Comparative proteomics of adult Paragonimus kellicotti excretion/secretion products released in vitro or present in the lung cyst nodule.

Paragonimus kellicotti is a zoonotic lung fluke infection, the agent of North American paragonimiasis, and an excellent model for other Paragonimus infections. The excretory/secretory proteins (ESP) released by parasites and presented at the parasite-host interface are frequently proposed to be useful targets for drugs and/or vaccines In vitro culture conditions may alter ESP compared to those produced in vivo. In order to investigate ESPs produced in vivo we took advantage of the fact that adult P. kellicotti reproduce in the lungs of experimentally infected gerbils in tissue cysts. We performed a mass-spectrometric analysis of adult P. kellicotti soluble somatic protein (SSPs) extracts, excreted/secreted proteins (ESPs) produced by adult worms during in vitro culture, and lung cyst fluid proteins (CFPs) from experimentally infected gerbils. We identified 2,137 P. kellicotti proteins that were present in at least two of three biological replicates and supported by at least two peptides. Among those were 1,914 proteins found in SSP, 947 in ESP and 37 in CFP. In silico analysis predicted that only 141 of the total 2,137 proteins were secreted via classical or non-classical pathways. The most abundant functional categories in SSP were storage and oxidative metabolism. The most abundant categories in ESP were proteins related to metabolism and signal transduction. The 37 parasite-related proteins in CFP belonged to 11 functional categories. The largest groups were proteins with unknown function, cytoskeletal proteins and proteasome machinery. 29 of these 37 proteins were shared among all three sample types. To our knowledge, this is the first study that compares in vitro and in vivo ESP for any Paragonimus species. This study has provided new insights into ESPs of food-borne trematodes that are produced and released in vivo. Proteins released at the host-parasite interface may help the parasite evade host immunity and may represent new targets for novel treatments or diagnostic tests for paragonimiasis.

Paragonimus and paragonimiasis in West and Central Africa: unresolved questions.

Paragonimiasis, human lung fluke disease, is a foodborne anthropozoonosis caused by the trematodes assigned to Paragonimus and is regarded by the World Health Organization as a Neglected Tropical Disease (NTD). The life cycle of this medically important parasite centres on a complex freshwater biological community that includes two intermediate hosts: a mollusc and a decapod, usually a brachyuran. Although there is a perception that the biology, symptoms, diagnosis and treatment of Paragonimus is well understood, in reality, this is not the case, especially in Africa. Much remains unknown concerning the life-cycle of the parasite, its transmission, the current epidemiology of the disease, diagnosis and the effectiveness of treatment. Furthermore, cases of paragonimiasis may be misdiagnosed as resistant tuberculosis (TB) because of the similar pulmonary symptoms and no remission after anti TB therapy. The endemic foci of human paragonimiasis in Africa have been reported mainly in the forest zones of Upper Guinea (Liberia, Guinea and Ivory Coast) and Lower Guinea (Nigeria, Cameroon, Equatorial Guinea and Gabon). Despite the perceived medical importance of paragonimiasis, relatively little attention has been paid to this NTD since its discovery in Africa in the 1960s. This review focuses on the current understanding of the life cycle and transmission of Paragonimus in Africa, discusses its diagnosis and public health importance and highlights many outstanding gaps in the knowledge that still exist for this NTD.

Morphological and molecular characterization of Paragonimus caliensis Little, 1968 (Trematoda: Paragonimidae) from Medellin and Pichinde, Colombia.

