日本血吸虫和曼氏血吸虫成虫31/32kD蛋白的纯化及在诊断中的应用

本研究以前染蛋白为指示,将制备型SDS—PAGE和电渗析技术相结合,分离、纯化日本血吸虫和曼氏血吸虫成虫31/32kD蛋白,用于ELISA中诊断血吸虫病。结果:纯化日本血吸虫成虫31/32kD蛋白(Psj31/32)与急、慢性日本血吸虫病和曼氏血吸虫病人血清反应的阳性率分别为100％、100％和98.4％;纯化曼氏血吸虫成虫31/32kD蛋白(Psm31/32)与上述病人血清反应的阳性率均为100％。与NHS和其它寄生虫病人血清反应的特异性较高。Psj31/32和Psm31/32检测同种血清,它们的OD值具有高度的正相关关系,但OD值与曼氏血吸虫病人粪便中虫卵数量(EPG)无相关关系。结果提示:两种纯化的31/32kD蛋白在血吸虫病诊断中具有较高的敏感性和特异性,并具有共同抗原决定簇存在,不仅可用于诊断急、慢性日本血吸虫病,而且Psj31/32也可作为诊断曼氏血吸虫病的候选抗原,用于我国输入性曼氏血吸虫病的诊断。     

Characterization of three types of Schistosoma mansoni soluble egg antigen and determination of their immunodiagnostic potential by western blot immunoassay

On the search of highly sensitive and specific antigenic components for use in serological tests, the serologic activities of the various protein fractions of three types of Schistosoma mansoni soluble egg antigen (SEA) were compared in an immunoblot analysis for their ability to detect schistosomiasis mansoni infections . Three types of soluble egg antigen (SEA) were prepared from three suspensions of Schistosoma mansoni eggs; namely living SEA (L-SEA), dead SEA (D-SEA) and mixed SEA (M-SEA). The three antigens were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A total of 80 Egyptian individuals were enrolled in the present study. After being screened by clinical examination, urine and stool analysis, sigmoidoscopy rectal snip examination, abdominal ultrasonography and indirect haemagglutination test (IHAT), the study population were grouped into an active intestinal schistosomiasis group (group I, n=20), a schistosomiasis seropositive group by IHA test (group II, n=20), a parasite control group including 10 patients with hydatidosis & 10 patients with fascioliasis (group III, n = 20) and a normal control group (group IV, n=20). Sera of all subjects were studied by immunoblotting for the presence of IgG antibodies against the various protein fractions of the three prepared types of SEA. Several protein bands from the 3 types of SEA reacted with the schistosomiasis patients' sera in a heterogenous manner. However, a 31-32 kilo daltons (kDa) protein fraction of L-SEA reacted with 80% (16/20) of group I sera, 40% (8/20) of group II sera, one hydatidosis serum, but no reaction occurred with normal sera. Also, in the active intestinal schistosomiasis group, the 31-32 kDa fraction of L-SEA was more recognized by patients with early active intestinal schistosomiasis without organomegaly (100%, 12/12) than in those with organomegaly (50%, 4/8) (P < 0.05). On the other hand, a 80-82 kDa band of M-SEA was recognized by 70% (14/20) of group 1, 30% (6/20) of group II & sera from 3 hydatidosis and 2 fascioliasis cases, but not by normal human sera. So, it can be concluded that the 31-32 kDa protein fraction of L-SEA is highly immunogenic, with the least cross reaction with other parasitic infections, and may be a useful serologic marker for diagnosing and differentiating between early and chronic schistosomiasis mansoni infection.     

