Seropositivity and identification of paramyosin for sparganosis in the Kangwon and Incheon provinces of the Republic of Korea.

Sparganosis is one of the top three tissue-dwelling heterologous helminthic diseases, along with cysticercosis and paragonimiasis, in Korea. Due to a lack of effective early diagnosis and treatment methods, this parasitic disease is regarded as a public health threat. This study evaluated reactivity, against sparganum extracts, of sera from inhabitants of Cheorwon-gun, Goseong-gun and Ongjin-gun in Korea. The sera from 836 subjects were subjected to enzyme-linked immunosorbent assay and immunoblot analysis. The sera from 18 (5.8%) and 15 (5.1%) inhabitants in Cheorwon-gun (n = 312) and Goseong-gun (n = 294), respectively, exhibited highly positive reactions to the sparganum antigen, whereas only two (0.9%) inhabitants in Ongjin-gun (n = 230) showed positivity. We sought antigenic proteins for serodiagnosis of positive sera by immunoproteomic approaches. Total sparganum lysates were separated by two-dimensional electrophoresis and then subjected to immunoblot analysis with mixed sparganosis-positive sera. We found seven antigenic spots and identified paramyosin as an antigenic protein by liquid chromatography-mass spectrometry. By two-dimensional (2D)-based mass analysis and immunoblotting against sparganosis-positive sera, paramyosin was identified as a candidate antigen for serodiagnosis of sparganosis.

Serodiagnosis of sparganosis by ELISA using recombinant cysteine protease of Spirometra erinaceieuropaei spargana.

The Spirometra erinaceieuropaei cysteine protease (SeCP) gene encoding a 36 kDa protein was expressed in Escherichia coli, and the potential of recombinant SeCP protein (rSeCP) as an antigen for the serodiagnosis of sparganosis was investigated by ELISA and compared with those of ELISA with sparganum excretory-secretory (ES) antigens. The sensitivity of rSeCP-ELISA and ES-ELISA was 96.67 % (29/30) and 100 % (30/30) respectively, for the detection of anti-sparganum IgG antibodies in sera of the experimentally infected mice (P > 0.05), and the specificities of both ELISA were 100 % (77/77). In heavily, moderately, and lightly infected mice (five, three, and one larvae per mouse), anti-sparganum antibodies were firstly detected by rSeCP-ELISA at 10-12 days post-infection (dpi), respectively, and then continued to increase with a detection rate of 100 % at 14-22 dpi. In three groups of infected mice, the anti-sparganum antibody levels at different times after infection were statistically different (P < 0.05). The results showed that the rSeCP might be a potential candidate antigen for early and specific serodiagnosis of sparganosis. But, it needs to be further evaluated with sera of the patients with sparganosis and other helminthiasis.

Identification of early diagnostic antigens from Spirometra erinaceieuropaei sparganum soluble proteins using immunoproteomics.

In order to identify early specific diagnostic antigens of Spirometra erinaceieuropaei (syn. S. erinacei or S. mansoni) sparganum, soluble proteins were analyzed by two-dimensional electrophoresis (2-DE) and western blotting probed with immune sera from infected mice at 14 days post-infection. From a total of approximately 462 proteins spots mainly distributed in pI range of 5-6.6 and with molecular mass of 25-48 kDa, 6 immuno-reactive protein spots with molecular mass of 31.8-38.3 kDa were characterized by MALDI-TOF/TOF-MS. Three proteins were identified as S. erinaceieuropaei cysteine protease, Toxoplasma gondii hypothetical protein and Pecten spp actin, while the remaining were unidentified. The cysteine protease from S. erinaceieuropaei soluble proteins recognized by early infection sera might be developed as diagnostic reagent for early detection of sparganosis.

A case of inguinal sparganosis mimicking myeloid sarcoma.

We report here a case of inguinal sparganosis, initially regarded as myeloid sarcoma, diagnosed in a patient undergone allogeneic hematopoietic transplantation (HSCT). A 56-year-old male patient having myelodysplastic syndrome was treated with allogeneic HSCT after myeloablative conditioning regimen. At day 5 post-HSCT, the patient complained of a painless palpable mass on the left scrotum and inguinal area. Pelvic magnetic resonance imaging and computed tomography revealed suspected myeloid sarcoma. Gun-biopsy was performed, and the result revealed eosinophilic infiltrations without malignancy. Subsequent serologic IgG antibody test was positive for sparganum. Excisional biopsy as a therapeutic diagnosis was done, and the diagnosis of sparganosis was confirmed eventually. This is the first report of sparganosis after allogeneic HSCT mimicking myeloid sarcoma, giving a lesson that the physicians have to consider the possibility of sparganosis in this clinical situation and perform adequate diagnostic and therapeutic approaches.

