Multiplexed CRISPR-Cas9 protocol for large transgene integration into the Schistosoma mansoni genome.

Precise, on-target CRISPR-Cas9 genome editing has been shown in Schistosoma mansoni, involving both non-homology end joining and homology-directed repair pathways. Here, we present a multiplexed CRISPR-Cas9 protocol for large transgene integration into the S. mansoni genome. We describe steps for deploying multiplexed ribonucleoprotein complexes (RNPs) and donor DNA preparation. We then detail procedures for introducing RNPs into schistosome eggs by square-wave electroporation in the presence of a 5' phosphorothioate-modified double-stranded donor transgene. For complete details on the use and execution of this protocol, please refer to Ittiprasert et al. (2023).1.

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.

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

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.

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.

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm's excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

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.

An Innovative Test for the Rapid Detection of Specific IgG Antibodies in Human Whole-Blood for the Diagnosis of Opisthorchis viverrini Infection.

Chronic human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis can last for decades and cause liver and biliary diseases, including life-threatening pathology prior to cholangiocarcinoma (CCA). CCA generally has a poor prognosis. Serological diagnosis can support parasitological examination in diagnosing disease and screening for the risk of CCA. Here, we present an improved and innovative lateral flow immunochromatographic test (ICT) kit that uses whole-blood samples (WBS) rather than serum to diagnose human opisthorchiasis, which also successfully diagnosed human clonorchiasis. This ICT includes a soluble worm extract of O. viverrini adults and colloidal-gold-labeled conjugates of the IgG antibody to evaluate the diagnostic values with simulated WBS (n = 347). Simulated WBS were obtained by the spiking infection sera with red blood cells. The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy for detecting opisthorchiasis were 95.5%, 87.0%, 80.5%, 97.2%, and 90.1%, respectively. For clonorchiasis, these findings were 85.7%, 87.0%, 53.6%, 97.2%, and 86.8%, respectively. Combined for both diseases, they were 93.2%, 87.0%, 84.0%, 94.6%, and 89.6%, respectively. The ICT kit can possibly replace the ICT platforms for antibody detection in serum samples in field surveys in remote areas where sophisticated equipment is not available.

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.

Adherence of Helicobacter pylori to Opisthorchis viverrini gut epithelium and the tegument mediated via L-fucose binding adhesin.

Recent reports implicate both the liver fluke Opisthorchis viverrini as a reservoir of Helicobacter pylori within the human gastrointestinal tract and H. pylori in the pathogenesis of opisthorchiasis-associated cholangiocarcinoma. We postulated that adherence of bacterial ligands to host receptors initiates colonization of the live fluke by H. pylori and here we aimed to assess the molecular interaction between O. viverrini and H. pylori by investigating host receptors for H. pylori in the fluke. Several known receptors of H. pylori including Lewis B, sialyl-Lewis X, Toll-like receptor 4 and L-fucose were detected immunohistochemically and histochemically by focusing analysis on the gut epithelium and tegument of the adult stage of the fluke. The frequency of detection of Lewis B, sialyl-Lewis X, TLR4 and L-fucose in 100 individual worms was 3, 3, 19 and 70%, respectively. Detection of H. pylori by a diagnostic ureA gene-based PCR assay revealed the presence of H. pylori in individual O. viverrini worms in 41 of 49 (79%) worms examined. In addition, numbers of bacteria decreased in a dose- and time-dependent fashion following exposure to fucosidase. These findings suggested that L-fucose represents a tractable receptor for H. pylori that can mediate bacterial colonization of the gut of O. viverrini.

Chromosome-level genome of Schistosoma haematobium underpins genome-wide explorations of molecular variation.

Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.

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.

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

MIxS-SA: a MIxS extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms.

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MIxS) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MIxS standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MIxS-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

Rapid assessment of Opisthorchis viverrini IgG antibody in serum: A potential diagnostic biomarker to predict risk of cholangiocarcinoma in regions endemic for opisthorchiasis.

BACKGROUND: Opisthorchiasis is caused by an infection with fish-borne liver flukes of the genus Opisthorchis. Opisthorchiasis frequently leads to chronic inflammation in the biliary tract and is classified as a group 1 biological carcinogen by the International Agency for Research on Cancer: a definitive risk for cholangiocarcinoma (CCA). METHODS: We used the rapid immunochromatographic test (ICT) to detect anti-Opisthorchis viverrini IgG and IgG4 subclass antibodies in sera of patients with CCA. The ICT kits were developed based on soluble antigens excreted and secreted by O. viverrini adult worms. RESULTS: ICT indicated sera was positive for IgG and IgG4 antibodies, respectively, in 22 (61.1%) and 15 (41.6%) participants of the 36 study participants diagnosed with CCA (P > 0.05). Our study also included groups with other cancers and with liver cirrhosis, where the IgG ICT and IgG4 ICT kits were 27.7% (13/47) and 25.5% (12/47) positive, respectively (P > 0.05). Neither total the IgG ICT nor the IgG4 ICT yielded positive results in a control group of 20 healthy participants. Moreover, the percentage positivity rate using the ICT for total IgG between the CCA group and the other cancers and liver cirrhosis group was significantly different (P < 0.05). By contrast, no significant difference between these groups was apparent in the ICT for IgG4 antibody. The CCA group was 6.53 times more likely to have positive anti-O. viverrini IgG antibody (odds ratio 6.53, P < 0.001) and 3.27 times more likely to have positive anti-O. viverrini IgG4 antibody (odds ratio 3.27, P = 0.010) than the non-CCA group. CONCLUSION: This information is of potential value for the development of a diagnostic biomarker to predict risk for O. viverrini infection-associated CCA.

A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection.

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly lethal adenocarcinoma of the hepatobiliary system, which can be classified as intrahepatic, perihilar and distal. Each anatomic subtype has distinct genetic aberrations, clinical presentations and therapeutic approaches. In endemic regions, liver fluke infection is associated with CCA, owing to the oncogenic effect of the associated chronic biliary tract inflammation. In other regions, CCA can be associated with chronic biliary tract inflammation owing to choledocholithiasis, cholelithiasis, or primary sclerosing cholangitis, but most CCAs have no identifiable cause. Administration of the anthelmintic drug praziquantel decreases the risk of CCA from liver flukes, but reinfection is common and future vaccination strategies may be more effective. Some patients with CCA are eligible for potentially curative surgical options, such as resection or liver transplantation. Genetic studies have provided new insights into the pathogenesis of CCA, and two aberrations that drive the pathogenesis of non-fluke-associated intrahepatic CCA, fibroblast growth factor receptor 2 fusions and isocitrate dehydrogenase gain-of-function mutations, can be therapeutically targeted. CCA is a highly desmoplastic cancer and targeting the tumour immune microenvironment might be a promising therapeutic approach. CCA remains a highly lethal disease and further scientific and clinical insights are needed to improve patient outcomes.