胶体染料试纸条试剂盒检测曼氏血吸虫病的初步研究

目的探索日本血吸虫胶体染料试纸条试剂盒(DDIA)用于检测曼氏血吸虫病的可行性。方法采用日本血吸虫DDIA检测从埃及尼罗河地区曼氏血吸虫病流行区采集的曼氏血吸虫病人血清及当地健康人血清。结果34份曼氏血吸虫病人血清DDIA均呈阳性,14份当地健康者血清均为阴性。与用曼氏血吸虫IHA和ELISA法检测结果相同。结论日本血吸虫DDIA也可用于检测曼氏血吸虫病。     

Efficacy of sodium metaperiodate (SMP)-ELISA for the serodiagnosis of schistosomiasis mekongi

Schistosomiasis mekongi is an important public health issue in endemic countries. In this study, we evaluated an indirect immunodiagnostic ELISA method using Schistosoma mekongi soluble egg antigen. Sodium metaperiodate (SMP)-ELISA was utilized in order to remove the glycosylated epitopes responsible for false positive reactions and the results using this method were compared with those using conventional ELISA (conv-ELISA). Forty-two serum samples from schistosomiasis mekongi egg-positive patients and 100 serum samples from schistosomiasis-negative Cambodian subjects were tested using both ELISA methods. The ranges of ELISA values for positive and negative sera were distinct on SMP-ELISA, but the ranges of the two groups of sera overlapped on conv-ELISA. Therefore, diagnostic criteria may be established based on the highest ELISA value on negative sera and the lowest ELISA value on positive sera. In the present study, both the sensitivity and specificity of SMP-ELISA reached 100% using the criteria in which an ELISA value > or = 0.2 was positive.     

Serodiagnosis of human intestinal schistosomiasis

We developed an enzyme linked-immunosorbent assay (ELISA) for serodiagnosis of Schistosoma mansoni infection using a purified immunogenic fraction from schistosome adult worm, obtained by SDS-polyacrylamid gel electrophoresis. Sera from patients with active schistosomiasis (egg passers; n=10); inactive schistosomiasis previously treated with praziquantel (not passing eggs; n=10); fascioliasis, hydatosis (n=5); and healthy controls (n=10) were examined. Western blot analysis revealed that the Sm 31/32 KDa fraction of Schistosoma mansoni is recognized by sera from of both active and inactive schistosomiasis. ELISA IgG reactivity (optical density, OD) to Sm 31/32 KDa fraction by ELISA was significantly higher in sera of schistosomiasis patients (active and inactive), (p<0.001) compared to normal controls, while no significant difference was detected between active (OD=0.79 +/- 0.23) & inactive (OD=0.87 +/- 0.37) patients. No reactivity was detected using facioliasis or hydatosis sera. The overall level of specificity and sensitivity attained was 90% and 93%, respectively. It is concluded that the developed Sm 31/32 KDa ELISA may be of value in serodiagnosis of active and inactive intestinal Schistosoma mansoni infection in humans.     

Serological differences between acute and chronic schistosomiasis mansoni detected by enzyme-linked immunosorbent assay (ELISA).

Sera from patients with acute and chronic schistosomiasis mansoni, and from laboratory-infected monkeys, were examined by an enzyme-linked immunosorbent assay technique using antigens prepared from eggs, cercariae, and adult worms. Sera from patients with acute schistosomiasis and from monkeys 2 months post-infection reacted more positively to cercarial antigen than to adult worm antigen whereas sera from both patients with chronic schistosomiasis and monkeys infected for longer than 4 months reacted more positively to adult worm antigen. These differential responses to antigen serologically differentiated between acute and chronic schistosome infections.     

Diagnosis of human schistosomiasis by detection of circulating cathodic antigen with a monoclonal antibody.

Monoclonal antibody (MAb) 5H11 reacted with repeating epitopes on Schistosoma mansoni circulating cathodic antigen (CCA) and detected CCA in sera of Egyptian S. mansoni-infected patients. MAb 5H11 was both capture and biotinylated detection antibody in a sandwich ELISA of trichloroacetic acid-pretreated serum samples. Sera of patients with 7-500 eggs/g of stool were positive by MAb 5H11-CCA sandwich ELISA. Stool egg counts and CCA serum levels correlated (r = .52), and CCA levels decreased by 4 weeks after praziquantel treatment in patients with pretreatment egg counts of greater than or equal to 50/g of stool (P less than .05). Sera of Schistosoma haematobium-infected patients, uninfected individuals, and most patients with other helminths were negative in this assay. The MAb 5H11-CCA sandwich ELISA appears sensitive and specific for immunodetection of active schistosomiasis mansoni and useful for monitoring its chemotherapy.     

