibodies for diagnosing urogenital schistosomiasis: a biomarker identification study.

BACKGROUND: Sensitive diagnostics are needed for effective management and surveillance of schistosomiasis so that current transmission interruption goals set by WHO can be achieved. We aimed to screen the Schistosoma haematobium secretome to find antibody biomarkers of schistosome infection, validate their diagnostic performance in samples from endemic populations, and evaluate their utility as point of care immunochromatographic tests (POC-ICTs) to diagnose urogenital schistosomiasis in the field. METHODS: We did a biomarker identification study, in which we constructed a proteome array containing 992 validated and predicted proteins from S haematobium and screened it with serum and urine antibodies from endemic populations in Gabon, Tanzania, and Zimbabwe. Arrayed antigens that were IgG-reactive and a select group of antigens from the worm extracellular vesicle proteome, predicted to be diagnostically informative, were then evaluated by ELISA using the same samples used to probe arrays, and samples from individuals residing in a low-endemicity setting (ie, Pemba and Unguja islands, Zanzibar, Tanzania). The two most sensitive and specific antigens were incorporated into POC-ICTs to assess their ability to diagnose S haematobium infection from serum in a field-deployable format. FINDINGS: From array probing, in individuals who were infected, 208 antigens were the targets of significantly elevated IgG responses in serum and 45 antigens were the targets of significantly elevated IgG responses in urine. Of the five proteins that were validated by ELISA, Sh-TSP-2 (area under the curve [AUC]serum=0·98 [95% CI 0·95-1·00]; AUCurine=0·96 [0·93-0·99]), and MS3_01370 (AUCserum=0·93 [0·89-0·97]; AUCurine=0·81 [0·72-0·89]) displayed the highest overall diagnostic performance in each biofluid and exceeded that of S haematobium-soluble egg antigen in urine (AUC=0·79 [0·69-0·90]). When incorporated into separate POC-ICTs, Sh-TSP-2 showed absolute specificity and a sensitivity of 75% and MS3_01370 showed absolute specificity and a sensitivity of 89%. INTERPRETATION: We identified numerous biomarkers of urogenital schistosomiasis that could form the basis of novel antibody diagnostics for this disease. Two of these antigens, Sh-TSP-2 and MS3_01370, could be used as sensitive, specific, and field-deployable diagnostics to support schistosomiasis control and elimination initiatives, with particular focus on post-elimination surveillance. FUNDING: Australian Trade and Investment Commission and Merck Global Health Institute.

Orally Administered Bacillus Spores Expressing an Extracellular Vesicle-Derived Tetraspanin Protect Hamsters Against Challenge Infection With Carcinogenic Human Liver Fluke.

BACKGROUND: The human liver fluke Opisthorchis viverrini is a food-borne trematode that causes hepatobiliary disease in humans throughout Southeast Asia. People become infected by consuming raw or undercooked fish containing metacercariae. Development of a vaccine to prevent or minimize pathology would decrease the risk of severe morbidity, including the development of bile duct cancer. METHODS: We produced an oral vaccine based on recombinant Bacillus subtilis spores expressing the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (Ov-TSP-2), a protein that is abundant on the surface of O. viverrini secreted extracellular vesicles (EVs). Recombinant spores expressing Ov-TSP-2-LEL were orally administered to hamsters prior to challenge infection with O. viverrini metacercariae. RESULTS: Vaccinated hamsters generated serum IgG as well as bile IgG and IgA responses to Ov-TSP-2-LEL, and serum IgG from vaccinated hamsters blocked the uptake of fluke EVs by a human bile duct epithelial cell line. Vaccinated hamsters had 56% reductions in both adult flukes and fecal eggs compared to the control group. CONCLUSIONS: These findings indicate that oral vaccination of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL is efficacious at reducing infection intensity and could form the basis of a vaccine for control of carcinogenic liver fluke infection in humans.

Impact of geography and time on genetic clusters of Opisthorchis viverrini identified by microsatellite and mitochondrial DNA analysis.

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.