Paragonimiasis is a subacute to chronic inflammatory granulomatous lung disease caused by the genus Paragonimus. In Latin America Paragonimus mexicanus Miyazaki & Ishii, 1968 is the only confirmed species to cause human infections. Paragonimus caliensis Little, 1968 is an uncommon species often regarded as a synonym of P. mexicanus. Recently, the study of two types of Paragonimus metacercariae from Costa Rica has provided new molecular and morphological evidence that P. caliensis is a separate species from P. mexicanus. In the present study, molecular, morphological and phylogenetic tools have been used to characterize two populations of Paragonimus located at west of Medellin, Antioquia and at Pichinde, Valle del Cauca (type locality of P. caliensis), Colombia. Adults and metacercariae obtained from Medellin, and metacercariae from Pichinde were analyzed. For morphological observations we used light microscopy and scanning electron microscopy (SEM). Morphology of metacercariae and adults matched with the holotype of P. caliensis. The number and arrangement of sensory papillae in the acetabulum region differs from the morphotypes reported for P. caliensis in Costa Rica. Two morphotypes in branching patterns of ovary and two morphotypes in branching patterns of testes were identified. The main morphological differences between P. caliensis and P. mexicanus corresponded to the size of gonads and their relative positions in the body, and the occasional presence of a cyst wall in P. caliensis metacercariae. The molecular and phylogenetic analyses (using nuclear ribosomal ITS2 and partial cytochrome c oxidase subunit 1 CO1 sequences) confirmed that P. caliensis from the type locality is the same species from Medellin and Costa Rica. Furthermore, these analyses also suggest genetic as well as geographical separation of P. caliensis populations between Colombia and Costa Rica. Currently, P. mexicanus and P. caliensis are sympatric in the Colombian Pacific bioregion, and specific diagnosis based on their egg size is not possible. Therefore, it is necessary to determine the biogeographic distribution ranges of both species and to implement molecular techniques to establish the role of P. caliensis in human paragonimiasis in Colombia.

Conservation and diversification of the transcriptomes of adult Paragonimus westermani and P. skrjabini.

BACKGROUND: Paragonimiasis is an important and widespread neglected tropical disease. Fifteen Paragonimus species are human pathogens, but two of these, Paragonimus westermani and P. skrjabini, are responsible for the bulk of human disease. Despite their medical and economic significance, there is limited information on the gene content and expression of Paragonimus lung flukes. RESULTS: The transcriptomes of adult P. westermani and P. skrjabini were studied with deep sequencing technology. Approximately 30 million reads per species were assembled into 21,586 and 25,825 unigenes for P. westermani and P. skrjabini, respectively. Many unigenes showed homology with sequences from other food-borne trematodes, but 1,217 high-confidence Paragonimus-specific unigenes were identified. Analyses indicated that both species have the potential for aerobic and anaerobic metabolism but not de novo fatty acid biosynthesis and that they may interact with host signaling pathways. Some 12,432 P. westermani and P. skrjabini unigenes showed a clear correspondence in bi-directional sequence similarity matches. The expression of shared unigenes was mostly well correlated, but differentially expressed unigenes were identified and shown to be enriched for functions related to proteolysis for P. westermani and microtubule based motility for P. skrjabini. CONCLUSIONS: The assembled transcriptomes of P. westermani and P. skrjabini, inferred proteins, and extensive functional annotations generated for this project (including identified primary sequence similarities to various species, protein domains, biological pathways, predicted proteases, molecular mimics and secreted proteins, etc.) represent a valuable resource for hypothesis driven research on these medically and economically important species.

[Clinical analysis of 17 cases of paragonimiasis].

OBJECTIVE: To investigate the clinical features, diagnosis and treatment of paragonimiasis, so as to improve the prevention and treatment of it. METHODS: The clinical data of paragonimiasis patients were collected and retrospectively analyzed. RESULTS: Totally 17 patients were diagnosed as paragonimiasis and the main clinical features of 11 patients were cough, chest pain and fever, and the pleural effusion was found in 13 cases. Peripheral blood eosinophil percentages of all patients were significantly increased, and the detections of antibody IgG againstParagonimus parasite of ELISA method were positive in all patients. All the patients were cured after praziquantel treatment and no recurrence found in the follow-up visit. CONCLUSIONS: The clinical features of paragonimiasis patients are diverse, and pleural effusion is quite common in imaging examinations. The eosinophil percentages and antibody detections have important values for the diagnosis of paragonimiasis. Praziquantel is an effective medicine in the treatment.

Ultrastructure of spermatogenesis in the testis of Paragonimus heterotremus.