Biological implications of the phenotypic plasticity in the Schistosoma mansoni-Nectomys squamipes model

The water-rat Nectomys squamipes is mostly important non-human host in schistosomiasis mansoni transmission in Brazil, due to its susceptibility, high abundance and water-contact pattern. During experimental infection of N. squamipes with Schistosoma mansoni, adult worms show phenotypic plasticity. This finding led us to investigate whether biological behavior is also affected. This was assessed comparing the biological characteristics of four S. mansoni strains: BE (State of Belém do Pará), CE (State of Pernambuco), CMO (State of Rio Grande do Norte) and SJ (State of S?o Paulo) using laboratory-bred N. squamipes. The infection was monitored by determination of the pre-patent period, fecal egg output, egg viability, intestinal egg count and, infectivity rate. No biological modification was observed in these parameters. Overall results highlight that N. squamipes was susceptible to several S. mansoni strains, suggesting that it might contribute to the maintenance of schistosomiasis mansoni in Brazil.     

Familial resemblance in humoral immune response to defined and crude Schistosoma mansoni antigens in an endemic area in Brazil.

This study addressed whether the humoral immune response to crude and defined Schistosoma mansoni antigens aggregates within families. The sample included 155 siblings from 42 nuclear families in Brazil. Sera examined by ELISA for antibody isotypes reactive to defined schistosome antigens and crude schistosome antigens (soluble adult worm antigen preparation and soluble egg antigen) demonstrated that there was a difference in sibling-pair correlations between defined and crude S. mansoni antigens. In contrast to the finding with crude antigens, egg-positive sibling pairs showed significant familial resemblance for all IgG subclasses and IgE to adult-stage antigens Smp20.8 and Smp50. Only the IgE and IgG4 isotypes showed familial resemblance to the egg-stage antigen, Smp40. Egg-negative sibling pairs showed significant familial resemblance only for IgE and IgG4 to Smp40. That both the IgE and IgG4 response to defined S. mansoni antigens showed familial resemblance is interesting in light of the converging evidence for the role of IgE and IgG4 in human susceptibility and resistance to reinfection.     

Emergence of infections with Schistosoma mansoni in the Dhofar Governorate, Oman

Infections with Schistosoma mansoni were identified in an area of Dhofar (Oman), where this parasite had been virtually absent during recent years and was reported only very sporadically before 1992. In the present survey, performed late in 2001, between 1 and 13% of children (n=519) were found to excrete eggs (one Kato-Katz-smear) in four schools, from four different villages, but no infections were detected in additional five schools (n=281). Infections were light (<72 eggs/g of faeces) in 19 of the 36 children found infected. Serologic examination of sera (n=511) was done by ELISA (based on soluble worm antigen) and immunofluorescence tests (IFT, based on cryostat sections of adult S. mansoni). The prevalence according to serological tests was between 3 and 43% in the four schools with infected children. Positive test results were taken to reflect active infections, since false positive reactions could largely be excluded. According to ultrasound (US) examinations performed on 96 individuals (children and adults) from the four villages, livers were normal in all except three cases of mild pathology, which could be assigned to schistosomiasis mansoni (pattern C, ages 32-40 years). All data suggest that transmission of S. mansoni has been re-introduced only recently in Dhofar and that this emergence of schistosomiasis is limited to at most a few foci.     

Identification of a genus-specific Schistosoma mansoni soluble egg antigen reactive with the serum of infected patients

The ability of serum samples obtained from humans with schistosomiasis mansoni to recognize Schistosoma mansoni soluble egg antigens (SmSEA) was assessed by the enzyme-linked immunotransfer blot (EITB) method. The sera from 15 infected patients before and 2 years after treatment with oxamniquine (n = 30) recognized bands ranging in molecular weight from 9 to 200 Kd. A 31 Kd component of SmSEA was recognized by all infection sera, but not by normal human serum. The sera from 2 humans infected with S. haematobium and 2 with S. japonicum also recognized the 31 Kd band present in SmSEA, whereas those from 2 humans infected with Fasciola hepatica did not. The 31 Kd antigen was isolated by electrophoresing SmSEA through a 15% SDS-acrylamide gel, followed by excision and electroelution of the 31 Kd band. The purified 31 Kd was used in conjunction with ELISA at a concentration of 1 microgram/ml to confirm the apparent genus specificity of this protein. Thus, the sera of patients infected with S. mansoni, S. haematobium, or S. japonicum were clearly reactive in ELISA, whereas normal human serum or sera from patients with F. hepatica were negative. In conclusion, the 31 Kd protein appears to be a good candidate for developing a screening assay for the immunodiagnosis of schistosomiasis.     

