This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.
Animals ; Antibodies, Helminth/immunology ; Antibodies, Monoclonal/*immunology ; Antigens, Helminth/*immunology ; Clonorchis sinensis/immunology ; Cross Reactions/immunology ; Fasciola hepatica/immunology ; Female ; Helminth Proteins/*immunology ; Heterophyidae/*immunology ; Immunologic Tests ; Mice ; Mice, Inbred BALB C ; Paragonimus westermani/immunology ; Trematode Infections/*diagnosis/immunology

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.
Adult ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*diagnostic use ; Clinical Laboratory Techniques/*methods ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; Immunoglobulin G/*blood ; Paragonimiasis/*diagnosis ; Paragonimus/*immunology ; Parasitology/*methods ; Predictive Value of Tests ; Sensitivity and Specificity

A 94-year-old female with end-stage renal disease presents with fever, fatigue, and hematochezia. She had previously resided in Hunan Province, China, and Myanmar, and she immigrated to Taiwan 30 years ago. Colonoscopy revealed a colonic ulcer. Biopsy of the colonic ulcer showed ulceration of the colonic mucosa, and many Paragonimus westermani-like eggs were noted. Serum IgG antibody levels showed strong reactivity with P. westermani excretory-secretory antigens by ELISA. Intestinal paragonimiasis was thus diagnosed according to the morphology of the eggs and serologic finding. After treatment with praziquantel, hematochezia resolved. The present case illustrates the extreme manifestations encountered in severe intestinal paragonimiasis.
Aged, 80 and over ; Animals ; Anthelmintics/therapeutic use ; Antibodies, Helminth/blood ; Antigens, Helminth/immunology ; Colonic Diseases/complications/drug therapy/*pathology ; Colonoscopy ; Enzyme-Linked Immunosorbent Assay ; Female ; Gastrointestinal Hemorrhage/complications/drug therapy/*pathology ; Humans ; Intestinal Diseases, Parasitic/complications/drug therapy/parasitology/*pathology ; Kidney Failure, Chronic/complications ; Paragonimiasis/complications/drug therapy/parasitology/*pathology ; Paragonimus westermani/*immunology ; Praziquantel/therapeutic use ; Taiwan ; Ulcer/complications/drug therapy/*pathology

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.
Animals ; Benzimidazoles/*therapeutic use ; *Desensitization, Immunologic ; Drug Hypersensitivity/etiology/immunology/*therapy ; *Drug Resistance ; Female ; Humans ; Middle Aged ; Paragonimiasis/*drug therapy/immunology/parasitology ; Paragonimus/isolation & purification/physiology ; Praziquantel/*adverse effects/*immunology/therapeutic use ; Treatment Outcome

Seroprevalence of the IgG antibodies for Clonorchis sinensis, Paragonimus westermani, Taenia solium metacestode (cysticercus), and Spirometra erinacei plerocercoid (sparganum) was measured using enzyme-linked immunosorbent assay (ELISA) in sera of patients in Korea from 1993 to 2006. A total of 74,448 specimens referred nationwide from 121 hospitals revealed an IgG positive rate of 7.6% for the 4 parasites. The IgG positive rate (18.7%) for the 4 parasites in 1993 decreased gradually to 6.6% in 2006. Individual positive rate decreased from 5.2% (1993) to 1.6% (2006) for C. sinensis, from 2.8% (1993) to 1.1% (2006) for P. westermani, from 8.3% (1993) to 2.2% (2006) for cysticercus, and from 2.6% (1993) to 1.6% (2006) for sparganum. The positive rate was highest (21.2%) in the group of patients who ranged in age from 50-59 yr old, and in the group that was referred from the Seoul area (55.9%). In conclusion, our results suggest that tissue invading parasitic infections should always be included in differential diagnosis for patients with eosinophilia associated lesions of the central nervous system, liver, and lungs in Korea.
Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Animals ; Antibodies, Helminth/*blood ; Child ; Child, Preschool ; Clonorchiasis/diagnosis/*epidemiology ; Clonorchis sinensis/immunology/isolation & purification ; Cysticercosis/diagnosis/*epidemiology ; Cysticercus/immunology/isolation & purification ; Diagnosis, Differential ; Enzyme-Linked Immunosorbent Assay ; Eosinophilia/immunology ; Female ; Humans ; Immunoglobulin G/blood ; Infant ; Male ; Middle Aged ; Paragonimiasis/diagnosis/*epidemiology ; Paragonimus westermani/immunology/isolation & purification ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Sparganosis/diagnosis/*epidemiology ; Sparganum/immunology/isolation & purification

