Idiopyrgus souleyetianus (Gastropoda: Tomichiidae) as an intermediate host of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) in Brazil.

Despite the importance of fish-borne trematodes of the family Opisthorchiidae as causative agents of human liver fluke disease, studies on these parasites outside Asia are relativally scarce. In South America, human focus of amphimerosis is known in Ecuador since the mid-20th century, and Amphimerus spp. have also been reported in wild and domestic mammals. Nevertheless, the knowledge on the snails that act as the first intermediate host of these potentially zoonotic parasites are scarce. Herein, a new cercaria of the pleurolophocercous morphotype found in the freshwater snail Idiopyrgus souleyetianus from Brazil was subjected to morphological and molecular studies. Multigene phylogenetic analyses based on 28S, 5.8S-ITS-2 and Cox-1 sequences enabled the identification of Amphimerus sp., a species distinct from that reported in humans from Ecuador. This cercariae was morphologically compared with other opisthorchiid cercariae known. The possible occurrence of human amphimerosis in Brazil is discussed.

Bithyniid snails as hosts of Opisthorchiidae and Notocotylidae in the south of Western Siberia, Russia.

Opisthorchiasis and related infections are persistent and substantial public health burdens from eastern Europe to southeastern and northern Asia. Snails of the family Bithyniidae act as first intermediate hosts not only for the trematodes of the family Opisthorchiidae but also for those of the family Notocotylidae. There are certain morphological similarities between the aforementioned trematode cercariae. In order to find natural local foci of opisthorchiasis, which are established primarily according to the presence of infected bithyniid snails at the area under examination, it is crucial to correctly identify the rediae and cercariae of the trematodes. The aim of our investigation was to evaluate the role of bithyniid snails in the transmission of Opisthorchiidae and Notocotylidae in ecosystems in the south of Western Siberia. We have been studying the process of bithyniid snail dissemination in Western Siberia and examining their infection by trematodes from 1994 until now. A total of 16,213 bithyniid snails in 23 water bodies (in 28 localities) of four major basins situated in the Novosibirsk region were inspected for trematode infestation. Long-term research has been conducted in the Kargat river estuary and the Ob river floodplain for 15 and 25 years, respectively. In both water bodies, the prevalence of notocotylid and opisthorchiid trematodes was positively correlated with the global land-ocean temperature index. Trematode parthenitae were identified if there were mature cercariae capable of leaving the shell of the host snail on their own. Identification to the species of opisthorchiid cercariae was verified using molecular genetic analysis methods targeting ITS2 locus. Opisthorchis felineus and Metorchis bilis, two opisthorchiid species that are potentially perilous to human health, were found in bithyniids in the Novosibirsk region. The mean prevalence of infection with notocotylid trematodes in bithyniid snails was higher than the corresponding prevalence of infection with opisthorchiid trematodes. The results of this study can be used to identify and predict natural foci of epidemiologically and/or epizootically dangerous diseases.

Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador.

Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2-gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.

A review of molecular identification tools for the opisthorchioidea.

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.

Amphimerus lancea as a Potential Etiological Agent of Human Amphimerosis in South America: A Morphological Analysis Based on Literature Data.

PURPOSE: Despite recent advances in the study of amphimerosis, aspects related to the taxonomy of the opisthorchiid species involved in human infection in Ecuador are not completely known. In the present study, previous morphological descriptions of Amphimerus sp. found in human beings and animals from Ecuador were re-studied, aiming to the identification of the parasite. METHODS: The morphological traits and measures of isolates of Amphimerus from Ecuadorian foci of transmission previously reported by different authors were critically analyzed and used to achieve the specific identification of the parasite. Morphological and morphometric data, including measures of structures, ratio between suckers, and disposition of vitellaria, were used for taxonomic identification based on taxonomic keys, reviews, and descriptive works. RESULTS: The morphological study based on literature data reveals that Amphimerus lancea (Diesing, 1850) is a species potentially involved in human amphimerosis in Ecuador. The main characteristics here used for differential diagnoses of this species is the larger size of the ventral sucker, which results in an oral sucker/ventral sucker ratio in isolates here considered as A. lancea (1.8-2.7) higher than those verified in other seven species of the genus Amphimerus reported in South America (0.5-1.3). The relative space that the ventral sucker occupies in relation to body width (at the level of ventral sucker) is also greater in A. lancea (49-64% vs 15-38%). CONCLUSION: Amphimerus lancea is at least one of the species involved in human amphimerosis in Ecuador. The parasite distribution and animal reservoirs are updated and the possibility of new areas of occurrence of human diseases in South America is highlighted. Future integrative taxonomic studies using material properly fixed is encouraged, which can corroborate the morphological identification here achieved and result in progress in the complex taxonomy of Amphimerus spp.

