Nuclear ribosomal transcription units in Asian Paragonimus species (Paragonimidae: Platyhelminthes): genetic characteristics, polymorphism, and implications for intersuperfamilial phylogeny.

The entire transcribed sequences (from the 5' terminus of 18S to the 3' terminus of 28S rRNA genes) of the ribosomal transcription units (rTU*) of five Asian Paragonimus species were obtained and characterized. The rTU* length was 7661 bp for P. heterotremus (LC strain, Vietnam), 7422 bp for P. iloktsuenensis (Amami strain, Japan), 6932 bp for P. skrjabini miyazakii (OkuST1 strain, Japan), 7422 bp for P. ohirai (Kino strain, Japan), and three strains of P. westermani: 8616 bp (Megha strain, India), 7292 bp (Bogil strain, South Korea), and 7052 bp (QT2 strain, Vietnam) without intergenic spacer region (IGS). All seven Asian Paragonimus strains' genetic characteristics were described, including the length of individual genes/regions, repeat polymorphism, base composition, and skewness. To investigate the superfamilial relationships in the Xiphidiata, with a focus on the Troglotrematoidea and its associated superfamilies, we used the PhyML software package to create three comprehensive maximum-likelihood phylogenies. The datasets used were 83 concatenated 28S + 18S, 83 single complete 18S, and 157 single, partial 28S rDNA sequences, respectively, from entire rTUs and/or accessible ribosomal sequences of the same species from the suborders Xiphidiata, Echinostomata, and Haplosplanchnata, with a Schistosoma sequence as an outgroup. Three phylogenetic trees revealed that Echinostomata and Haplosplanchnata are monophyletic, while Xiphidiata is polyphyletic and contains the monophyletic Troglotrematoidea. The concatenated 18S + 28S and single 18S phylogenies revealed well-bootstrap supported seven superfamilies (Troglotrematoidea, Haploporoidea, Gorgoderoidea, Brachycladioidea, Microphalloidea, Plagiorchioidea, and Opecoeloidea) that are monophyletic in the Xiphidiata. The Haploporoidea was a basal superfamily nested close to the Gorgoderoidea and Troglotrematoidea and was not supported as a distinct suborder Haploporata. Six of seven xiphidiatan superfamilies were monophyletic in the partial 28S phylogeny, with the exception of Opecoeloidea, which was separated into two different subclades: Opecoelidae and Stenakridae/Zdzitowieckitrematidae. The monophyletic Haploporoidea/Haploporata was separated from the Gorgoderoidea associates and placed in a marginal group in Xiphidiata. There were two notable clusters in the Paragonimidae: mixed-Paragonimus, which included a fairly compact group of P. heterotremus strains, and P. westermani/siamensis, which was divided into geographical/country strain groups. In conclusion, combined ribosomal rDNA sequences were more effective than single rDNA markers in resolving interfamilial and familial relationships. The ribosomal datasets presented here will be useful for taxonomic reassessment, as well as evolutionary and population genetics research in the Troglotrematoidea and other superfamilies in the Xiphidiata and the class Trematoda.

Paragonimus heterotremus Chen et Hsia, 1964 (Digenea: Paragonimidae): species identification based on the biological and genetic criteria, and pathology of infection.

As a result of the experimental infection of rats with metacercariae of Paragonimus heterotremus Chen et Hsia, 1964 from crabs (Potamiscus tannanti) caught in Yen Bai province, Vietnam, it was found that worms migrated into the lungs, to the liver and less frequently to the tissue that lines body cavities of the hosts, where they reached the adult stage, but in the muscles, worms stayed at the larval stage. Studies have shown that for P. heterotremus, rats can simultaneously play the role of the final and paratenic host; herewith, an infection with the trematode of this species can lead to the development of three forms of paragonimiasis: pulmonary, hepatic and muscular. Eggs from the adult worms localised in the liver, unlike eggs from the adult worms localised in the lungs, were not excreted into the external environment, but accumulated inside the organ. Histology and description of changes, which take place on the external surface of organs affected with P. heterotremus, are given in this study. Based on the behavioural characteristics of worms during rat infection and molecular genetic data, we established that worms from Vietnam and India should be assigned to different species of Paragonimus. P. heterotremus distribution is limited to the territory of the Southeast China, Northern Vietnam, Laos and Thailand.

Paragonimiasis: Neglected, Re-Emerging or Expanding Frontiers. Lessons from a Case Presenting in Jos, Plateau State.

