Circumoval Precipitation Research Articles

Immunodiagnosis and its role in schistosomiasis control in China: a review

Parasitological techniques (stool examination and/or urine filtration) are still the diagnosis of choice in national schistosomiasis control programmes the world over. However, the success of current control efforts, mainly due to the large-scale administration of praziquantel, emphasises the need for a more sensitive approach. In addition, microscopy is labour-intensive and time-consuming, while declining compliance rates after repeated chemotherapy make good coverage for the long-term increasingly problematic. China's success in the control of schistosomiasis is contributing to an enhanced understanding of the need for better and more sensitive screening methods. Immunodiagnostic techniques have a high sensitivity, are easy to perform and are an excellent epidemiological tool for the screening of target populations in schistosome-endemic areas. These assays are also useful for the surveillance of cure after chemotherapy, and for periodic control of transmission of the infection after it has been eliminated in an area. A succinct historical background of using immunodiagnosis for schistosomiasis japonica in China is given, together with a review and evaluation of the relative efficacy of the main techniques applied, i.e. the intradermal test, the circumoval precipitation test, the indirect hemagglutination assay, the dye dipstick immunoassay and different kinds of enzyme-linked immunosorbent assay applications. The important role of immunodiagnosis in the screening for schistosomiasis in China is discussed.

Serological studies on shistosomiasis mansoni in the Northeast Brasil

Sera from the patients (N = 10) with schistosomiasis mansoni of the hospital of Federal University of Pernambuco, the Schistosoma mansoni egg-positive (N = 51) and -negative (N = 452) inhabitants in Cabo City area, out-patients (N = 37) of the IMIP hospital and Japanese immigrants (N = 127) in Petrolina City area of northeast Brazil as well as Japanese healthy subjects (N = 30) were examined by serological tests including an enzyme-linked immunosorbent assay with antigens prepared from eggs (ELISA-egg) and adult worms (ELISA-adult). The ELISA with egg or adult antigen correctly identified 100% of the uninfected individuals lived in non-endemic area of schistosomiasis. Moreover, when examined cross-reactivity of our ELISA with sera isolated from 78 subjects infected with various intestinal parasitic infections, only one of these sera reacted with the egg and adult antigens. On the examination of 51 sera from the egg-positive subjects, the ELISA-egg revealed the highest sensitivity (98.0%), whereas a large number of false negative reactions of ELISA-adult, Ouchterlony method using adult antigen, circumoval precipitation and immediate intradermal skin test were observed. A low sensitivity of these serologic tests except for ELISA-egg appears to be primarily due to their inability to detect antibody in the sera from egg-positive infantiles. There was no positive correlation between the absorbance values of these two types of ELISA among the sera isolated from ELISA-positive subjects. Rather, by the reactivity of these sera to egg or adult antigen, they could be divided into two subgroups; one reacted more positively with egg antigen and the other with adult antigen. Moreover, it was confirmed that the sera from young subjects (under 20 years old) appear to be highly reactive to the egg antigen than did aged ones. These data suggest that the ELISA with egg antigen, but not with the adult antigen, appears to be useful for the serological survey of schistosomiasis mansoni in the endemic area of northeast Brazil.

Serological evaluation of ultrasound examination for chronic schistosomiasis japonica in a previously endemic area—the Chikugo River Basin, Japan

Ultrasound diagnosis of chronic schistosomiasis japonica was assessed by comparison with the results of serological tests using enzyme-linked immunosorbent assay (ELISA) with egg and adult worm antigen, and the circumoval precipitation test. The subjects were persons resident in the Chikugo River Basin in Kyushu, southwest Japan, where schistosomiasis used to be highly prevalent. Of 93 individuals with a past history of infection, 37 (39.8%) presented the characteristic echo pattern of the disease (network, sieve, mottled or mixed pattern). In the serological tests, the ultrasound (US)-positive subjects showed a significantly higher IgG antibody-positive rate in ELISA (81.1% for egg-ELISA), than the US-negative subjects (37.5% for egg-ELISA). Individuals bearing dead Schistosoma japonicum eggs proven by tissue biopsy showed a similar antibody-positive rate (76.5% for egg-ELISA) to that of the US-positive subjects. Sera of a group which had no schistosomiasis characteristic echo pattern, but had liver fibrosis, hepatomegaly or liver cirrhosis as shown by US, were also highly positive (71.4% for egg-ELISA). The present serological studies thus confirmed the usefulness of ultrasound diagnosis for chronic schistosomiasis japonica. Furthermore, we were able to determine certain indications of liver abnormality currently undefined in the normally utilized US classifications.