Eosinophil degranulation plays a crucial role in tissue inflammatory reactions associated with helminth parasitic nfections and allergic diseases. Paragonimus westermani, a lung fluke causing human paragonimiasis, secretes a large amount of cysteine proteases, which are involved in nutrient uptake, tissue invasion, and modulation of hos's immune responses. There is, however, limited information about the response of eosinophils to direct stimulation by cysteine proteases (CP) secreted by P. westermani. In the present study, we tested whether degranulation and superoxide production from human eosinophils can be induced by stimulation of the 2 CP (27 kDa and 28 kDa) purified from excretory-secretory products (ESP) of P. westermani newly excysted metacercariae (PwNEM). A large quantity of eosinophil-derived neurotoxin (EDN) was detected in the culture supernatant when human eosinophils isolated from the peripheral blood were incubated with the purified 27 kDa CP. Furthermore, the 27 kDa CP induced superoxide anion production by eosinophils in time- and dose-dependent manners. In contrast, the purified 28 kDa CP did not induce superoxide production and degranulation. These findings suggest that the 27 kDa CP secreted by PwNEM induces superoxide production and degranulation of human eosinophils, which may be involved in eosinophil-mediated tissue inflammatory responses during the larval migration in human paragonimiasis.
Animals ; Astacoidea/parasitology ; *Cell Degranulation ; Cysteine Endopeptidases/*immunology/isolation & purification ; Eosinophils/*immunology/metabolism ; Helminth Proteins/*immunology/isolation & purification ; Humans ; Paragonimiasis/*immunology/metabolism ; Paragonimus westermani/*enzymology/isolation & purification ; Superoxides/*immunology

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.
Sequence Alignment ; Recombinant Proteins/biosynthesis/genetics ; Phylogeny ; Paragonimus westermani/*enzymology/genetics ; Molecular Sequence Data ; Hemoglobins/metabolism ; Escherichia coli/enzymology/genetics ; DNA, Complementary/genetics ; Cysteine Endopeptidases/*genetics/immunology/*metabolism ; Astacoidea/parasitology ; Antigens, Helminth/genetics/immunology/metabolism ; Animals ; Amino Acid Sequence

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.
Animals ; Antigens, Helminth/*analysis/isolation & purification ; Chromatography, Ion Exchange ; Immunohistochemistry/methods ; Microscopy, Immunoelectron ; Paragonimus/*immunology ; Support, Non-U.S. Gov't

In order to observe the antigenic fractions in saline extract of adult Paragonimus westermani, proteins in the crude extract were separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) in reducing conditions. The separated protein fractions were transferred to nitrocellulose paper on which 20 sera from human paragonimiasis were reacted and immunoblotted. Out of 15 stained protein bands in SDS-PAGE, 7 reacted with the sera. Of 14 reacted bands, 30 kilodalton(kDa) band was the most frequently reacted (95%) and was a strong antigen. Protein bands of 23 and 46 kDa were also strong antigens. Bands of over 150 kDa, 120 kDa, 92 kDa, 86 kDa, 74 kDa, 62 kDa, 51 kDa, 32 kDa, 28 kDa, 16.5 kDa and 15.5 kDa were also reactive but their frequencies of the reaction were variable.
parasitology-helminth-trematoda ; Paragonimus westermani ; immunology ; antigen ; electrophoresis

Enzyme-linked immunosorbent assay (ELISA) using crude and affinity-purified antigens of adult worms of Paragonimus westermani was performed for infected cat sera with different worm burden, from preinfection to 18th week after infection. Crude antigen was used with supernatant of homogenated worms by freezing-thawing method, and the supernate was centrifuged for l hour at 10,000 rpm at 4C. Affinity-purified antigen (antibody-bound antigen) was prepared from fractions (bound and unbound) of crude antigen by affinity chromatography on CNBr-activated sepharose 4B, and IgG as a ligand was prepared from paragonimiasis cat serum (6 months infected) obtained by ammonium sulfate (40-45 per cent saturated) precipitation method. By SDS-PAGE, crude antigen showed 22 polypeptide fractions while purified antigen showed 4 fractions: 36, 400, 34,700,27,600 and 11,500 in molecular weights. All cats were divided into five groups(G1-G5) by different worm burdens. The mean of recovered worms (+/-SD) and the number of cats in each group are as follows:G1, 2 worms(0) and 4 cats; G2, 4.75(+/-0.66) and eight; G3, 10.75(+/-1.92) and four; G4, 25.20(+/-3.43) and five; G5, 48(+/-12.63) and five cats. The results were summarized as follows: The antibody levels(OD value) increased by worm burden in G1 to G4 generally. However, individual antibody levels were not exactly related with worm burden in all groups, especially there was a wide difference in G4 and G5. These results suggested that the worm burden in G4 (about 20 - 30 worms) is enough to produce antibody maximum in cats of 2~3 kg weight. The antibody levels increased significantly (p<0.05) compared to control sera at the 3rd week in G1 and G2, at the 2nd week in G3, and at the 1st week in G4 and G5. Especially in the 4th week, OD value increased more in G1(p<0.001) and in G2 to G5(p<0.01). In the pattern of antibody levels by ELISA in each group, OD in G1 increased to the 18th week continuously, in G2 OD was maintained same after the 16th week, but in G3 it decresed after the 16th week, and it was maintained same in G4 and G5 after the 14th week. The antibody levels by ELISA with the affinity-purified antigen were higher than those with crude antigen in all groups generally. Especially, the difference of OD values between two antigens was larger from the 4th to the 10th week. In G1 and G2 OD with purified antigen was higher than that with crude one to the 18th week. It was also higher in G3 than that with crude antigen to the 16th week and OD of G4 and G5 were higher before the 14th week than that with crude antigen, however became lower at the 16th week. Consequently, the antibody level in ELISA with affinity-purified antigen was more sensitive at the early weeks after infection and in light infection groups than that with crude antigen.
parasitology-helminth-trematoda ; immunology ; enzyme-linked immunosorbent assay ; Paragonimus westermani ; paragonimiasis ; cat