BACKGROUND AND OBJECTIVES: Paragonimiasis is endemic in Eastern Nigeria. An upsurge was recorded after the Nigeria/Biafra war as protein lack in Biafra forced people to eat fresh water crabs. Its protean manifestations create confusion with several diseases. Elimination was assumed after a while and suspicion index fell. The interest in reporting this case follows its presentation outside the traditional endemic zone. RESULTS: The patient, though living in Eastern Nigeria and manifesting several pointers of Paragonimiasis, was treated as tuberculosis despite negative sputum AFB; without improving. He then presented up-country in Jos where history led to suspicion and confirmation of Paragonimiasis. By this time he had severe cor-pulmonale and died despite treatment. CONCLUSION: In this current economic downturn in Nigeria which may drive people to cheaper protein sources, a high index of suspicion should be raised for paragonimiasis when a patient presents with chronic cough productive of AFB-negative sputum and haemoptysis.

Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus.

Cathepsin L is a cysteine protease belonging to the papain family. In parasitic trematodes, cathepsin L plays essential roles in parasite survival and host-parasite interactions. In this study, cathepsin L of the lung fluke Paragonimus pseudoheterotremus (PpsCatL) was identified and its molecular biological and immunological features characterized. A sequence analysis of PpsCatL showed that the gene encodes a 325-amino-acid protein that is most similar to P. westermani cathepsin L. The in silico three-dimensional structure suggests that PpsCatL is a pro-enzyme that becomes active when the propeptide is cleaved. A recombinant pro-PpsCatL lacking the signal peptide (rPpsCatL), with a molecular weight of 35 kDa, was expressed in E. coli and reacted with P. pseudoheterotremus-infected rat sera. The native protein was detected in crude worm antigens and excretory-secretory products and was localized in the cecum and in the lamellae along the intestinal tract of the adult parasite. Enzymatic activity of rPpsCatL showed that the protein could cleave the fluorogenic substrate Z-Phe-Arg-AMC after autocatalysis but was inhibited with E64. The immunodiagnostic potential of the recombinant protein was evaluated with an enzyme-linked immunosorbent assay (ELISA) and suggested that rPpsCatL can detect paragonimiasis with high sensitivity and specificity (100 and 95.6 %, respectively). This supports the further development of an rPpsCatL-ELISA as an immunodiagnostic tool.

Molecular identification of the trematode Paragonimus in faecal samples from the wild cat Prionailurus bengalensis in the Da Krong Nature Reserve, Vietnam.

Conventional identification of Paragonimus species and their natural definitive hosts is based on the morphological features of adult parasites isolated from the lungs of wild mammalian hosts. However, wild animals are protected by strict regulations and sampling is not always possible. Recently, molecular techniques have been developed to identify the internal transcribed spacer (ITS) sequences of Paragonimus eggs in faeces/sputum of human patients. Also, mammalian hosts can be identified using the D-loop sequence of mitochondrial DNA in faecal samples. In this study, we used molecular techniques on faeces from wild animals collected in Da Krong Nature Reserve, Quang Tri province, central Vietnam, where Paragonimus metacercariae are highly prevalent in mountain crabs, to identify Paragonimus species and their natural definitive hosts. The results indicated that wild cats, Prionailurus bengalensis, were infected with at least three different Paragonimus species, P. westermani, P. skrjabini and P. heterotremus. Because all of these species can infect humans in Asian countries, human paragonimiasis should be considered in this area.

Paragonimiasis acquired in the United States: native and nonnative species.

Paragonimiasis is a parasitic lung infection caused by lung flukes of the genus Paragonimus, with most cases reported from Asia and caused by P. westermani following consumption of raw or undercooked crustaceans. With the exception of imported P. westermani cases in immigrants, in travelers returning from areas of disease endemicity, and in clusters of acquired cases following consumption of imported Asian crabs, human paragonimiasis caused by native lung flukes is rarely described in the United States, which has only one indigenous species of lung fluke, Paragonimus kellicotti. Clinicians should inquire about the consumption of raw or undercooked freshwater crabs by immigrants, expatriates, and returning travelers, and the consumption of raw or undercooked crayfish in U.S. freshwater river systems where P. kellicotti is endemic when evaluating patients presenting with unexplained fever, cough, rales, hemoptysis, pleural effusions, and peripheral eosinophilia. Diagnostic evaluation by specific parasitological, radiological, serological, and molecular methods will be required in order to differentiate paragonimiasis from tuberculosis, which is not uncommon in recent Asian immigrants. All cases of imported and locally acquired paragonimiasis will require treatment with oral praziquantel to avoid any potential pulmonary and cerebral complications of paragonimiasis, some of which may require surgical interventions.