Competitive radioimmunoassays using hybridoma and anti-idiotype antibodies in identification of antibody responses to, and antigens of, Schistosoma japonicum.

A hybridoma-derived monoclonal antibody, I.134, with apparent specificity for Schistosoma japonicum adult worms has immunodiagnostic potential for schistosomiasis japonica in the Philippines. Sera from known infected individuals will inhibit the binding of 125I-labelled I.134 to a crude adult worm extract (AWE) in a competitive radioimmunoassay (CRIA), although a 10% false negative rate has been noted. Another monoclonal antibody, P.41, which produces circumoval precipitation (COP) reactions with S. japonicum eggs was shown previously not to be useful in identification of heavily infected individuals using a CRIA with extracted egg antigens (EA). However, the 125I-P.41/EA assay has now been demonstrated to be capable of detecting a small subset of infected persons. Thus, four infected individuals with serum COP reactions shown to be consistently of the bleb type only (and with no segmented precipitates seen in the optimized COP test) have serum inhibitory activity in the P.41 CRIA. These same sera are negative in the I.134 CRIA. Unsuccessful attempts have been made to substitute a large pool of affinity-purified anti-I.134 idiotypic (Id) antibodies for antigen (i.e. AWE) in the immunodiagnostic I.134 CRIA. However, anti-Id CRIAs have been shown to be useful in monitoring the isolation of target antigens of hybridoma anti-schistosome antibodies. The availability of a range of hybridoma antibody-based CRIAs will greatly facilitate the quantitative analysis of anti-S. japonicum antibody specificities in sera from clinically defined patient groups and the isolation of antigens of immunoparasitological interest.

Control of schistosomiasis japonica in the Nagatoishi area of Kurume, Japan.

A pilot experiment using NaPCP as a molluscicide to control schistosomiasis japonica was initiated in 1950--51 in Nagatoishi-cho, Kurume, Japan, where 1,050 persons lived behind dikes built to prevent flooding from the Chikugo River. They farmed 60 ha of rice, and 72.9% of them were infected with schistosomiasis. At the end of 2 years, 99.5% of the snails (Oncomelania h. nosophora) were controlled and new cases dropped from 30--35 per year to 5 and 0 at the end of the 1st and 2nd year, respectively. Mollusciciding was continued by Japanese, and paddy irrigation ditches were lined with concrete by 1958. Land reclamation, involving conversion of cultivated land in the river bottom to a golf course, resulted in virtual elimination of infected snails from this area and, when found, snails were uninfected. Using immunologic tests (intradermal, complement fixation, and circumoval precipitation) plus multiple stool examinations in 1972 and 1978, it was possible to demonstrate a marked decrease in cases of schistosomiasis in children 15 years of age or younger. These results were significantly different (P less than 0.001) from those found in similar tests run in 1940 and 1966. By 1978 all children in the Nagatoishi area of Kurume, Japan, were negative for Schistosoma japonicum infection.