Foodborne zoonotic parasites of the family Opisthorchiidae.

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

Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador.

Amphimerus sp. is a fluke that dwells in the biliary tracts of vertebrate definitive hosts including humans, domestic, and wild mammals in Latin America. Opisthorchiid liver infections are rarely studied in the Americas confirming its status as a neglected tropical disease. In Ecuador, small trematode eggs were reported in human cases from the province of Manabí in 1949, and recently, Amphimerus sp. adults were recovered from human and reservoir hosts in the province of Esmeraldas. Due to the lack of research on the infectious sources of Amphimerus sp. in the continent, we have developed a series of epidemiological studies with parasitological and molecular techniques to elucidate the endemicity of opisthorchiid fluke infections. We developed a cross-sectional study in three communities at Pedro Pablo Gómez parish in the province of Manabí, Ecuador. We examined a total of 176 fecal samples to detect opisthorchiid eggs, and four fish species to find opisthorchiid metacercariae. To study adult worms, we treated and purged seven patients in a family and dissected the livers of a dog and a cat infected. We observed morphological features of adults and metacercariae and used polymerase chain reaction with restricted fragment length polymorphism (PCR-RFLP) and DNA sequencing of a section of the ITS2 gene for identification. Small trematode eggs were detected in 63 (35.8%) out of 176 fecal samples of residents in the three study sites. Adult opisthorchiid flukes were recovered from human patients, a dog and a cat, and they were morphologically and molecularly identified as Amphimerus sp. Opisthorchiid metacercariae were also identified molecularly as Amphimerus sp. in four fish species, i.e., Rhoadsia altipinna, Bryconamericus bucay, Andinoacara rivulatus, and Piabucina aureoguttata. Metacercariae of the heterophyid Haplorchis pumilio were also found in the four fish species examined. This is the first study to confirm the current endemicity of Amphimerus sp. in Pedro Pablo Gómez, Manabí, Ecuador. The adult worms isolated here shared morphological characteristics with previous Amphimerus sp. descriptions and were molecularly similar to Amphimerus sp. described in the province of Esmeraldas. Moreover, this study is the first to document four fish species as infection sources of Amphimerus sp. detected via a molecular protocol targeting the metacercariae of the parasite. Fish species identified here should be targeted for public health campaigns to avoid further human liver-fluke infections by Amphimerus sp. or potential intestinal-fluke infections by H. pumilio or others.

Morphological and molecular data for highly pathogenic avian parasite Erschoviorchis anuiensis sp. n. and phylogenetic relationships within the Opisthorchiidae (Trematoda).

Adult worms of Erschoviorchis anuiensis sp. n., parasites of the pancreas and liver of birds, were found by feeding the Muscovy ducks Cairina moschata dom. with freshwater fish (Phoxinus percnurus) from the Amur River basin (Russia). The trematodes obtained differ from the only previously known representative of the genus, E. lintoni by the large size of the ventral sucker, testes and ovary, the shape of the ovary (three-lobed vs irregular oval for E. lintoni), and the degree of vitellarium development (well-developed vitellarium with numerous follicles vs weakly developed vitelline fields for E. lintoni). In addition, genetic data were obtained for E. anuiensis sp. n., including nucleotide sequences of the ITS region and the 28S rRNA gene of nuclear DNA, and the mitochondrial сох1 gene. These data show that the genus Erschoviorchis is a sister group to the representatives of the genera Opisthorchis, Clonorchis, and Metorchis. At the same time, it did not cluster with species of Amphimerus, in which E. lintoni has sometimes been placed. The results of the study indicated that E. anuiensis sp. n., as well as E. lintoni, when it occurs in the pancreas, leads to significant associated pathological changes, manifested in an increase in size, changes of structure and tissue density.