Metacercarial polymorphism and genetic variation of Paragonimus heterotremus (Digenea: Paragonimidae), and a re-appraisal of the taxonomic status of Paragonimus pseudoheterotremus.

Paragonimus heterotremus, which is an important pathogen for human paragonimiasis in Asia, is recognized as having the smallest metacercariae (maximum diameter < 300 µm) of any previously reported Paragonimus species. Recently, P. pseudoheterotremus has been described from Thailand as a new species having metacercariae (about 200 µm) slightly smaller than those of Thai P. heterotremus. In fact, the small size of P. pseudoheterotremus metacercariae is compatible with those of P. heterotremus from India and China. In this study in Vietnam, we found variably sized small metacercariae which are expected to consist of both P. heterotremus and P. pseudoheterotremus. Contrary to expectation, the adult flukes obtained by separate infection of experimental cats with different sized metacercariae were all identified as P. heterotremus, using both morphological and molecular characteristics. The molecular analyses of an extensive collection of P. heterotremus/P. pseudoheterotremus isolates from Asian countries also indicated that genetic distances between different populations of P. heterotremus are even larger than that between P. pseudoheterotremus and P. heterotremus. The haplotype network showed that all P. heterotremus and P. pseudoheterotremus isolates formed a P. heterotremus complex consisting of three groups with strong geographical origins. In addition, the Indian P. heterotremus group is the root of the other P. heterotremus and P. pseudoheterotremus populations. Based on the observed metacercarial polymorphisms and genetic variation in P. heterotremus, P. pseudoheterotremus should be considered a geographically isolated population of the P. heterotremus complex.

Current status of Paragonimus and paragonimiasis in Ecuador.

A review of national and international publications on paragonimiasis in Ecuador, epidemiological records from the Ministry of Public Health and unpublished research data was conducted to summarise the current status of the parasite/disease. The purpose of the review is to educate physicians, policy-makers and health providers on the status of the disease and to stimulate scientific investigators to conduct further research. Paragonimiasis was first diagnosed in Ecuador 94 years ago and it is endemic to both tropical and subtropical regions in 19 of 24 provinces in the Pacific Coast and Amazon regions. Paragonimus mexicanus is the only known species in the country, with the mollusc Aroapyrgus colombiensis and the crabs Moreirocarcinus emarginatus, Hypolobocera chilensis and Hypolobocera aequatorialis being the primary and secondary intermediate hosts, respectively. Recent studies found P. mexicanus metacercariae in Trichodactylus faxoni crabs of the northern Amazon. Chronic pulmonary paragonimiasis is commonly misdiagnosed and treated as tuberculosis and although studies have demonstrated the efficacy of praziquantel and triclabendazole for the treatment of human infections, neither drug is available in Ecuador. Official data recorded from 1978-2007 indicate an annual incidence of 85.5 cases throughout the 19 provinces, with an estimated 17.2% of the population at risk of infection. There are no current data on the incidence/prevalence of infection, nor is there a national control programme.

Infection and biogeographical characteristics of Paragonimus westermani and P. skrjabini in humans and animal hosts in China: A systematic review and meta-analysis.

BACKGROUND: Paragonimiasis, primarily caused by Paragonimus westermani and P. skrjabini in China, is a common food-borne parasitic zoonosis. However, the national distribution of Paragonimus spp. infection and its associated environmental determinants remain poorly understood. In this paper, we summarize the infection of P. westermani and P. skrjabini and describe key biogeographical characteristics of the endemic areas in China. METHODS: Data on Paragonimus infection in humans and animal hosts were extracted from eight electronic databases, including CNKI, CWFD, Chongqing VIP, SinoMed, Medline, Embase, PubMed, and Web of Science. A random-effects meta-analysis model was used to estimate the pooled prevalence. All survey locations were georeferenced and plotted on China map, and scatter plots were used to illustrate the biogeographical characteristics of regions reporting Paragonimus infection. RESULTS: A total of 28,948 cases of human paragonimiasis have been documented, with 2,401 cases reported after 2010. Among the 11,443 cases with reported ages, 88.05% were children or adolescents. The pooled prevalence of P. skrjabini is 0.45% (95% CI: 0.27-0.66%) in snails, 31.10% (95% CI: 24.77-37.80%) in the second intermediate host, and 20.31% (95% CI: 9.69-33.38%) in animal reservoirs. For P. westermani, the pooled prevalence is 0.06% (95% CI: 0.01-0.13%) in snails, 52.07% (95% CI: 43.56-60.52%) in the second intermediate host, and 21.40% (95% CI: 7.82-38.99%) in animal reservoirs. Paragonimus are primarily distributed in regions with low altitude, high temperature, and high precipitation. In northeastern China, only P. westermani infections have been documented, while in more southern areas, infections of both P. westermani and P. skrjabini have been reported. CONCLUSIONS: Paragonimiasis remains prevalent in China, particularly among children and adolescents. Variations exist in the intermediate hosts and geographical distribution of P. westermani and P. skrjabini. Additionally, altitude, temperature, and precipitation may influence the distribution of Paragonimus.