Enzyme immunoassay of the level of parasite-specific IgE antibodies in schistosomiasis using the monoclonal antibody BL-IgE 9

Sera from patients with chronic schistosomiasis (Schistosoma mansoni or Schistosoma haematobium) were examined for the presence of parasite specific IgE antibodies by means of ELISA technique using tegument antigen prepared from adult worms of Schistosoma mansoni and using the monoclonal antibody BL-IgE 9. Individuals from tropical countries who had no schistosomiasis and blood donors from GDR were studied for comparison. Significantly higher levels of specific IgE antibody were given by sera from patients with schistosomiasis than by the controls. These differential responses serologically differentiated between patients with chronic schistosome infections and noninfected individuals.     

Immunodiagnostic tests for bilharziasis. IV. Demonstration of antibody in blood and serum eluates collected on filter paper discs (author's transl)]

Serum and blood samples dried on filter paper discs and normal sera were collected from 142 Liberians with proven schistosomiasis and 25 Liberians without schistosomiasis. These samples were tested by the indirect hemagglutination test (IHA) employing lyophilized sheep erythrocytes sensitized with either cercarial or adult Schistosoma mansoni antigens. The following results were obtained: Out of the 142 sera collected from the schistosomiasis patients 130 (91.5%) reacted with the cercarial antigen and only 89 (62.6%) reacted with the adult S. mansoni antigen. None of the 25 sera from persons free of schistosomiasis reacted with either antigen. The sensitized lyophilized erythrocytes showed no loss of antibody activity after laboratory storage at 4 degrees C for 4 months which was interrupted twice for 16 hours each during shipment. Filter paper serum and blood samples were stored at -25 degrees C up to 84 days with 16 hours interruption during shipment. 77.6% of the eluates from blood showed the same antibody titer levels as in normal serum; 10% showed lower antibody titers and 11.5% had negative results compared with normal serum. Results obtained with eluates from dried serum were somewhat better. An additional storage of the dried blood specimens for ten days at room temperature resulted in a rise of the proportion of negative titers from 11.5% to 34.1%. This loss of antibody activity was found in two thirds of the samples with low antibody titers. The results indicate that blood and serum samples dried on filter paper platelets should be stored at low temperatures (-25 degrees C) to preserve the antibody response.     

The detection of antibodies against Schistosoma mansoni soluble egg antigens (SEA) and CEF6 in ELISA, before and after chemotherapy

Circulating IgG antibody reactivity and excreted egg counts were investigated in 489 Kenyans given chemotherapy for schistosomiasis mansoni. Antibody reactivity was measured in ELISA, using either unfractionated aqueous soluble constituents of Schistosoma mansoni eggs (SEA) or CEF6 (a soluble fraction of S. mansoni eggs containing two cationic antigens) as the antigen source. Antibody reactivity for each antigen source was strongly associated with egg counts, both pre- and post-treatment. Approximately 6 months after chemotherapy, egg counts were zero in 84% of the subjects. The mean optical densities (OD) measured in the post-treatment ELISA were 60% (CEF6) or 45% (SEA) lower than the pre-treatment values, the reduction in the OD with CEF6 as antigen source being significantly greater than that observed with SEA (P <0.001). The usefulness of an assay for antibody reactivity in monitoring the effects of the treatment of schistosomiasis is discussed.     