Comparative analyses of complete ribosomal DNA sequences of Clonorchis sinensis and Metorchis orientalis: IGS sequences may provide a novel genetic marker for intraspecific variation.

Both Clonorchis sinensis and Metorchis orientalis are the fish-borne zoonotic trematodes, and have a wide distribution of southeastern Asia, especially in China. Due to the similar morphology, life cycle, and parasitic positions are difficult to differentiate between both metacercariae. In the present study, the complete rDNA sequences of five C. sinensis and five M. orientalis were obtained and compared for the first time. And the IGS rDNA sequences were tested as a genetic marker. The results showed complete rDNA lengths of C. sinensis were range from 8049 bp to 8391 bp, including 1991 bp, 1116 bp, 3854 bp, and 1088-1430 bp belonging to 18S, ITS, 28S and IGS, respectively. And the complete rDNA lengths of M. orientalis were range from 7881 bp to 9355 bp, including 1991 bp, 1077 bp, 3856 bp, and 957-2431 bp belonging to 18S, ITS, 28S and IGS, respectively. Comparative analyses reveal length difference main in IGS, which has higher intraspecific and interspecific variations than other ribosomal regions. Forty four repeat (forward and inverted) sequences were found in the complete rDNAs of C. sinensis and M. orientalis. The phylogenetic analyses showed that the sequences of ITS1, ITS2, 18S and 28S could be used as different level genetic markers. In IGS phylogenetic tree, Opisthorchiidae, Paramphistomidae, Dicrocoeliidae, and Schistosomatidae formed monophyletic groups, and the same length sequences were clustered together in the same species. These findings of the present study provide the new molecular data for studying the complete rDNA of C. sinensis and M. orientalis, and indicate IGS sequences may used as a novel genetic marker for studying intraspecific variation in trematodes.

Description of a new species, Cryptocotyle lata sp. nov., and discussion of the phylogenetic relationships in Opisthorchioidea.

Adult Cryptocotyle lata sp. nov. worms were obtained from experimental studies. In the Russian southern Far East, the life cycle of this parasite is carried out using freshwater snails (Boreoelona ussuriensis), freshwater fish, and birds as the first intermediate, second intermediate, and definitive hosts, respectively. The morphological indices of C. lata sp. nov. are closest to Cryptocotyle concava; however, these two species differ in terms of their sizes of body, oral and ventral suckers, eggs, and the shape of their testes and ovaries. Analysis of the life cycles of the Cryptocotyle representatives suggested that C. concava were at least two cryptic species, one of which circulates using brackish water Hydrobia snails, and the other using freshwater Amnicola snails as the first intermediate hosts. Molecular data (i.e., the 28S gene and the second internal transcribed spacer (ITS2) of rDNA) were used to analyze the phylogenetic relationships of C. lata sp. nov. and other representatives of Opisthorchioidea. The long repeats and secondary structure of the ITS1 region were studied. Representatives of the Opisthorchiidae and several species from Heterophyidae (including the genus Cryptocotyle) were found to have molecular features that suggested that these species belonged to Opisthorchiidae. At the same time, the genetic relatedness of worms, which are united in common clusters on phylogenetic trees, is consistent with the use of the first intermediate hosts from different taxonomic groups in their life cycles; namely, snails of the Truncatelloidea are hosts of trematodes from a cluster with Opisthorchiidae and a number species of the family Heterophyidae, while snails of the Cerithioidea are hosts of worms from a cluster that includes only the Heterophyidae. In addition, the results of genetic studies indicate that Clonorchis sinensis, Metorchis ussuriensis, Metorchis bilis, Metorchis xanthosomus, and Metorchis orientalis should be included in the genus Opisthorchis.

A comparison of Kato-Katz technique to three other methods for diagnosis of Amphimerus spp. liver fluke infection and the prevalence of infection in Chachi Amerindians of Ecuador.