Presence of three distinct genotypes within the Paragonimus westermani complex in northeastern India.

The name Paragonimus westermani (Kerbert, 1878) is commonly applied to members of a species complex that includes the well-known Asian lung fluke of medical and veterinary importance. Unambiguous molecular and morphological evidence showing the presence of a member of the complex in India has recently been published. In the present study we report the occurrence of 2 more members of the P. westermani complex in northeastern (NE) India. Surveys of the freshwater crabs Maydelliatelphusa lugubris in NE India revealed 2 morphologically distinct types of lung fluke metacercariae. Phylogenetic analyses, using DNA sequences from ITS2, 28S and cox1 gene regions indicate that these lung metacercariae belong to P. westermani complex. Type 1 metacercariae have a more basal position within the complex whereas type 2 metacercariae are closely related to the relatively derived forms of P. westermani from NE Asia (Japan, Korea, China) and Vietnam. A third type of metacercaria (type 3), detected in another crab host, Sartoriana spinigera in Assam, was phylogenetically close to P. siamensis, also a member of the P. westermani group. Molecular evidence has demonstrated the existence of 3 genotypes of lung flukes within the Paragonimus westermani complex in NE India. Two of these were previously unknown.

Paragonimus and paragonimiasis in Vietnam: an update.

Paragonimiasis is a food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Vietnam, research on Paragonimus and paragonimiasis has been conducted in northern and central regions of the country. Using a combination of morphological and molecular methods, 7 Paragonimus species, namely P. heterotremus, P. westermani, P. skrjabini, P. vietnamensis, P. proliferus, P. bangkokenis and P. harinasutai, have been identified in Vietnam. Of these, the first 3, P. heterotremus, P. westermani and P. skrjabini, are known to infect humans in other countries. However, in Vietnam, only P. heterotremus, found in some northern provinces, has been shown to infect humans. Even nowadays, local people in some northern provinces, such as Lai Chau and Yen Bai, are still suffering from P. heterotremus infection. In some provinces of central Vietnam, the prevalence and infection intensity of P. westermani metacercariae in freshwater crabs (the second intermediate hosts) are extremely high, but human cases have not been reported. Likewise, although P. skrjabini was found in Thanh Hoa Province, its pathogenicity to humans in Vietnam still remains uncertain. The results of molecular phylogenetic analyses of Vietnamese Paragonimus species provides new insights on the phylogeny and taxonomy of the genus Paragonimus. Comprehensive molecular epidemiological and geobiological studies on the genus in Vietnam and adjacent countries are needed to clarify the biodiversity and public health significance of the lung flukes.

Molecular variation in the Paragonimus heterotremus complex in Thailand and Myanmar.

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P. heterotremus, and it is likely that P. pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were PCR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P. heterotremus formed a sister group to sequences from P. pseudoheterotremus-type metacercariae. Sequences of P. heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P. heterotremus complex in Thailand and the form referred to as P. pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region.

Genetically variant populations of Paragonimus proliferus Hsia & Chen, 1964 from central Vietnam.

Among about 50 nominal Paragonimus species, Paragonimus proliferus is rather a rare species, found only in Yunnan province, China, until our recent discovery of this species in Lai Chau province, northern Vietnam close to Yunnan, China. Here we add Quang Binh province, central Vietnam as a new endemic area of P. proliferus. Large excysted metacercariae found in mountainous crabs, Potamiscus tannanti, were morphologically identified as P. proliferus, which was confirmed further by molecular analyses. Second internal transcribed spacer (ITS2) sequences of the P. proliferus population in Quang Binh province were completely (100%) identical with those of P. proliferus populations in Lai Chau province, northern Vietnam and Yunnan province, China. However, cytochrome oxidase subunit 1 (CO1) gene sequences of Quang Binh population were significantly different (5.6%) from that of previously reported northern Vietnam and Yunnan, China populations. A phylogenetic tree revealed that all CO1 sequences of P. proliferus Quang Binh population formed a distinct group, which was clustered with northern Vietnam and Yunnan, China populations with the bootstrap value of 75%. This is the first record of the genetically variant population of P. proliferus, distribution of which is geographically remote from the previously reported endemic areas in the border between northern Vietnam and Yunnan, China, suggesting that P. proliferus may be much more widely distributed in the Indochina peninsula (or South-East Asia) than expected.

