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The aim of this study was to describe the CT and sonographic findings of hepatic visceral larva migrans of Toxocara canis.Fifty-four patients (44 men, 10 women; age range, 30-80 years; mean age, 53 years) with serologically confirmed visceral larva migrans of Toxocara canis underwent evaluation of the liver with CT (n = 25), sonography (n = 48), or both. Two radiologists used consensus for retrospective evaluation of CT and sonographic findings. Correlation between the presence and severity of hepatic abnormalities on images and the degree of peripheral eosinophilia was assessed.Seventeen (68%) of 25 patients who underwent CT had single or multiple ill-defined, oval or elongated, small, low-attenuating lesions in the liver. Eighteen (38%) of 48 patients who underwent sonography had single or multiple small, poorly defined, oval or elongated, hypoechoic scattered focal lesions in the liver. In the 19 patients who underwent both CT and sonography, the two techniques had no significant difference in rate of detection of hepatic lesions (p = 0.375, McNemar test). The lesion numbers on CT and sonography showed excellent linear correlation (r = 0.844, p = 0.001) by Pearson's correlation test. An independent samples t test showed that eosinophil count and percentage in the peripheral blood were significantly higher in patients with hepatic lesions on CT and sonography than in patients without lesions.CT and sonographic findings of hepatic visceral larva migrans of T. canis are multiple, ill-defined, oval or elongated, small, nodular lesions scattered in the liver parenchyma. The presence of hepatic lesions on images was associated with higher peripheral eosinophil count and percentage.

The diagnostic applicability of the Clonorchis sinensis recombinant 7-kDa protein was evaluated. In enzyme-linked immunosorbent assays and immunoblots, the protein showed high sensitivities (81.3 and 71.9%, respectively) and specificities (92.6 and 89.7%, respectively) for sera obtained from various helminthic infections. Some paragonimiasis sera showed cross-reactions. The antigen might be valuable in the serodiagnosis of human clonorchiasis.

Purpose The aim of this study was to document the characteristic sonographic findings of clonorchiasis for the diagnosis of active infection in an endemic area. Methods In a village in northeastern China, residents underwent fecal examinations for detection of Clonorchis sinensis eggs. Shortly thereafter, residents were examined with abdominal sonography. An experienced radiologist performed the sonographic examinations and analyzed the findings. Subjects whose fecal examinations were positive for eggs were considered to have active clonorchiasis; those whose examinations were negative for eggs were used as control subjects. The distinguishing sonographic features of active clonorchiasis were identified by stepwise logistic regression analysis. Results The study population comprised 457 subjects; fecal examinations revealed C. sinensis eggs in 316 and no eggs in 141. Four sonographic findings distinguished subjects with active clonorchiasis from control subjects: increased periductal echogenicity (p < 0.001; R = 0.11; sensitivity, 35%; specificity, 91%), floating echogenic foci in the gallbladder (p < 0.001; R = 0.09; sensitivity, 28%; specificity, 94%), diffuse dilatation of the intrahepatic bile ducts (p < 0.01; R = 0.03; sensitivity, 67%; specificity, 48%), and gallbladder distention (p < 0.05; R = 0.02; sensitivity, 3%; specificity, 100%), in decreasing order of significance. Among these 4 sonographic findings, increased periductal echogenicity and floating echogenic foci in the gallbladder were more significantly associated with active infection than were the other 2. Conclusions Increased periductal echogenicity and floating echogenic foci in the gallbladder were identified as the 2 most significant findings for the sonographic diagnosis of active C. sinensis infection. © 2003 Wiley Periodicals, Inc. J Clin Ultrasound 32:17–23, 2004

We screened the genome of a trematode, Clonorchis sinensis, in order to identify novel retrotransposons and thereby provide additional information on retrotransposons for comprehensive phylogenetic study. Considering the vast potential of retrotransposons to generate genetically variable regions among individual genomes, randomly amplified polymorphic DNAs (RAPDs) detected by arbitrarily primed polymerase chain reactions were selected as candidates for retrotransposon-related sequences. From RAPD analysis, we isolated and characterized a novel retrotransposon in C. sinensis as the first member of uncorrupted long-terminal-repeat (LTR) retrotransposons in phylum Platyhelminthes. The retrotransposon, which was named Clonorchis sinensis Retrotransposon 1 (CsRn1), showed a genomewide distribution and had a copy number of more than 100 per haploid genome. CsRn1 encoded an uninterrupted open reading frame (ORF) of 1,304 amino acids, and the deduced ORF exhibited similarities to the pol proteins of Ty3/gypsy-like LTR retrotransposons. The mobile activity of master copies was predicted by sequence analysis and confirmed by the presence of mRNA transcripts. Phylogenetic analysis of Ty3/gypsy-like LTR retrotransposons detected a new clade comprising CsRn1, Kabuki of Bombyx mori, and an uncharacterized element of Drosophila melanogaster. With its high repetitiveness and preserved mobile activity, it is proposed that CsRn1 may play a significant role in the genomic evolution of C. sinensis.