Serodiagnosis of experimental sparganum infections of mice and human sparganosis by ELISA using ES antigens of Spirometra mansoni spargana.

We conducted a study of serodiagnosis of experimental sparganum infections of mice and human sparganosis by enzyme-linked immunosorbent assay (ELISA) using excretory-secretory (ES) antigens of Spirometra mansoni spargana and compared the sensitivity and specificity of crude and ES antigens for detecting the specific anti-sparganum IgG antibodies. By crude antigen ELISA and ES antigen ELISA, anti-sparganum IgG was detected in all of 30 serum samples of the infected mice; no cross-reactions were observed in serum samples of the mice infected with Trichinella spiralis, Schistosoma japanicum, Toxoplasma gondii, and normal mice. Anti-sparganum IgG was detected by ES antigen ELISA in sera of mice infected with one, two, four, six, and eight spargana at 3 weeks post-infection (wpi), with a detection rate of 100%, and lasted to 18 wpi when the experiment was ended. The difference in anti-sparganum antibody levels among five groups of the infected mice was statistically significant (F=245.296, p<0.05); the antibody levels were correlated with infecting doses of spargana (r=0.323, p<0.05). The sensitivity of both ELISA in detecting the serum samples of patients with sparganosis was 100% (20/20), but 96.72% (59/61) of specificity of ES antigen ELISA in detecting serum samples of patients with cysticercosis, echinococcosis, paragonimiosis, clonorchiosis, and schistosomiasis, and healthy persons was significantly greater than 72.13% (44/61) of crude antigen ELISA (χ (2) = 14.027, p<0.05). Our finding indicates that ELISA using ES antigens of S. mansoni spargana may be applied to the specific early serodiagnosis of sparganosis.

Seroprevalence of tissue invading parasitic infections diagnosed by ELISA in Korea.

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.

Sparganum mansoni parasitic infection in the lung showing a nodule.

Reported herein is a 57-year-old man infected by Sparganum mansoni, a kind of tapeworm, showing a solitary nodule of the middle lobe of the right lung. Because a transbronchial biopsy could not diagnose the nodule, a right middle lobectomy was performed on suspicion of malignant tumor. The lesion was diagnosed as sparganosis by histological and immuno-serological examinations. Histological examination revealed granulomatous inflammation with neutrophil and eosinophil infiltration around the worm and interstitial pneumonia surrounding the nodule. Moreover, vasculitis with foreign body giant cell was seen around the lesion. To the authors' knowledge this is the second case of sparganosis limited in the lung, and the current report presents the first detailed histological description of a pulmonary sparganosis case.

Eyelid sparganosis.

PURPOSE: To report a case of sparganosis in the muscle layer of the eyelid. DESIGN: Observational case report. METHODS: A 67-year-old man with migratory painful swelling on the eyelid that was unresponsive to medications was evaluated and treated surgically. RESULTS: Computed tomography showed a 1.5 x 1.5-cm sized, thick-walled lesion and ultrasound biomicroscopy showed hypoechoic tubular and cystic lesions. During surgery, a white, thread-like plerocercoid, 7 cm in length was detected in the orbicularis muscle of the lower eyelid. Histopathologic examination demonstrated the characteristic feature of the sparganum larva and foreign body granulomatous reaction. Serodiagnosis by enzyme-linked immunosorbent assay was positive. Three months postoperatively, the lesion resolved completely. CONCLUSION: Although rare, sparganosis should be suspected in a moving eyelid mass unresponsive to the medical treatment.

Enzymatic N-glycan analysis of 31 kDa molecule in plerocercoid of Spirometra mansoni (sparganum) and its antigenicity after chemical oxidation.

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

A case of breast sparganosis.

A 29-year-old Korean woman visited the Department of Surgery in MizMedi Hospital with a palpable itching mass on the right breast that had existed for the past 7 months. She had no history to eat either frogs or snakes, but had the history of drinking impure water. Sonography revealed a serpiginous hypoechoic tubular structure associated with partial fat necrosis in breast parenchymal layer and subcutaneous fat layer. It also revealed oval cystic lesions. At operation, an ivory white opaque ribbon-like worm that measured 16.5 cm in length and 0.5 cm in width was extracted. Anti-sparganum specific serum IgG level in the patient's serum (absorbance = 0.71), measured by ELISA, was found to be significantly higher than those of normal controls (cut off point = 0.21). Sonography and ELISA appear to be helpful to diagnose sparganosis. Breast sparganosis is rarely found throughout the world.

A neutral cysteine protease of Spirometra mansoni plerocercoid invoking an IgE response.