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.

[A new species of the genus Nanhaipotamon (Decapoda:Potamidae) serving as intermediate host of Paragonimus skrjabini].

OBJECTIVE: To describe a new species of the genus Nanhaipotamon. METHODS: Freshwater crabs were collected in the counties of Yongtai, Minqing, Youxi, Songxi, Zhenghe and Shouning, Fujian Province. The morphological characteristics of the crabs were described. The habitats were observed and crabs examined for the presence of Paragonimus metacerariae. RESULTS: A new species of freshwater crabs named as Nanhaipotamon fujianense sp. nov. was described: holotype (FJ6132-1): male, carapace length 18.44 mm, breadth 23.64 mm, thickness 12.61 mm; allotype (FJ6132-2): female, length 18.76 mm, breadth 25.25 mm, thicknes 14.31 mm, collected from Yongtai County in the middle of Fujian (N 25 degrees 44,778'; E118 degrees 32,278', and 232 m above sea lever). Distal segment of the first male pleopod with triangle convex inner-distal angle, and the axe-like expanded out-distal angle. The out-lateral border slightly sloped downwards. The segment length is 2.1 times as long as the subdistal segment. The crabs usually lived in the crevice of small stream. Paragonimus metacerariae were found in the crabs collected from Yongtai, Minqing, Youxi, Songxi and Zhenghe Counties. CONCLUSION: A new species of freshwater crab (Nanhaipotamon fujianense sp. nov.) has been recorded which serves as the intermediate hosts of Paragonimus skrjabini.

Ultrastructure of Spermatogenesis in the Testis of Paragonimus heterotremus

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.

A paragonimiasis patient with allergic reaction to praziquantel and resistance to triclabendazole: successful treatment after desensitization to praziquantel.

Paragonimiasis is an infectious disease caused by trematodes of the genus Paragonimus. This trematode can be treated successfully with praziquantel in more than 90% of the cases. Although praziquantel is generally well tolerated, anaphylactic reactions to this drug have been reported in a few cases. We report here a 46-year-old Korean female with paragonimiasis, presumed to be due to Paragonimus westermani, who displayed an allergic reaction to praziquantel and resistance to triclabendazole treatment. The patient was successfully treated with praziquantel following a rapid desensitization procedure. Desensitization to praziquantel could be considered when no alternative drugs are available.

[Two new species of freshwater crabs (Decapoda: Potamidae) serving as intermediate hosts of Paragonimus in Fujian, China].

OBJECTIVE: To investigate the natural resources of the freshwater crab which can serve as the intermediate hosts of Paragonimus in Pinghe and Youxi of Fujian Province. METHODS: Freshwater crabs were collected. The morphological characteristics of the crabs and the habitats were observed. The crabs were dissected and examined for the presence of Paragonimus metacercariae. RESULTS: Two new species of crabs were described, named as Sinopotamon zhangzhouense sp. nov. and Bottapotamon youxiense sp. nov.. S. zhangzhouense sp. nov., holotype: male, carapace length 35.9 mm, breadth 42.8 mm, thickness 18.6 mm, collected from Pinghe County in southwest of Fujian. (24 degrees 14.206' N, 117 degrees 12.594' E). Distal segment of the first pleopod of male tended flattish, and showed palm nest-shaped concave, which divided into two point leafs, and longitudinal crack clearly identified in back. The end half of distal segment reversed to ventral outwardly. This species usually lived in the sluggish stream. The infection rate of Paragonimus westermani and P. cenocopiosus in S. zhangzhouense sp. nov. was 44.9% (35/78). B. youxiense sp. nov., holotype: male, carapace length 13.35 mm, breadth 16.63 mm, thickness 7.20 mm, collected from Youxi County in central Fujian (26 degrees 10.558' N, 118 degrees 22.012' E). The first pleopod of male was in slightly flat shape, ample and developed, as bow-like uplift. This species usually lived in the relatively flat terrain of stream. The infection rate of P. skrjabini metacercariae in B. youxiense sp. nov. was 92.1% (58/63). CONCLUSION: Two new species of freshwater crabs (S. zhangzhouense sp nov. and Ryouxiense sp. nov.) serving as the intermediate hosts of Paragonimus have been described.