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adultParagonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed inEscherichia coliwas used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.

Neurocysticercosis (NCC) is an important cause of neurological disease worldwide. A 10-kDa antigen of Taenia solium metacestodes (TsMs) has been shown to be specific for immunodiagnosis of NCC. Screening of a TsM complementary DNA (cDNA) library isolated a cDNA encoding this protein. The cloned cDNA contained a 258-bp complete open-reading frame that encodes an 86-amino acid polypeptide with a calculated molecular weight of 9582 Da. It showed 73% homology with a 10-kDa antigen of T. crassiceps. The recombinant protein was expressed bacterially as a fusion protein at a high level. In immunoblot with recombinant protein, 97% (184/190) of sera from patients with active NCC showed strong reactivity, whereas 14% (4/29) of those from patients with chronic calcified NCC reacted weakly. In 180 sera from other patients with parasitic infections and from normal controls, it showed 98% specificity. A single recombinant TsM antigen has a high potential for serological differentiation of active NCC.

Rationale and objectives The authors sought to correlate the radiologic findings of hepatobiliary fascioliasis with pathologic features. Methods Serial ultrasound, computed tomography (CT), and magnetic resonance findings in seven rabbits with experimentally induced fascioliasis were obtained every other week. Direct cholangiogram was also obtained after the rabbits were killed. Radiology-pathology correlation was done in specimens. Results In the parenchymal phase (an acute phase of parenchymal invasion of a larva), CT showed subcapsular clustered areas of low attenuation. Magnetic resonance appearance was similar in shape but better than CT in characterizing the hemorrhagic nature of the lesion. Ultrasound findings were nonspecific in this phase. In the ductal phase (a stationary phase after residing in the bile duct), CT showed dilatation of central ducts with symmetric periportal hypoattenuation (periportal tracking). Magnetic resonance could not depict mild ductal dilatation. Ultrasound was most valuable in demonstrating the moving worm within the dilated duct. Pathologically, the hepatic parenchymal lesions consisted of a cluster of eosinophilic granulomas with hemorrhagic change (migratory tract of the flukes). Ductal changes were observed predominantly in the central bile ducts. Periportal lymphangiectasia was also noted. Conclusions Computed tomography or magnetic resonance can demonstrate the characteristic evolutionary pattern of fascioliasis that reflects the unique life cycle of Fasciola hepatica. The role of ultrasound, although limited in the parenchymal phase, was most useful in the ductal phase in that it demonstrated the moving worms themselves.

In 40 cases of human paragonimiases caused by Paragonimus westermani (20 cases), P. miyazakii (10 cases), and P. skrjabini (10 cases), responses of serum immunoglobulin G (IgG), IgG subclasses, and IgE were analyzed by immunoblotting with crude antigens prepared from egg, 4-week-old juvenile, and adult forms of P. westermani . The 32- and 35-kDa proteins in the adult extracts showed specific reactions regardless of the causative species (39 of 40 cases; 98%). Sera of patients infected with P. westermani and P. miyazakii reacted strongly with the 28-, 46-, and 94-kDa proteins of egg extracts, while those from patients infected with P. skrjabini reacted faintly. No sera from patients with other trematodiases (0 of 15 cases), cestodiases (0 of 20 cases), or lung cancer (0 of 5 cases) or from healthy controls (0 of 10 individuals) showed positive reactions. Analysis by IgG subclass revealed that IgG4 (33 of 40 cases; 83%) and IgG1 (29 of 40 cases; 73%) antibodies in the patient sera recognized the 32- and 35-kDa proteins predominantly. IgG3 reaction was found in 50% (10 of 20 cases) and 30% (3 of 10 cases) of the sera of patients infected with P. westermani and P. miyazakii , respectively. In an IgE immunoblot, 83% (33 of 40 cases) of the sera from paragonimiasis patients reacted with the 32- and 35-kDa proteins while no sera from patients with heterologous diseases and healthy controls showed a positive reaction. Both 32- and 35-kDa proteins in adult extracts of P. westermani were highly reliable for serodiagnosis of human paragonimiases.

In cestode parasites, calcareous corpuscles are thought to be associated with a number of intracellular physiologies by regulating the trafficking of mineral components. We previously separated these particular components by Ficoll, and their binding proteins of 10 kDa and 35 kDa in the Spirometra mansoni plerocercoid (sparganum). In the present study, we purified a 10 kDa protein employing octyl-Sepharose CL-4B affinity chromatography. Anti-serum raised against the purified protein showed specific reactions to the calcareous corpuscles of the worm section by immunohistochemistry, and recognized the 10 kDa protein by immunoblotting.