Discovery of Paragonimus skrjabini in Vietnam and its phylogenetic status in the Paragonimus skrjabini complex.

Two members of the Paragonimus skrjabini complex, P. skrjabini and P. miyazakii, are now considered as two sub-species, P. skrjabini skrjabini and P. skrjabini miyazakii. They are well known as important pathogens for human paragonimiasis in China and Japan. Recently, members of this species complex have been reported from India. Here we report the first discovery of P. skrjabini from freshwater crab hosts in Thanh Hoa province, Vietnam. For morphological and molecular phylogenetic studies, adult worms were obtained by experimental infection in cats and dogs. Molecular analyses of metacercariae and adults revealed that the P. skrjabini population from Thanh Hoa, Vietnam was almost completely identical with that from Yunnan province, China. Those populations from Thanh Hoa, Vietnam and Yunnan, China and those from Manipur, India were significantly different from P. skrjabini populations reported from other localities of China in cytochrome oxidase subunit 1 (CO1) gene sequences, indicating considerable genetic variation within the P. skrjabini complex. Moreover, low bootstrap values in the CO1 tree suggested that more variant genotypes belonging to P. skrjabini complex may be found in other Asian countries in between Vietnam and India, such as Myanmar, Laos and Thailand. Since P. skrjabini is known as a pathogen for humans, paragonimiasis cases caused by P. skrjabini might be found in Vietnam and other Asian countries.

Paragonimus kellicotti flukes in Missouri, USA.

Paragonimiasis is an infection caused by lung flukes of the genus Paragonimus. In Asia, P. westermani infections are relatively common because of dietary practices. However, in North America, cases of paragonimiasis, which are caused by P. kellicotti flukes, are rare. Only 7 autochthonous cases of paragonimiasis were reported during 1968-2008. In 2009, we reported 3 new case-patients with paragonimiasis who had been seen at our medical center over an 18-month period. Six additional case-patients were identified in St. Louis, Missouri, USA, and treated at Washington University-affiliated health centers in 2009-2010. We report detailed descriptions of these case-patients, which includes unusual clinical manifestations. We also describe public health interventions that were undertaken to inform the general public and physicians about the disease and its mode of transmission.

Paragonimus & paragonimiasis in India.

Ever since the discovery of the first indigenous case in 1981, paragonimiasis has gained recognition as a significant food borne parasitic zoonosis in India. The data available on the occurrence of paragonimiasis, until today, may be just the tip of an iceberg as the study areas covered were restricted to Northeast Indian States. Nevertheless, the results of research on paragonimiasis in India have revealed valuable information in epidemiology, life cycle, pathobiology and speciation of Indian Paragonimus. Potamiscus manipurensis, Alcomon superciliosum and Maydelliathelphusa lugubris were identified as the crab hosts of Paragonimus. Paragonimus miyazakii manipurinus n. sub sp., P. hueit'ungensis, P. skrjabini, P. heterotremus, P. compactus, and P. westermani have been described from India. P. heterotremus was found as the causative agent of human paragonimiasis. Ingestion of undercooked crabs and raw crab extract was the major mode of infection. Pulmonary paragonimiasis was the commonest clinical manifestation while pleural effusion and subcutaneous nodules were the common extra-pulmonary forms. Clinico-radiological features of pulmonary paragonimiasis simulated pulmonary tuberculosis. Intradermal test, ELISA and Dot-immunogold filtration assay (DIGFA) were used for diagnosis and epidemiological survey of paragonimiasis. Phylogenitically, Indian Paragonimus species, although nested within the respective clade were distantly related to others within the clade.

Comparative genomics and transcriptomics of 4 Paragonimus species provide insights into lung fluke parasitism and pathogenesis.