Schistosoma mansoni cationic egg antigens (CEF6): immunoserology with oxamniquine-treated patients and involvement of CEF6 in the circumoval precipitin reaction

The serologic activity of a cationic fraction (denoted CEF6) of Schistosoma mansoni soluble egg antigen was compared in an ELISA with that of the unpurified soluble egg antigen for the ability to detect human infections and for the prediction of chemotherapeutic success in patients followed up to 5 years post-treatment with oxamniquine. The cationic fraction correctly identified 100% of 20 patients as infected with S. mansoni; moreover, 50% (10 of 20) seroconverted to negative by 2 years post-treatment and 100% of 15 patients tested were negative 5 years post-treatment. In general, the cationic fraction was superior to the unpurified soluble egg antigen for the detection of infection and for the prediction of chemotherapeutic success. The cationic fraction also exhibited greater immunologic specificity over the unpurified soluble egg antigen in that the latter exhibited higher titers than the former to antibodies against heterologous parasite antigens (Fasciola hepatica, Clonorchis sinensis, Paragonimus westermani adult worms; Trichinella spiralis larvae). Moreover, rabbit antisera raised against egg antigens isolated from the precipitation formed when fresh S. mansoni eggs are incubated with S. mansoni infection of immunization sera (known as circumoval precipitin reactions or COP) reacted strongly with the cationic fraction in ELISA. In addition, antiserum to the cationic fraction as well as antisera against either of the two antigenic components of this fraction, known as antigens alpha 1 and omega 1, all give positive COP reactions when incubated with fresh S. mansoni eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the enzyme linked immunosorbent assay (ELISA) test for Schistosoma mansoni infections

Extensive studies of the use of the ELISA test for the detection of antibodies in Schistosoma mansoni infections are described. A method has been evolved for the determination of the optimum value for the reference serum endpoint. In chimpanzees infected with S. mansoni a crude egg antigen detected antibodies earlier in the infection than did a worm antigen and was generally more reactive. The ELISA test, using the egg antigen, has been applied to sera from populations infected with S. mansoni, with other human schistosomes, or with helminth infections other than schistosomiasis. The ELISA test was as sensitive as the IFAT and CFT, but more specific. However, many cross-reactions occurred in infections with other human (and with animal) schistosomes, although to a lesser extent with other helminths. In surveys in the Sudan the use of blood collected on absorbent paper was only slightly less sensitive for the detection of antibodies than sera, and this technique showed that the prevalence of infection was higher than that measured by stool examination alone. Possible future developments are discussed with a view to improving sensitivity and specificity both for clinical and epidemiological work.     

Serodiagnosis of Schistosoma mansoni infections in an endemic area of Burkina Faso: performance of several immunological tests with different parasite antigens

The performance of indirect haemagglutination assays (IHA), enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescent antibody tests (IFAT) were compared with 450 sera from a Schistosoma mansoni-endemic area in Burkina Faso. All participants in this survey provided at least one sample each of stool, urine and serum. From those with an egg-negative Kato-Katz thick smear, a second stool sample was examined. IHA was based on either extracts of adult S. mansoni worms (SmIHA) or S. japonicum egg antigen (SjIHA). For ELISA, three antigen preparations were used, namely: (i) soluble S. mansoni adult worm antigens (SWAP); (ii) soluble S. mansoni egg antigens (SEA); and (iii) a cationic exchange fraction of S. mansoni eggs (CEF6). IFAT was performed with S. mansoni male worm sections. Among the egg-excretors, the sensitivity of ELISA was high and egg antigens performed slightly better (SEA, 96%; CEF6, 97%) than worm antigen (94%). Sensitivity of IHA was satisfactory with homologous (Sm, >85%), but not heterologous (Sj, 56%) parasite antigen. In IFAT, the parenchyma-associated fluorescence showed high sensitivity (95%), but gut-associated fluorescence, which is known to be a sensitive diagnostic marker for schistosome-infected European travelers, was observed only in 76% of a sub-sample of 100 of the endemic sera. Among sera from egg-negative individuals, many gave positive reactions in several or all of the tests employed. These reactions (formally "false positive") are considered to represent true infections, since chemotherapy had not yet been delivered to this population. For the purpose of further surveys in Burkina Faso or other resource-poor settings, we suggest IHA as an accurate diagnostic test and propose to further improve its performance by including egg rather than worm antigens.     