BACKGROUND: Recently, a high prevalence of infection by the liver fluke Amphimerus spp. has been documented in the Chachi Amerindians of Ecuador. For diagnosis, no studies exist that compare the sensitivity of different coproparasitological detection techniques. The present study compares the Kato-Katz technique with three other coproparasitological methods for detecting eggs of Amphimerus in stools, as well as determines the prevalence of infection in Chachi residents in a Tropical rain forest area in the northwest coast of Ecuador. METHODOLOGY/RESULTS: A total of 105 samples, utilizing the Kato-Katz technique (KK), the spontaneous sedimentation technique in tube (SSTT), the formalin-ether concentration technique (FEC), and direct smear microscopy (DM), were examined. Combining the four methods (fixed "gold" standard), 38 samples were positive with a prevalence of infection of 36.2%. The sensitivities of individual methods were 71%, 58%, 50% and 3% for KK, SSTT, FEC, and DM respectively. Our results indicated that KK alone had the best performance, detecting 27 (71%) of the 38 positive samples. The combination of KK and SSTT detected amphimeriasis in 36 (95%) samples, and KK and FEC in 31 (82%) samples. CONCLUSIONS: DM showed the lowest sensitivity, which raises concern for its value, because it is the standard technique for stool examination for detection of parasites in both public and private laboratories in Ecuador. SSTT alone detected eggs in 22 samples (58%) and would be recommended for field studies because of its simplicity. Performing two techniques on a single sample enhances the detection of Amphimerus infection. Its sensitivity is relative to a fixed "gold" standard, determined as the combined results of the four techniques performed. This study confirms the high prevalence of human infection by Amphimerus in the indigenous Chachi group where the first human cases were described.

Diagnosis of amphimeriasis by LAMPhimerus assay in human stool samples long-term storage onto filter paper.

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas.

De novo assembly and functional annotations of the transcriptome of Metorchis orientalis (trematoda: Opisthorchiidae).

Metorchis orientalis is a neglected zoonotic parasite, living in the gallbladder and bile duct of poultry and some mammals as well as humans. In spite of its economic and medical importance, the information known about the transcriptome and genome of M. orientalis is limited. In this study, we performed de novo sequencing, transcriptome assembly and functional annotations of the adult M. orientalis, obtained about 77.4 million high-quality clean reads, among which the length of the transcript contigs ranged from 100 to 11,249 nt with mean length of 373 nt and N50 length of 919 nt. We then assembled 31,943 unigenes, of which 20,009 (62.6%) were annotated by BLASTn and BLASTx searches against the available database. Among these unigenes, 19,795 (62.0%), 3407 (10.7%), 10,620 (33.2%) of them had significant similarity in the NR, NT and Swiss-Prot databases, respectively; 5744 (18.0%) and 4678 (14.6%) unigenes were assigned to GO and COG, respectively; and 9099 (28.5%) unigenes were identified and mapped onto 256 pathways in the KEGG Pathway database. Furthermore, we found that 98 (1.08%) unigenes were related to bile secretion and 5 (0.05%) to primary bile acid biosynthesis pathways category. The characterization of these transcriptomic data has implications for the better understanding of the biology of M. orientalis, and will facilitate the development of intervention agents for this and other pathogenic flukes of human and animal health significance.

High prevalence of zoonotic trematodes in roach (Rutilus rutilus) in the Gulf of Finland.

The intention to increase roach (Rutilus rutilus) consumption is in focus for ecological and economic reasons in Finland. However, its safety as food has not been considered comprehensively. We collected and artificially digested 85 roach halves originating from the south-eastern coast of Finland, and found trematode metacercariae in 98.8% of the samples. Based on polymerase chain reaction (PCR) and sequencing of amplicons generated from the ITS2 gene region, zoonotic parasites of the family Opistorchiidae were identified as Pseudamphistomum truncatum and Metorchis bilis, and also non-zoonotic Holostephanus dubinini (family Cyathocotylidae) and Posthodiplostomum spp. (family Diplostomidae) were identified. The species identity of other trematodes found is currently being investigated. Mixed infections of several trematode species were common. The prevalence of morphologically identified zoonotic P. truncatum was 46%, and zoonotic M. bilis was found in one sequence sample. The high prevalence of zoonotic trematode metacercariae in roach from the Gulf of Finland is alarming. Only thoroughly cooked roach products can be recommended for human or animal consumption from the area.

LAMPhimerus: A novel LAMP assay for detecting Amphimerus sp. DNA in human stool samples.

BACKGROUND: Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed. METHODOLOGY/PRINCIPAL FINDINGS: A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively. CONCLUSIONS/SIGNIFICANCE: We have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans.