When immunoglobulin G (IgG) was incubated withSpirometra mansoniplerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, ofMr27 (± 0·8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory–secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5·7 with 0·1 M sodium acetate but was active over the pH range 4·5–8·0. Its activity was inhibited completely by 10−5M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2(5 × 10−5M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted–secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

SUMMARYIn the pathogenesis of sparganosis, proteases have been considered to play important roles in tissue migration and parasite feeding. Several bands of proteolysis were observed when crude extracts ofSpirometra mansoniplerocercoid (sparganum) were examined using gelatin substrate gel at neutral pH, of which two proteases of 198 and 104 kDa were purified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE–anion exchange chromatography. All the purified proteases exhibited optimal activity at pH 7·5 and 0·1 M Tris–HCI. Proteolytic activities at 198 and 104 kDa were inhibited specifically by serine protease inhibitors, and 4-(amidinophenyl)methansulfonyl fluoride (APMSF, 0·5 m) andN-α–p–tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was inhibited byN-tosyl-l-phenylalanine chloromethyl ketone (TPCK, I mM) and chymostatin (0·1 mM), and was potentiated in 10 mM Ca2+which showed that the protease had a chymotrypsin-like property. All the proteases were Schiff(PAS) positive. Proteases of 198 and 104 kDa degraded collagen completely within 24 h. The 36 kDa enzyme cleaved human recombinant interferon-γ (rIFNγ) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody responses in the infected patients.

A mu-class glutathione S-transferase (Cs26GST) of molecular mass 26 kDa was characterized from Clonorchis sinensis. In adult C. sinensis, the distribution of the Cs26GST was investigated by immuno-histochemistry and electron microscopy. Cs26GST was localized to the tegument and parenchyma. Immunogold labeling was strong in the tegumental cell bodies and moderate in the tegument and ova in the oviduct. It is suggested that Cs26GST plays a role in the metabolism and fecundity of C. sinensis.

Fourteen magnetic resonance (MR) images of 11 patients with cerebral sparganosis were reviewed retrospectively and compared with 13 computed tomographic (CT) scans of 10 of the patients. All patients underwent both nonenhanced and contrast material-enhanced MR imaging. All white matter degenerations, which were slightly hypointense on T1-weighted images and were hyperintense on T2-weighted images, were seen as areas of low attenuation on CT scans. Better contrast between normal and degenerated brain tissues was seen at MR, and two lesions were seen that were missed at CT. The parasitic granuloma was isointense to brain parenchyma on images obtained with all pulse sequences. On two follow-up MR images, location or shape of the enhancing lesions changed, suggesting migration of the worm. A new finding of hyperintensity on T1-weighted images and hypointensity on T2-weighted images in subcortical areas probably due to petechial hemorrhages was demonstrated in eight patients only at MR. Small calcific foci, which were important diagnostic clues of cerebral sparganosis, were detected in four patients only at MR, while they were seen in eight patients at CT.

Prevalence survey of neurocysticercosis was made in a mixed epilepsy patients of Changmi Club in Korea. From February 1987 to July 1990, a total of 2,667 randomly selected patients at 27 local centers was tested for their serum levels of anti-Cysticercus antibody (IgG) by enzyme-linked immunosorbent assay. Positive rate of the antibody was 4.0% in the examined patients. The standardized antibody positive rate by provincial population was 3.1%. The rate was the highest in patients living in Cheju Do (8.4%). The patient age brackets of 0 approximately 9 years and over 50-year showed higher positive rates of the antibody. In 750 normal persons who checked up routine physical examination, the antibody positive rate was 2.1% (standardized rate was 1.8%). These seroepidemiological data disclosed for the first time the prevalence of cysticercosis in epileptic patients and in population.

In 1959, the Korean Society for Parasitology was founded by clinical scientists, specialists of public health, and 5 core parasitologists with experience in American science and medicine. The Society this year celebrates its 50th anniversary. Due to public health importance at the time of foundation, medical parasitology was the main stream for next 3 decades. Domestic problems of niche parasitic diseases, unlisted in 6 tropical diseases of major importance, had been studied by own efforts. To cope with the demand of parasite control, evaluation system for control activity was built up. Control activity against soil-transmitted nematodes, conducted for almost 3 decades, was evaluated as a success. Evaluation of praziquantel efficacy for clonorchiasis, paragonimiasis, and neurocysticercosis, population dynamics of Ascaris lumbricoides infection in a situation of continuous reinfections, diagnostic modalities of antibody tests combined with brain imaging developed for helminthiasis of the central nervous system and researches on intestinal trematodes were achievements in the first 30 years. During the recent 2 decades, science researches, such as cell and molecular biology of parasites and immunology of parasitic infections have been studied especially on parasitic allergens and proteolytic and anti-oxidant enzymes. Experiences of international cooperation for world health have been accumulated and would be expanded in the future.

Summary Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue-invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory–secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory–secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans-epoxysuccinyl-l-leuciloamido-(4-guanidino) butane (E-64) inhibited this process dose-dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion–secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host’s tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.