BACKGROUND: Paragonimus spp. (lung flukes) are among the most injurious foodborne helminths, infecting ∼23 million people and subjecting ∼292 million to infection risk. Paragonimiasis is acquired from infected undercooked crustaceans and primarily affects the lungs but often causes lesions elsewhere including the brain. The disease is easily mistaken for tuberculosis owing to similar pulmonary symptoms, and accordingly, diagnostics are in demand. RESULTS: We assembled, annotated, and compared draft genomes of 4 prevalent and distinct Paragonimus species: Paragonimus miyazakii, Paragonimus westermani, Paragonimus kellicotti, and Paragonimus heterotremus. Genomes ranged from 697 to 923 Mb, included 12,072-12,853 genes, and were 71.6-90.1% complete according to BUSCO. Orthologous group analysis spanning 21 species (lung, liver, and blood flukes, additional platyhelminths, and hosts) provided insights into lung fluke biology. We identified 256 lung fluke-specific and conserved orthologous groups with consistent transcriptional adult-stage Paragonimus expression profiles and enriched for iron acquisition, immune modulation, and other parasite functions. Previously identified Paragonimus diagnostic antigens were matched to genes, providing an opportunity to optimize and ensure pan-Paragonimus reactivity for diagnostic assays. CONCLUSIONS: This report provides advances in molecular understanding of Paragonimus and underpins future studies into the biology, evolution, and pathogenesis of Paragonimus and related foodborne flukes. We anticipate that these novel genomic and transcriptomic resources will be invaluable for future lung fluke research.

Molecular identification of morphologically similar microcercous cercariae of two trematode families, Paragonimidae and Troglotrematidae, concurrently found in the same snail species of the subfamily Triculinae.

Microcercous cercariae possess a very short tail and are produced by digenean species of several families including medically important species, such as members of the genera Paragonimus Braun, 1899, Nanophyetus Chapin, 1927 and Troglotrema Odhner, 1914. During our survey of cercariae of Paragonimus spp. in Vietnam, we found microcercous cercariae from ten (0.29%) out of 3,400 snails of Triculinae gen. sp. 2. They were morphologically and molecularly analysed for species identification. The molecular analysis, based on ITS2 sequences, revealed two distinct species: four specimens were identical to Paragonimus proliferus Hsia et Chen, 1964 (Paragonimidae Dollfus, 1939), and the other six specimens were closest to members of the family Troglotrematidae Odhner, 1914 and were temporarily named Troglotrematidae gen. sp. Morphologically, cercariae of the two species found in this study are similar to each other in their gross characteristics but can be distinguished from one another by subtle morphological details. The cercaria of P. proliferus has an I-shaped excretory bladder and does not have mucous gland cells. In contrast, that of Troglotrematidae gen. sp. has a Y-shaped excretory bladder and mucous gland cells. Besides, the redia of P. proliferus is elongate with a short intestine and contains 5-6 cercariae whereas that of Troglotrematidae gen. sp. is more round with a longer intestine and harbours 3-4 cercariae. Our results have shown the importance of the shape of the excretory bladder and the presence/absence of mucous gland cells of the cercaria as well as the shape and size of the redia, and its intestinal length as valuable taxonomic characters of intramolluscan trematode larvae. In addition, the finding of similar microcercous cercariae of different species in the same snail species suggests that careful attention to morphological details is required in the differentiation of Paragonimus cercariae and those of closely related species.

Paragonimus and paragonimiasis in Asia: An update.

Paragonimiasis, or lung fluke disease, is a typical food-borne parasitic zoonosis caused by infection with trematodes belonging to the genus Paragonimus. More than 50 species of Paragonimus have been reported throughout the world, of which seven valid species infect humans, an estimated one million people annually worldwide. Among the seven species, P. westermani, P. heterotremus, and P. skrjabini/P. s. miyazakii, distributed in Asia, are the most important species as the cause of paragonimiasis. Humans acquire infection through the ingestion of raw, pickled or undercooked freshwater crustaceans, 2nd intermediate hosts, or consuming raw meat of wild boar or deer, paratenic hosts. Infections often occur clustered in foci where dietary habits allow transmission of the parasites. Paragonimiasis typically causes a subacute to chronic inflammatory disease of the lungs. The symptoms, including chronic cough, chest pain, dyspnea and hemoptysis, mimic those of tuberculosis and lung cancer. Serologic tests are commonly used for the diagnosis of paragonimiasis, and Praziquantel is the treatment of choice. In this review, the current status of Paragonimus and paragonimiasis in Asia is outlined based on the latest information and findings. We also summarize current trends of paragonimiasis in Japan, which is one of the most endemic area of paragonimiasis in the world, for the better understanding and control of paragonimiasis.