Schistosomiasis Mansoni in Baboons

Abstract Six baboons were each repeatedly exposed to a total of 700 cercariae, divided into small (trickle) doses, and four of the six received an additional challenge dose of 1,000 cercariae. The levels of various antibodies were measured over the course of their 3- to nearly 4-year infections by the following assays: enzyme-linked immunosorbent assay (ELISA) with anti-baboon µ- and γ-chain conjugates against a soluble adult worm antigen, slide flocculation with cercarial antigen, circumoval precipitation (COP), and “long-term” passive cutaneous anaphylaxis (PCA) with adult worm antigen. Some additional multiply infected baboons were also followed for opsonizing antibodies to schistosomules and by “short-term” PCA for anti-adult worm IgG reaginic antibodies. With respect to anti-adult response measured by ELISA, IgM class antibodies appeared by 4–6 weeks post-infection (p.i.) and peaked at 7–9 weeks (p.i.) Anti-adult IgM class antibodies were maintained over a long span of time, and at higher levels in multiply exposed baboons, in contrast to what was observed (in the accompanying report) in singly exposed baboons. They showed a tendency to increase with accumulated cercarial exposures. Neither IgM nor IgG anti-adult antibodies showed dramatic post-challenge responses. Flocculating cercarial antibodies were unrelated to anti-adult antibodies and were transient in appearance, confirming their unsuitability for diagnosing active infections. COP antibodies were generally present throughout the infection period. Antibodies capable of opsonizing schistosomules in vitro for complement-mediated, baboon peripheral blood leukocyte adherence and damage were measured in three baboons. Their titers were unrelated to anti-adult IgM or IgG class antibodies or to immunity status. IgE and IgG class anti-adult anaphylactic antibodies, measured by long-term and short-term PCA, respectively, were erratic in appearance and persistence. IgE, but not IgG, anti-adult anaphylactic antibodies were related to dermal hypersensitivity as measured by direct skin tests with adult worm antigen. Cercarial dermatitis occurred in baboons which had had prior exposure to the parasite, and its severity was related to the number of cercariae penetrating the site.

Immunoperoxidase localization by electron microscopy of soluble egg antigen and human IgG in circumoval precipitin reactions around Schistosoma mansoni eggs.

Soluble egg antigen and human IgG were localized by the unlabelled antibody enzyme technique for electron microscopy on Schistosoma mansoni eggs having circumoval precipitin reactions. Reaction products indicating the presence of soluble egg antigen were on portions of the circumoval precipitate outside the egg, between the eggshell and vitelline membrane, and between the vitelline membrane and miracidium inside the egg. Reaction products indicating human IgG were only found on portions of the circumoval precipitate external to the eggshell.

Analysis of the immunoglobulins responsible for the circumoval precipitation reaction.

Two morphologically distinct types of circumoval precipitates (COP) have been observed in human Schistosoma japonicum infections. An elongated segmented COP occurs in chronic human infections. An unsegmented "reaction of recent infection" (RRI) occurs in serum from humans with recently acquired infections and is morphologically similar to the reaction observed in the sera of mice infected with S. japonicum. Sera from infected mice and humans were separated by G-200 chromatography to determine whether the unsegmented "RRI" was due to IgM antibody and the segmented COP reaction due to IgG. There was an elevation of the 19S fraction of sera of mice with 10 and 16 week infections. In addition, the murine 7S fraction was elevated in the 16 week infections. The COP activity was confined to the 7S fraction in the murine sera. Sera from Philippine patients which produced reactions of recent infection (acute sera), segmented COP reactions (chronic sera), and mixed reactions (believed to be from a transition stage between acute and chronic schistosomiasis) were tested. All human sera had elevation of both the 19S and 7S fractions of the acute serum. However, COP-reactive antibodies were confined to the 7S fraction of sera from the transition stage and acute stage infections. The results suggest that although IgM antibodies do in certain cases participate in the COP and produce reactions of recent infection, antibody class is not responsible for the different morphology of this reaction.

The major serological antigen (MSA1) from Schistosoma mansoni eggs is a "circumoval" precipitinogen.

A monoclonal hybridoma antibody to the major serological egg antigen of Schistosoma mansoni (anti-MSA1) reacted with schistosome eggs, forming a circumoval precipitate. This precipitate was seen when anti-MSA1 was incubated with S. mansoni, S. haematobium, and S. japonicum eggs.