Paragonimus westermani causes inflammatory lung disease in humans. The parasite excretes a host of biologically active molecules, which are thought to be involved in pathophysiological and immunological events during infection. Analyses of the 2-DE protein profiles of the excretory-secretory products (ESP) of adult P. westermani revealed approximately 147 protein spots, at least 15 of which were identified as cysteine proteases (CPs), at pHs between 4.5 and 8.5, and molecular weights (MWs) between 27 and 35 kDa. An additional three CPs (designated as PwCP-3, -8 and -11) were newly recognized by TOF/TOF MS. Their molecular biological information, which shared a high level sequence homology, was elucidated. The majority of the CPs reacted strongly with sera from paragonimiasis patients. When we observed the chronological changes in the antibody responses of the respective CPs against canine sera collected serially at 1, 3, 5, 7, 11 and 14 wk after experimental infection, these molecules exhibited a multiplicity of distinct immune recognition patterns. Our results clearly showed that P. westermani adult ESP were principally composed of excretory-secretory CPs, and that these CPs may exert effects not only on host tissue degradation and nutrient uptake, but also on the immune-regulating cells via synergistic and independent interactions.

This prospective study was performed to evaluate the diagnostic usefulness of sonography in terms of the intensity of Clonorchis sinensis infection. Total 1,384 residents were subjected to this study at an endemic area in China, in which a clonorchiasis control program had been performed. History taking, fecal examination, and sonography were performed, and 87.8% of the subjects were found to be egg positive. Sonography showed intrahepatic bile duct dilatation (IHDD) in 68.4% of the subjects, increased periductal echogenicity (IPDE) in 24.5%, and gallbladder sludge (GBS) in 20.8%. Moreover, the grades of these sonographic findings were higher in subjects with heavier infections as determined by egg counts. IHDD was found to be the most accurate sonographic indicator of any infection degree (76.3), whereas IPDE and GBS were useful only in cases of heavy infection. Subjective symptoms were found to be only poorly correlated with sonographic findings or infection intensity.

Acephalic cysticercus (Ac), a rarely developed multilobulated and nonencysted form of larvalTaenia, causes hydrocephalus or adhesive arachnoiditis in the ventricles and subarachnoidal space that often lead to fatal outcome in affected patients. Ac has been proposed to originate fromT. soliumon the basis of morphological features, while no molecular data supporting the presumption have been available. In the present study, we investigated the immunological properties as well as molecular characteristics of Ac that was obtained surgically from 6 patients. Immunoblotting of the cyst fluid from Ac samples demonstrated the constitutive expression of aT. soliummetacestode (TsM) 10 kDa protein. Specific antibodies against the truncated 10 kDa protein, which appears to be species specific for TsM cysticercosis, were detected in both serum and cerebrospinal fluid samples of Ac patients. Nucleotide sequences of mitochondrial cytochromecoxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes of Ac were almost identical to those ofT. soliumbut differed substantially from those of the otherTaeniaspecies. In phylogenetic analysis, Ac clustered withT. soliumin a well-supported clade. Our results strongly suggest that Ac may have originated fromT. solium.

Bacterially expressed recombinant 10-kDa protein of Taenia solium metacestode (TsM) was previously found to be reliable in the diagnosis of active stage neurocysticercosis (NCC) by immunoblotting but not by ELISA. In this study, we evaluated the diagnostic feasibility of detecting eukaryote-expressed recombinant 10-kDa protein of TsM by ELISA (rTsM10-ELISA) in the serum and cerebrospinal fluid (CSF) from NCC patients. In 45 cases of active NCC, 91.1 and 97.8% cases showed positive reactions for serum and CSF by rTsM10-ELISA. ELISA employing the crude cyst fluid antigen (CF-ELISA) also revealed a similar result. Negligible cross-reactions were observed in serum samples from control subjects and from subjects with other helminthic diseases by rTsM10-ELISA (5/139 cases, 3.6%). By contrast, CF-ELISA demonstrated a high degree of cross-reactivity (24/139, 17.3%) especially from those patients with alveolar and cystic echinococcoses. The overall sensitivity and specificity of rTsM10-ELISA were 94.3 and 96.4%; and those of CF-ELISA were 95.7 and 84.5%, for serum and CSF, respectively. Antibody responses to rTsM10 were detected as early as 3 months after experimental infection of T. solium eggs in pigs. Our results show that ELISA with rTsM10 could be highly applicable in the serodiagnosis of NCC from early stage of infection.

A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6.5 and 7.5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52-76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.

Ferritin is an intracellular protein involved in iron metabolism. A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates. Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center. PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus. Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity. Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture. Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P. westermani lysate. PwYF-1 protein was localized to vitelline follicles and the eggs of P. westermani. Collectively, PwYF-1 protein was identified as a P. westermani yolk ferritin.