[Paragonimus skrjabini infection in animal reservoir hosts and questionnairing in residents at a village of Hubei Province].

Freshwater crabs (Sinopotamon denticulatum) were examined for metacercariae. Cats and dogs were also examined for Paragonimus infection. Questionnairing was carried out on health knowledge and behaviors among local residents in a village of Baokang County, Hubei Province. Results showed that the infection rate of Paragonimus skrjabini metacercariae in Sinopotamon denticulatum was 20.5% (46/214), with 15.6% (20/128) in a mining area and 30.2% (26/86) for the non-mining area respectively (chi2 = 6.5, P < 0.05). The prevalence in cats and dogs was 25.0% (6/24) and 17.6% (6/34) respectively (chi2 = 0.46, P > 0.05). Questionnairing showed that dogs and cats were with the habit of foraging and defecating at streams and children had the habits of eating raw or under-cooked crabs. The natural and ecological environments are in favor of the life cycle of P. skrjabini.

Paragonimiasis: an important food-borne zoonosis in China.

The lung fluke, Paragonimus westermani, is of major socioeconomic importance in Asia. The parasite is transmitted via snails to freshwater crabs or crayfish, then to humans and other mammals, such as cats and dogs, and causes paragonimiasis. This review provides a background on the parasite and its life cycle; summarizes key aspects regarding the pathogenesis, diagnosis and treatment of paragonimiasis; describes the geographic distribution and prevalence of paragonimiasis; and makes some recommendations for future research and the control of this important disease in China.

Excretory-secretory products secreted by Paragonimus westermani delay the spontaneous cell death of human eosinophils through autocrine production of GM-CSF.

BACKGROUND: Eosinophils play important roles in tissue inflammatory responses associated with helminth infections. Excretory-secretory products (ESP) produced by tissue-invasive helminths contain a large quantity of proteolytic enzymes that can modulate the host's immune responses. However, little is known regarding the roles of worm-derived products that are responsible for eosinophilic inflammatory responses in helminth infections. OBJECTIVE: In the present study, we investigated whether ESP produced by Paragonimus westermani, which cause pulmonary or extrapulmonary paragonimiasis in human beings, regulates both cell survival and death of human eosinophils. METHODS: The ESP was obtained from P. westermani newly excysted metacercariae (PwNEM). Eosinophils were purified from peripheral blood of healthy donors, and the purified eosinophils were incubated with or without the ESP secreted by PwNEM. The viability of eosinophils was assessed by staining with propidium iodide using the flow cytometer. RESULTS: When eosinophils were incubated with a low concentration of the ESP produced by PwNEM, which totally consists of proteolytic enzymes, eosinophil cell death was delayed compared with results for cells incubated with medium alone. In fact, the ESP at a low concentration stimulated eosinophils to produce detectable levels of GM-CSF that can delay eosinophil cell death. In contrast, eosinophil cell death was dose-dependently accelerated when cells were incubated with high concentrations of the ESP. To see whether the dose-dependent biphasic survival effect of the ESP on eosinophils is primarily due to the protease activity contained in the ESP, a high dose of the ESP was treated with heat at 56 degrees C for 30 min before being added to eosinophils. Attenuating protease activity in a high dose of the ESP by heat treatment reversed the ESP-afforded eosinophil cell death. This prolonged survival of eosinophils induced by the heated ESP was remarkably inhibited by anti-GM-CSF-neutralizing mAb and Jak2 kinase inhibitor AG-490. CONCLUSION: These results suggest that the proteases in the ESP secreted by PwNEM are able to regulate eosinophil survival through the autocrine production of GM-CSF. Thus, the enhanced eosinophil survival induced by Paragonimus-secreted products may contribute to the elicitation of eosinophilic inflammatory responses at the worm-infected lesion in human paragonimiasis.