Immunization against Schistosoma mansoni in rhesus monkeys and the requirement of activation of both cell-mediated and humoral mechanisms.

When groups of rhesus monkeys were pretreated with BCG plus hyperimmune serum from monkeys with chronic schistosomiasis or with dialyzable transfer factor from uninfected monkeys plus hyperimmune serum and were challenged with 1,500 cercariae of Schistosoma mansoni, the mean worm burdens were significantly lower than that of untreated controls. Pretreatment with neither BCG alone nor Corynebacterium parvum plus a membrane antigen of adult worms of S. mansoni affected susceptibility. Neither lymphocyte proliferation in the presence of mitogens or schistosome antigen nor serological responsiveness (as measured by gel diffusion, Cercarienhüllenreaktion, circumoval precipitation, or enzyme-linked immunoabsorbent assay) correlated with the degree of resistance of the animals to S. mansoni. The pretreatment procedures used did not cause any abnormal histopathological responses and did not alter the characteristic host response to schistosome eggs in the lungs, liver, mesenteric lymph nodes, and colon.

Study of the circumoval precipitate in Schistosomiasis japonica by immunofluorescent technic.

The 48-hour circumoval precipitates formed by infected human and rabbit sera on lyophilized Schistosoma japonicum eggs were stained with the corresponding fluorescein-labelled monospecific goat antisera for study under a fluorescent microscope. The three major Ig classes were found to participate in the formation of the precipitate, with major components being IgG and IgM; IgA participated least. IgE and C3 also contributed to the precipitate. The circumoval precipitate on S. japonicum eggs is therefore a complex of different immunoglobulins reactive with antigen(s) from the egg.

Schistosoma mansoni: Immunity in Macaca mulatta

Twenty-nine rhesus monkeys were used to investigate immunity to Schistosoma mansoni. Infection with 1000 cercariae conferred complete protection to reinfection; this was demonstrated by failure of challenge cercariae to develop into egg-producing adults. Sensitization of monkeys with adult worm extracts did not result in any protection against subsequent exposure. Humoral response was assayed by indirect hemagglutination, cercarienhüllenreaktion, and circumoval precipitation, bentonite flocculation, and complement fixation tests. Infection or sensitization stimulated reactivity in all except the circumoval precipitation test. In this test reactivity was only induced by infection. Reinfection stimulated marked anamnestic responses detectable by all tests except the circumoval precipitation. Skin tests and passive cutaneous anaphylaxis reactions revealed reaginic type antibody in infected monkeys. Delayed-type skin reactivity was not observed. Antilymphocytic serum, Cytoxan and total-body irradiation were unable to alter the actively acquired immunity.

The Hoeppli phenomenon in schistosomiasis. Comparative pathology and immunopathology.

Summary Review of the comparative pathology of the Hoeppli phenomenon shows that it is correlated with substantial egg loads, appears during the later part of the acute primary state of infection, arises around mature eggs, occurs selectively in organs with large egg loads and affects a fraction of eggs not exceeding 10 percent at any time. Immunofluorescent studies in Mastomys coucha show that the Hoeppli corona contains both egg antigen and fixed host globulin, and is therefore an antigen-antibody complex. Within the complex, there is a gradient of antigen-antibody concentration, with antibody excess in the periphery. The precipitate pursues a sequence of maturation and degradation concomitant with the evolution of the pseudotubercle, during which additional host and parasite components may be incorporated into it. The characteristics of the Hoeppli phenomenon in three schistosome species, its relationship to the in vitro circumoval precipitate, and to stellate precipitates in other granulomatous diseases are discussed, and it is postulated that the Hoeppli phenomenon is an auxiliary mechanism of antigen sequestration invoked by the heavily infected host when antigen production against certain egg fractions attains a critical concentration and ratio resulting in precipitation of an antigen-antibody complex in vivo.