In order to investigate the epidemiological situation of intestinal parasite infections in Laos, parasitological surveys were carried out on a national scale including 17 provinces and the Vientiane Municipality. A total of 29,846 stool specimens were collected from primary schoolchildren from May 2000 to June 2002 and examined once with the cellophane thick smear technique. The cumulative egg positive rate for intestinal helminths was 61.9%. By species, the rate for Ascaris lumbricoides was 34.9%, hookworm 19.1%, Trichuris trichiura 25.8%, Opisthorchis viverrini 10.9%, Taenia spp. 0.6% and Hymenolepis spp. 0.2%. The northern mountainous regions such as Phongsaly, Huaphan or Saysomboune Province showed a higher prevalence (over 70%) of soil- transmitted helminths. The regions along the Mekong River such as Khammuane, Saravane or Savannakhet Province showed a higher prevalence (over 20%) of fish-borne parasites. On the other hand, Schistosoma mansoni eggs were detected in 1.7% of schoolchildren only in Champassak Province, a previously endemic area. The highest prevalence was noted in Phongsaly Province (96.0%) and the lowest in Bolikhamxay Province (27.5%). An additional small-scale survey by cellophane anal swab detected Enterobius vermicularis eggs in 35.7% of 451 schoolchildren aged 6-8 years in Khammuane, Vientiane, Champassak Province and the Vientiane Municipality. Meanwhile, the mean blood haemoglobin level of hookworm-infected children was not lower than that of children not infected with hookworm, suggesting that nutritional factors are more important than parasite infection per se. Nevertheless, the above results indicate that a nationwide parasite control project is necessary to reduce possible morbidity due to parasitic diseases in the country.

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.

Taenia solium neurocysticercosis (NCC) represents one of the major public health problems associated with several neurological manifestations worldwide. We previously identified a recombinant 10-kDa protein of T. solium metacestode (CyDA) specific to active NCC. Immunoblottings with sera from active NCC patients and from animals experimentally infected with larval T. solium (pig), T. saginata (pig), T. asiatica (pig), and T. crassiceps (mouse) strongly recognized CyDA, while sera from patients infected only with adult worms did not. Mapping of antigenic sites using deletion mutants revealed that amino acids (aa) residues 30–34, Asn-Met-Thr-Val-Met (NMTVM), reacted only with sera from active stage T. solium cysticercosis cases. Recognition of CyDA aa 30–34 resided almost exclusively in the IgG4 isotype. Competitive immunoprecipitation with synthetic peptides confirmed the specificity of anti-sera for this penta-peptide. These results demonstrated that aa residues NMTVM in CyDA comprise the core sequence for an active stage NCC-related antigenic determinant. Index Descriptors and Abbreviations: Taenia solium metacestode; Cestode; Neurocysticercosis; Specific antigen; Epitope; Larval stage; Genus-/species-specificity; Hydrophobic ligand binding protein; Neurocysticercosis, NCC; T. solium metacestode, TsM; 10-kDa protein of TsM, CyDA; Amino acids, aa; Central nervous system, CNS; Hydrophobic ligand binding protein, HLBP; Cyst fluid, CF; Pooled serum of 10 active NCC patients, serum-pool.

The characteristics of vivax malaria epidemics along the demilitarized zone (DMZ) in the Republic of Korea has been established by the early surveillance data. To further characterize the epidemic, data of civilian patients microscopically diagnosed with malaria from 1995 through 2000 were analyzed in Yonchon-gun (county). Malaria incidence was greater in male civilians > 30 years of age (p < 0.05). The annual parasite index was significantly higher in those living in the administrative areas (Myeon) traversed by DMZ than those living in Myons not traversed by DMZ (p < 0.05). Analysis according to the distance (4 to 14 km) from DMZ showed that people living in villages close to DMZ had higher annual parasite indices than those living in villages remote from DMZ (p for trend < 0.05). Civilians living in Myeons with plains and located in northwestern part of the county had higher annual parasite indices than those living in hilly Myeons located in southeastern part of the county (p for trend < 0.05). These findings suggest that the contraction of vivax malaria is related with night-time outdoor activities, and that the distance from DMZ is a risk factor. In this area, the flying distance of infected vector mosquitos can explain the annually repeating occurrence of civilian cases.

A 28-kDa glutathione S-transferase (Cs28GST) was purified from a Clonorchis sinensis cytosolic fraction through anion-exchange and glutathione-affinity column chromatographies. A monoclonal antibody raised against Cs28GST reacted specifically to the C. sinensis antigen among trematode proteins. A putative peptide of 212 amino residues deduced from a cDNA clone appeared homologous with 28-kDa GST of trematodes, and its secondary structural elements predicted a GSH-binding site. Recombinant Cs28GST showed GST enzyme activity with CDNB substrate and was sensitive to the model inhibitors. The recombinant Cs28GST was antigenically indistinguishable from the native form and was recognized specifically by C. sinensis-infected human sera. The Cs28GST was localized in the tegument and underlying mesenchymal tissues. It is suggested that Cs28GST may play significant physiological roles against bioreactive molecules and be a useful reagent for serodiagnosis of clonorchiasis.