Idiotypic/anti-idiotypic interactions in schistosomiasis and Chagas' disease

Immunoaffinity-purified antibodies against soluble Schistosoma mansoni egg antigens (SEA) were isolated from the sera of patients with schistosomiasis mansoni. Similarly, antibodies against Trypanosoma cruzi epimastigote antigens were obtained from sera of patients with Chagas' disease. These antibody preparations were used in culture to demonstrate the presence of anti-idiotypic T lymphocytes in peripheral blood mononuclear cell preparations from patients with either schistosomiasis mansoni or Chagas' disease, or with both of these infections. Only cells from patients with schistosomiasis or both infections proliferated upon exposure to the anti-SEA antibodies. Conversely, only cells from patients with Chagas' disease or both infections responded to anti-epimastigote antibodies. Western blot analysis of SEA and epimastigote antigens, developed by patients' sera or by immunoaffinity-purified antibody preparations, substantiated that anti-SEA immunoaffinity-purified antibodies only reacted with components of SEA, and anti-epimastigote immunoaffinity-purified antibodies only reacted with components of epimastigote antigenic preparation. These studies demonstrate the presence of anti-idiotypic T lymphocytes in the peripheral blood of patients with schistosomiasis or Chagas' disease which are specific for idiotypes generated during these infections.     

Isolation and characterization of Schistosoma haematobium egg antigens

Schistosoma haematobium soluble egg antigen (ShSEA) was prepared from eggs isolated from the livers of hamsters or mice infected for at least 3 months. Immunoaffinity purified S. haematobium egg antigens (ShSh) were isolated by first passing ShSEA through a column containing anti-S. mansoni hamster IgG coupled to CNBr-activated Sepharose 4B, and recycling the unbound fraction until no more bound material could be eluted with an acid wash. The unbound fraction was then filtered through a second antibody affinity column containing anti-S. haematobium hamster IgG, and in the acid eluate the ShSh antigens were obtained. This antigenic preparation was shown by PAGE to contain at least 6 distinct bands ranging in molecular weight (Mr) from 116 to less than 31 Kd. A 40 Kd polypeptide was identified by both silver staining and EITB as specific for S. haematobium eggs. In addition, a 55 Kd worm-egg shared antigen was identified as a prominent band in EITB expressed during a primary S. haematobium hamster infection. The sera from hamsters harboring patent S. haematobium or S. mansoni infections were reacted by ELISA with ShSh antigens. The anti-Sh sera showed significantly higher absorbance values than the anti-Sm sera, demonstrating that only a minor population of S. mansoni cross-reactive egg antigens is still present in the ShSh antigens. Sera collected weekly for 13 weeks from hamsters with a primary infection of S. haematobium were then tested by ELISA against ShSh, ShSEA and SmSEA antigens. Antibody levels against both ShSEA and SmSEA were shown to increase early in infection (2 weeks). Moreover, antibody levels to ShSh did not increase until week 5 post-infection. These findings suggest that the purification procedure utilized results in the elimination of most of the S. mansoni worm antigens cross-reactive with S. haematobium eggs. The ShSh antigens had shown a high degree of sensitivity and stage-species specificity also suggesting their potential as antigens for the immunodiagnosis of schistosomiasis haematobia.     

The enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of schistosomiasis.

The enzyme-linked immunosorbent assay (ELISA) done with soluble egg antigens (SEA) of Schistosoma mansoni was utilized for the detection of infections with schistosomes. The method readily detected experimental infections in NIH outbred, New Zealand black, and New Zealand white mice by 6-10 weeks post-exposure to S. mansoni cercariae. In addition, the test was negative when the sera from 11 Puerto Rican normal controls were examined and was positive in 8 of 10 serum samples from humans with schistosomiasis mansoni. However, extensive cross-reactivity was seen when using serum from humans with fascioliasis, trichinosis, cysticercosis, and echinococcosis. Thus the ELISA test done with SEA as antigen lacks immunologic specificity. For the method to be an effective seroepidemiological tool in areas where these parasites are endemic further purification of the antigen and more extensive understanding of its components are needed.