Antibody response to immunization with Schistosoma mansoni egg antigen-antibody complex

1. 1. Rabbits were immunized with Schistosoma mansoni egg-precipitate complex (EPC); with eggs incubated in normal human sera (NHS); with clean eggs alone; and with human circumoval positive sera (COS). 2. 2. Immunization with the antigen-antibody complex resulted in the development of antibodies against both the antigen ( S. mansoni egg) and the antibody (human globulin) fractions of the complex. 3. 3. The antibodies formed in response to immunization with EPC however, possessed different biological and serological properties than the antibodies resulting from immunization with COS alone. These differences are visualized by the formation of extra bands of precipitation in agar-gel double diffusion and immunoelectrophoresis; and the highly specific affinity of anti-complex sera towards circumoval precipitate formed on eggs incubated in human COS. 4. 4. The interpretation given to these observations is that changes might have occurred in the antigenicity of the bound heterologous globulin fraction of the EPC as compared to the free globulin in COS. 5. 5. Mention is made of the possibility that homologous antibody may likewise be rendered antigenic following union with antigen, and of the significance such findings would have.

Detection of circumoval precipitins by the fluorescent antibody technic.

Summary1. Whole sera and globulins from the blood of individuals infected with Schistosoma mansoni were labeled with fluorescein isothiocyanate. Eggs of S. mansoni were incubated in these labeled globulins with subsequent formation of circumoval precipitate. Examination of this precipitate by the direct and indirect FA technics revealed a bright greenish-yellow fluorescence. 2. The fluorescent circumoval precipitin technic demonstrated excellent diagnostic performance. Positive reactions were obtained in 30 sera from individuals with known infections. Negative reactions were obtained in 20 sera from individuals not infected with S. mansoni and in the sera of 25 children, heavily parasitized with intestinal helminths, but not with S. mansoni. 3. The sensitivity and specificity of the circumoval reaction, in addition to the advantages introduced by the fluorescent antibody technic, might make the fluorescent circumoval precipitin technic highly reliable for serological diagnosis of schistosomiasis.

Behavior of different geographic strains of Schistosoma japonicum as determined by the circumoval test.

Discussion and conclusionAlthough the circumoval reaction is species specific for S. japonicum, the results of the present studies show that with this test, there were cross reactions among the 4 geographic strains. A significantly larger proportion of eggs showing positive circumoval precipitation, however, was seen when the eggs were incubated with the antisera of the heterologous strains. This indicates that the geographic strains of S. japonicum have already become partly distinct in their serological characteristics.

Electrophoretic serum protein studies in chronic human Manson's schistosomiasis.

Summary The serum proteins of 67 patients with chronic schistosomiasis and of 37 controls were studied by paper electrophoresis. Compared to the controls, slight but significant changes consisting of decrease of serum albumin and increase of gamma globulin were found in the infected group. The changes were more pronounced in patients with hepatosplenomegaly than in those without hepatosplenomegaly, but the differences were not significant. No significant relationship was found between the protein abnormalities and the hepatic functional impairment determined by the bromsulfalein retention test. The protein abnormalities had no relation to previous treatment with antimonial agents. The changes were significantly more marked in patients who had an abnormal cephalin flocculation test than in those whose cephalin flocculation test was normal. The gamma globulin was significantly higher in those sera that produced a large amount of precipitate (2+ to 4+) in the circumoval precipitation test than in those that produced either no or a small amount of precipitate (0 to 1+). However, no correlation between the circumoval precipitation test and the cephalin flocculation test was found. These findings indicate that decrease of serum albumin and increase of serum gamma globulin represent a protein response by the human host to chronic infection with Schistosoma mansoni. In view of the known hepatic involvement which occurs in this disease it would seem reasonable to suspect that the protein changes result from this involvement. However, in the present study they could not be correlated at an acceptable level of confidence either with abnormal bromsulfalein retention or with hepatosplenomegaly. The evidence presented supports the view that these changes are due substantially to an immunologic mechanism. This mechanism probably produces a group of antibodies, one of which is the circumoval precipitin.