In biliary passages, Clonorchis sinensis causes epithelial hyperplasia and is assumed to promote carcinogenesis. Glutathione S-transferase (GST) is an antioxidant enzyme involved in phase II defense in trematodes. A clone (pcsGSTM1) encoding a GST was identified by screening a C. sinensis cDNA library with a PCR-synthesized cDNA probe. The predicted amino acid sequence encoded by pcsGSTM1 cDNA had a high degree of sequence identity and folding topology similar to the mu-class GSTs. The estimated molecular mass of the protein, 26 kDa, was consistent with an expression by pcsGSTM1 cDNA. The bacterially expressed recombinant csGSTM1 protein possessed an enzymatic GST activity and conjugated GSH to reactive carbonyls of lipid peroxidation. The recombinant csGSTM1 protein did not share antigenic epitope(s) with GSTs of Fasciola hepatica, Paragonimus westermani and Schistosoma japonicum. The csGSTM1 was identified to a mu-class GST in C. sinensis.

A recombinant protein of Paragonimus westermani yolk ferritin was bacterially produced from a previously cloned complementary DNA and was used as an antigen for an enzyme-linked immunosorbent assay (ELISA) against paragonimiasis- and other helminth-infected sera to evaluate its serodiagnostic potential. The ELISA revealed that paragonimiasis westermani had 88.2% sensitivity and 100% specificity. The positive and negative predictive values of the ELISA were calculated to be 100% and 97.1%, respectively. Sera from cats experimentally infected with P. westermani began to produce immunoglobulin G antibodies against the yolk ferritin at 13 weeks after infection, which suggests that the corresponding antigen was derived from the vitellaria in accordance with maturation of P. westermani. These results indicate that the recombinant P. westermani yolk ferritin is a potent serodiagnostic reagent for paragonimiasis westermani from an early stage of the infection.

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.

The 27 kDa cathepsin L-like cysteine protease of Spirometra erinacei plerocercoid is known to play an important function in tissue penetration, nutrient uptake and immune modulation in human sparganosis. In the present study, the expression of this enzyme was examined at different developmental stages of S. erinacei including immature egg, coracidium, plerocercoid in tadpole and rat, and adult. Proteolytic activity against carboxybenzoyl-phenylalanyl-arginyl-7-amino-4-methylcoumarin was detected in the extracts of coracidia and plerocercoid while no activity was observed in those of immature egg and adult. The specific activity in coracidial extracts was lower than that in the plerocercoid. Reverse transcription-polymerase chain reaction and Northern blot analysis demonstrated that the gene was expressed in the coracidium and plerocercoid but not in immature egg and adult. These results suggest that the 27 kDa cysteine protease is only expressed in the stages involving active migration of the parasite in the host tissue.

Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4- and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4- and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment.

The diagnostic value of a 10 kDa subunit of 150 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was evaluated. Immunoblot analysis revealed that most sera from patients with neurocysticercosis recognized the 10 kDa subunit strongly (209/217 cases, 84.6%), while a few sera from individuals with other parasitic diseases including alveolar echinococcosis (AE, 2/20, 10%) and cystic echinococcosis (CE, 2/25, 8%) showed faint reactions. Sera of cases with other parasitic diseases, especially AE and CE, exhibited cross reactions against other bands in CF. Both differential immunoblot and immunoprecipitation analyses showed that the 10 kDa subunit was the most specific to cysticercosis and highly antigenic, whereas other components at 20–40, 64, 95 and 106 kDa in CF were cross-reactive. IgG subclass ELISA and immunoblot demonstrated that both IgG4 and IgG1 reactions were predominant in neurocysticercosis and recognized the 10 kDa.

Diagnosis of early paragonimiasis is difficult because parasitological evidence is not easily obtained. Antibody tests have been proposed as a good substitute for classical diagnostic techniques. Using the crude extracts of Paragonimus westermani eggs, metacercariae, 4- and 7-week juveniles, and 16-week adults as antigens, we observed the early antibody responses. Sera were obtained from 4 experimental cats, fed 50 metacercariae each, at intervals until 13 weeks post-infection. Antibody (IgG) responses were identified by ELISA using extracts of 4-week juveniles, followed by those of 7- and 16-week worms. Antibody responses were minimal against the metacercarial extracts. Antibodies to P. westermani egg extracts were elevated after 10 weeks post-infection. In immunoblot analysis, more than nine protein bands in 4-week juveniles reacted with the early infection sera. Antigenic proteins in adult worms were different from those of juveniles. After four weeks of infection, 32 and 35 kDa bands in the adult extracts were increasingly reactive. Egg specific proteins at 28, 46 and 94 kDa were reactive only after 10 weeks. Antigenic components reacting to the early infection sera changed during the maturation stages of P. westermani; almost all juvenile antigens were replaced by adult antigen components.