Paragonimiasis of the breast. Report of a case diagnosed by fine needle aspiration.

BACKGROUND: Paragonimiasis infestation is endemic mostly to Asia, Africa and South and Central America and commonly found in the lung. It is extremely rare in soft tissue. CASE: A case of paragonimiasis occurred in a 66-year-old woman who presented with a left, painful, 2-cm-diameter breast mass and had a history of eating uncooked freshwater crabs. On mammography, an ill-defined, round, isodense mass was detected without calcification. Hemorrhagic material was aspirated by fine needle aspiration (FNA) of the breast mass, which contained many parasitic eggs. They were about 90 microns long, ovoid, yellowish brown, transparent and thick shelled, with a flattened operculum at 1 end. The breast mass was completely excised. Many collapsed eggs of Paragonimus infiltrated the lobules and fibroadipose tissue, with accompanying abscess formation. To confirm the diagnosis, serologic testing for paragonimiasis was performed; it was positive for Paragonimus antibody. CONCLUSION: Paragonimiasis very rarely presents as a breast mass and can be diagnosed by FNA.

Cavitary mass lesion and recurrent pneumothoraces due to Paragonimus kellicotti infection: North American paragonimiasis.

North American paragonimiasis is well described in omnivorous and carnivorous animals on this continent. Humans are rarely infected, largely because of dietary customs, but are at risk for infection if raw or undercooked crayfish are consumed. We describe a patient with a pleuropulmonary infection due to Paragonimus kellicotti that presented as recurrent pneumothoraces and a cavitary lesion. This is the first case of North American paragonimiasis in which the diagnosis was based on the morphology of the eggs present in histologic sections.

Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187.

The involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.

[A case of Paragonimus miyazakii with migrating infiltrates: use of ELISA in diagnosis and treatment].

A 47-year-old man was referred to our clinic with abnormal chest radiographs. He was free of symptoms, but his chest CT demonstrated transient migrating infiltrates. Peripheral blood revealed eosinophilia and eosinophilic pneumonia was suspected. However, the bronchoalveolar lavage fluid showed no increase in eosinophils. Later, the patient's pleural effusion did contain eosinophils. His total IgE level was continuously elevated despite the absence of allergic disease, and so parasitic infection was suspected. Ouchterlony's double diffusion test after absorption of Paragonimus westermanii antigen was positive for Paragonimus miyazakii antigen. The ELISA values of both serum sample and pleural effusion were increased. As the ELISA value of the pleural effusion was higher than the serum one, we realised that pleural effusion was caused by Paragonimus miyazakii. Treatment with praziquantel for 3 days resulted in disappearance of the abnormal chest radiographic findings, and a reduced blood eosinophil level, IgE level and ELISA titre for Paragonimus miyazakii. We concluded that ELISA was useful in the diagnosis and assessment for treatment in Paragonimus miyazakii infections although there was no clearly relevant history.

Occurrence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea.

The biology, chromosome number, and karyotype of a lung fluke, Paragonimus westermani (Kerbert, 1878) collected in Haenam, Haenam-gun Chollanam-do, Korea were analyzed. We compared the size of metacercariae from Haenam with those taken from a crayfish collected at Youngam, Youngam-gun, Chollanam-do, Korea. The mean length of P. westermani metacercariae from Haenam was 300.3 microm and that from Youngam was 362.0 microm. Adult worms were recovered from the lungs of experimentally infected dogs. The mean egg sizes obtained from adult flukes were 72.1 x 46.8 microm from Haenam and 93.5 x 54.2 microm from Youngam. Semisulcospira tegulata collected in the Youngam area were found to be infected with cercariae of P. westermani, one of the snail-borne human lung fluke trematodes in Korea. Of 4218 snails studied, 5 (0.12%) harbored P. westrermani larvae. This is the first report of S. tegulata serving as the initial intermediate host of P. westermani. The chromosome numbers of P. westermani from Haenam and Youngam were 2n = 22 and 3n = 33. The diploid type of P. westermani has not been previously reported in Korea.