BACKGROUND: Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE: The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS: Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS: Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS: We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Chromosome morphometry in opisthorchiid species (Platyhelminthes, Trematoda).

Few existing studies have dealt with cytogenetics in trematodes, largely due to the attendant technical difficulty of chromosome preparation. We performed a comparative analysis of chromosomes in five opistorchiid species, including Opisthorchis felineus Rivolta, 1884, Opisthorchis viverrini Poirier, 1886, Clonorchis sinensis Cobbold, 1875, Metorchis xanthosomus Creplin 1846, and Metorchis bilis (Braun, 1790) Odening, 1962. For some of these species, no detailed morphometric description of their karyotypes has yet been published; for the karyotype of Metorchis bilis this is the first-ever description. We found that opisthorchiids, like other trematodes, are characterized by karyotypic conservatism (N=6-7) and karyotype asymmetry, although comparison of chromosome morphometric traits did reveal differences between the karyotypes of the species. Moreover, to address certain a methodological issue in trematode chromosome preparation, we analyzed how the source of chromosomal material (partenitae or mature flukes) and the chromosome preparation techniques used (air-drying and cell suspension methods) affected chromosome spreading and size, concluding that the most reliable comparative method involves comparing relative parameters (relative length, arm ratio, centromeric index) of chromosomes prepared using the same technique.

Multiple liver abscesses in a dog secondary to the liver fluke Metorchis conjunctus treated by percutaneous transhepatic drainage and alcoholization.

A 1-year-old German shepherd × husky cross dog was diagnosed with multiple liver abscesses and severe cholangitis secondary to the liver fluke Metorchis conjunctus. The dog was successfully treated with 2 percutaneous transhepatic drainage and alcoholization procedures, and a prolonged course of antibiotics and praziquantel.

Abcès hépatiques multiples chez un chien secondaire à une douve du foieMetorchis conjunctustraitée par drainage transhépatique percutané et alcoolisation. Un chien de race croisée Berger allemand et Husky âgé de 1 an a été diagnostiqué avec des abcès hépatiques multiples et une cholangite grave secondaire à la douve du foie Metorchis conjunctus. Le chien a été traité avec succès à l'aide de deux interventions de drainage transhépatique percutané et d'alcoolisation ainsi que d'un traitement prolongé aux antibiotiques et au praziquantel.(Traduit par Isabelle Vallières).

Integrative taxonomy of European parasitic flatworms of the genus Metorchis Looss, 1899 (Trematoda: Opisthorchiidae).

Metorchis spp. are flukes (Platyhelminthes: Digenea) that infect vertebrates, including humans, dogs, cats, poultry and wild game, with cyprinid freshwater fish serving as typical second intermediate hosts. In their definitive hosts, the Metorchis spp. are difficult to identify to species. We provide and analyze sequences of two nuclear (18S rDNA and ITS2) and two mitochondrial (CO1 and ND1) DNA loci of four morphologically identified European species of the Metorchis, namely Metorchis albidus, Metorchis bilis, Metorchis crassiusculus and Metorchis xanthosomus, and of another opisthorchiid, Euamphimerus pancreaticus. DNA analysis suggests that the Metorchis specimens identified morphologically as M. albidus (from Lutra lutra), M. bilis (from Phalacrocorax carbo) and M. crassiusculus (from Aquila heliaca and Buteo rufinus) represent a single species. Thus, M. albidus (Braun, 1893) Loos, 1899 and M. crassiusculus (Rudolphi, 1809) Looss, 1899 are recognized as junior subjective synonyms of M. bilis (Braun, 1790) Odening, 1962. We also provide comparative measurements of the Central European Metorchis spp., and address their tissue specificity and prevalence based on the examination of extensive bird cohort from 1962 to 2015. M. bilis and M. xanthosomus can be morphologically diagnosed by measuring the extent of genitalia relative to body length and by the size ratio of their suckers. They also differ in their core definitive hosts, with ducks (Anas, Aythya) and coots (Fulica) hosting M. xanthosomus, and cormorants (Phalacrocorax), the birds of prey (Buteo, Aquila, etc.), piscivorous mammals (Lutra, Vulpes, Ursus, etc.) and humans hosting M. bilis. Previous reports on the Metorchis spp. contain numerous suspected misidentifications.