Two outbreaks of acute toxoplasmosis involving 8 adult patients in Korea were linked to eating uncooked pork. In the first outbreak, 3 patients developed unilateral chorioretinitis within 3 months of eating a meal consisting of raw spleen and liver of a wild pig. In the second outbreak, 5 of 11 soldiers who ate a meal consisting of raw liver of a domestic pig developed lymphadenopathy. All 8 patients had high levels of IgG Toxoplasma gondii antibodies (> or = 1:1024) in the Sabin-Feldman dye test, modified agglutination test incorporating mercaptoethanol, and latex agglutination test. T. gondii IgM antibodies persisted in these patients for several months. Most patients had a favorable response to anti-T. gondii chemotherapy with pyrimethamine and sulfanomides.

Antigenic domain of major surface protein (p30) of Toxoplasma gondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (GST) fusion proteins. Fragments of p30 gene were as follows: T37, total p30 open reading frame (ORF); S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence: A19, N-terminal 2/3 parts of S28; P19, C-terminal 2/3 of S28; X9, N-terminal 1/3 part of S28; Y10, middle 1/3 of S28; and Z9, C-terminal 1/3 of S28, respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted into GST (26 kDa) expression vector, pGEX-4T-1 to transform into Escherichia coli (JM105 strain). GST fusion proteins were expressed with IPTG induction as 63, 54, 45, 45, 35, 36, and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with GST detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37, S28, and A19 but not those by P18, X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 1/3 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.

Protease activity was identified in crude extracts of Paragonimus westermani eggs which were purified from infected dog lungs, isolated on 14 weeks after metacercarial challenge. The eggs were used after removing possibly contaminated host or worm tissues on their shell surfaces. In the crude egg extracts, high proteolytic activities against carboxybenzoyl-phenylalanyl-arginyl-4-methoxy-beta-naphthylamide (Cbz-phe-arg-MNA) and Azocoll were detected whereas those against succinyl-alanyl-prolyl- phenylalanyl-p-nitroanilide (Suc-ala-pro-phe-pNA) were not revealed. The enzyme exhibited the maximal activity at pH 6. Its activity was inhibited by specific cysteine protease inhibitors, 10(-5) M 1-trans-epoxysuccinylleucylamido (4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA) while potentiated by 6.5-fold in the presence of 2.5 mM dithiothreitol (DTT). When the enzyme was purified partially by Sephacryl S-300 High Resolution gel filtration, it migrated as a single homogeneous band at 35 kDa. The 35 kDa cysteine protease has been recognized neither in the metacercariae nor in the adult. These findings indicated the presence of at least one protease of cathepsin family in immature eggs of P. westermani.

A 56 year-old Korean housewife/farmerlgoat keeper suffered from right upper quadrant pain and fever with chills. In the abdominal sonogram and computerized tomography, multiple, 2-3 cm, irregular shaped cavities were observed in the right lobe of liver. A liver biopsy revealed extensive central necrosis with Characot-Leyden crystals surrounded by palisading histiocytes, eosinophil-rich inflammatory infiltration. Worm was not observed. However, the serologic test for Fusciola-specific IgG antibody by micro-ELISA was positive. Prior antibody levels did not differ and eosionophilia persisted 6 and 16 months after praziquantel treatment although the cavitaxy lesions in the liver disappeared 6 months after the treatment. Reported herein is a human case of invasive fascioliasis diagnosed clinically by a combination of radiological, histopathological and serological studies . key words: Fosciolc sp. , fascioliasis, histology, radiologlr, serology, micro-enzyme linked immunosorbent assay (ELISA) , IgG

When tachyzoites (RH strain) of Toxoplasma gondii are injected intramuscularly, experimental mice survive up to 7 days, 1-2 days longer than those infected intraperitoneally. We observed sequential histopathological changes in inguinal lymph nodes after intramuscular injection of tachyzoites to thighs of specific pathogen free (SPF) mice. Initial findings on 1 or 3 days after the injection were reactive germinal centers, distended sinuses and epithelioid cell clusters in cortical and paracortical regions. Later on 5 days after the injection, however, effacement of nodal structure with depletion of cells and focal necrosis were observed. Necrotizing lymphadenitis in the experimental murine toxoplasmosis suggests the causal relation between T. gondii infection and the human disease.

In the definitive hosts, metacercariae of Parofonimn westermani excyst in host duodenum, penetrate intestinal wall, migrate peritoneal and thoracic cavities, and develop to sexual maturity in 8 weeks. This study was undertaken to examine the age of the maturing p. westernnni when their infectivity to the other definitive hosts was retained. On 3, 7, 10, 14,21 and 28 days after feeding the metacercariae to cats through a gastric tube, the developing worms were harvested. The juveniles of different age were fed again to other experimental cats. One to 12 weeks after the oral-transfer infections, the experimental cats were examined for establishment of infections. In the cats to which 3- day and 7-day old juveniles (grown up to 1.4 mm long) were fed, 31.4% and 22.6% of the transferred worms were found infected. The worms of 10-28 days old were not infective. Early maturing stages grown up to 7 days maintained their infectivity to the other definitive hosts. Key words: Pnrosoninur upestermani metacercariae, juvenile worm, transfer infectivity