When crude extracts of Spirometra mansoni plerocercoid (sparganum) were analysed by SDS-polyacrylamide gel electrophoresis (PAGE)/immunoblot using patients' sera, IgE antibodies reacted specifically with 21, 27 and 53 kDa proteins. The 21 and 27 kDa proteins have been previously characterized as cysteine proteases. In this study, the 53 kDa protein was confirmed, by immunoprecipitation, to induce a specific IgE response. The protein was purified by affinity chromatography using an IgG1 (kappa 2) type mAb. The protein was partially sensitive to peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (endo F) digestion. It exhibited an endoproteinase activity in a thiol-dependent manner preferentially degrading benzoyloxycarboxyl-phenylalanyl-arginyl-4- methoxy-beta-naphthylamide (Z-phe-arg-MNA) of a panel of substrates tested. This endoprotease activity was maximal at pH 6.5 and in 0.1 M sodium phosphate. The proteolytic activity was inhibited by 10(-5) M L-trans-epoxysuccinyl-L- leucylamido-(4-guanidino)butane (E-64) and 1 mM iodoacetamide (IAA), and potentiated by dithiothreitol (DTT, 5 mM).

Cleavage of immunoglobulin G by excretory-secretory cathepsin S-like protease of Spirometra mansoni plerocercoid.

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (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, of M(r) 27 (+/- 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(-5) M 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 x 10(-5) M). 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.

Immunodiagnosis of human sparganosis mansoni by micro-chemiluminescence enzyme-linked immunosorbent assay.

We compared a microcolorimetric enzyme-linked immunosorbent assay (colorimetric ELISA) and a microchemiluminescence enzyme-linked immunosorbent assay (chemiluminescence ELISA) for the detection of specific immunoglobulin G (IgG) in the serum of 9 patients with sparganosis mansoni and 9 healthy controls. The chemiluminescence ELISA was able to measure serum levels of specific IgG over a far wider range than the colorimetric assay, and its detection limit was at least 10-fold lower. An additional 5 sera from sparganosis patients and 5 more from healthy controls, together with sera from 28 patients with other parasitic diseases, were also examined by the chemiluminescence ELISA. All 14 patients with sparaganosis mansoni showed high levels of chemiluminescence (21,302 +/- 18,907 counts per second [cps]). All sera from the 14 healthy controls (1580 +/- 569 cps) and sera from 27 of the 28 patients with other parasites (4 with taeniasis saginata [1767 +/- 501 cps], 11 with diphyllobothriasis latum [1479 +/- 501 cps], 13 with cysticercosis cellulosae [2376 +/- 1437 cps]) showed chemiluminescence levels lower than those of any of the sparganosis mansoni patients. The exception was a patient with cysticercosis (5980 cps), who may have had a dual infection with Cysticercus cellulosae and Sparganum mansoni. Thus, the chemiluminescence ELISA demonstrated high sensitivity and specificity for human sparganosis mansoni.

Serodiagnosis of human sparganosis by a monoclonal antibody-based competition ELISA.

Competition ELISA test using sparganum specific monoclonal antibodies (Mab) was investigated to improve the diagnostic specificity of sparganosis. By cell fusion, one hybridoma clone secreting anti-sparganum specific Mab was selected (Sp-20), which reacted on bands of 32 kDa and 38 kDa. Sp-20 reacted on calcium corpuscles on IFA. By micro-ELISA, 16 of 17 sparganosis cases (95%) were found positive, but 1 of 18 clonorchiasis cases (5%), 4 of 16 cysticercosis cases (25%) and 2 of 16 normal controls (11%) showed false positive reactions. On the other hand, by competition ELISA using a sparganum specific Mab (Sp-20), 16 out of 17 (95%) of sparganosis cases were found positive, but 2 of 18 clonorchiasis cases (10%), 2 of 16 cysticercosis cases (12%), 3 of 16 paragonimiasis cases (18%) and 1 of 16 normal controls (6%) showed false positive reactions.

Human proliferative sparganosis in Venezuela: report of a case.

A case of human proliferative sparganosis, the first in the southern hemisphere, is reported. The patient had multiple nodular, papular, acne-like lesions, gynecomastia, and large subcutaneous abscesses. Laboratory findings showed that he also had severe anemia of the type associated with infection and chronic diseases, leukocytosis, eosinophilia, and severe hypoalbuminemia and hypergammaglobulinemia. His cellular and humoral immune responses were unaltered. New aspects on the functional repercussion of the parasitism of the human host by Sparganum proliferum are discussed. Treatment with mebendazole was ineffective; the patient's tolerance for the newer drug praziquantel was extremely poor.