A seroepidemiologic observation of anti-Spirometrc erinacei plerocercoid (sparganum) antibody (IgG) in serum was made in normal adult and epileptic patients in Korea from february, 1987 to September, 1990. Sera were tested by enzyme-linked immunosorbent assay (ELISA) for anti-sparganum antibody together with anti-Taenic soEiun metacestode, and anti-Parusonimus westermcni antibodies. Sera reacted positively to sparganum antigen only were considered. Positive rate for anti-sparganum antibody in 850 normal adults was 1.9% (standardized rate by provincial population was 1.7%). In 2,667 randomly selected patients of epilepsy at 28 local centers of the Changmi Club, positive rate was 2.5% (standardized rate: 2.3%). In both normal adult and patient groups, the higher antibody rates were observed in Kangwon and Chonnam Provinces. Positive rates were 10 times higher in male than in female in normal adults and 4.5 times in male epileptic patients. The rates were elevated especially with age over 30-year. odd ratio of the antibody was 1.32 which indicated an ambiguous etiologic factor for epilepsy Key words: Sparganum, sparganosis, seroepidemiology, epilepsy

We report the case of a 28-year-old male who had been ill with headache and motor weakness for a month and developed sudden pain and blindness of the right eye. During ophthalmoscopy, a worm was recognized penetrating the iris, occupying the anterior chamber for a brief period, returning back behind the iris, and leaving corneal edema with hyphyema. Enucleation was performed to prevent the worm's escape from the eye. The enucleated eye revealed areas of a focal degeneration of sclera and intraocular hemorrhage. Microscopic findings of an abrupt tissue defect and few inflammatory reactions in the uvea suggested very recent migration of a moving worm. The flatworm detected in the anterior chamber was identified to be a juvenile Fasciola sp. This case is presumed to be the first case of intraocular fascioliasis reported in the literature.

Diagnosis of cerebral paragonimiasis in its early active stage is important because curative chemotherapy is possible. Accordingly, this study was undertaken to evaluate the CT and MR features of early active cerebral paragonimiasis.We retrospectively reviewed the CT scans (n = 29) and MR images (n = 7) of the brain in 20 patients between 7 and 59 years old who had cerebral paragonimiasis in the early active stage. The diagnosis was based on a positive antibody test by enzyme-linked immunosorbent assay (ELISA) for paragonimiasis in serum and CSF. The diagnosis was confirmed surgically in seven patients.The most common and characteristic imaging finding was a conglomerate of ringlike enhancing lesions (grape-cluster appearance) with surrounding edema in one cerebral hemisphere in 11 patients (55%). Other nonspecific findings included a solitary ringlike lesion (n = 4) or irregular enhancing lesions (n = 2), localized hemorrhage with (n = 3) or without (n = 2) enhancing lesions, and a poorly defined non-hemorrhagic, nonenhancing lesion (n = 1).CT and MR findings of clustered ring-enhancing lesions, seen in approximately half of the cases of early cerebral paragonimiasis, are suggestive of cerebral paragonimiasis.

To ascertain that tegument of Paragonimus westermani has specific antigenic proteins, the tegumental fraction was isolated from 10-month-old worms by 0.1% digitonin solution, and subjected to SDS-PAGE and immunoblot. Component proteins of tegumental syncytium comprised of 94, 74 (76-66), 62, 54, 44, 42, 38, 28, 26, 25, 24, 17, 15.5 and 13. 5 kDa proteins. Of them, the 94, 44 and 42 kDa proteins were more specific to tegument, especially the 94 kDa protein was the most prevailing one. In immunoblot, antigens of the 94, 90, 78, 76, 74, 68, 65, 62, 60, 59 and 54 kDa proteins were commonly detected by 7 sera of 10 human paragonimiasis, but none of them reacted with 5 sera of clonorchiasis. In conclusion, the 94 kDa protein was the major tegumental protein, as well as the specific antigen. The 76 and 66 kDa proteins were the minor components of tegument, which were also specific antigens of P. westermani.

Specific antibody test in serum and cerebrospinal fluid (CSF) is still the main mode of serological diagnosis of cysticercosis. Of different techniques of antibody test, enzyme-linked immunosorbent assay (micro-ELISA) has widely been applied. This study was undertaken to observe whether diagnostic capability can be improved by applying more sensitive techniques such as Protein A-ELISA and avidin biotin complex ELISA (ABC-ELISA). When evaluated using 115 sera of human cysticercosis, the antibody positive rates were not significantly improved in Protein A-ELISA (82.6%) and in ABC-ELISA (86.1%) than in micro-ELISA (81.7%). The specificities, evaluated in 165 sera from other diseases and normal controls, were significantly improved (88.5% by micro-ELISA, 93.3% by Protein A-ELISA and 93.8% by ABC-ELISA). Antibody levels (absorbance, abs.) in individual serum were correlated well (r = 0.83-0.86) each other. An actual benefit of Protein A-ELISA and ABC-ELISA was that they needed smaller amount of test sample.