Quantitative determination of circulating antigens in human schistosomiasis mansoni using an indirect hemagglutination assay

In serum and urine specimens collected from a group of Schistosoma mansoni infected individuals from Makundju, Zaire, the schistosome circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA) were quantitatively determined using an indirect hemagglutination reaction with sheep erythrocytes sensitized with mouse IgM monoclonal antibodies directed against these circulating antigens. Levels of CAA in serum (up to 5 ng/ml) and CCA in serum and urine (up to 50 ng/ml) were strongly correlated with egg excretion and with each other. No correlation was found between egg excretion and antibody levels against the circulating antigens. Antigen was detectable only in patients excreting greater than 500 eggs per gram of feces.     

Circulating anodic and cathodic antigen in serum and urine of mixed Schistosoma haematobium and S. mansoni infections in Office du Niger, Mali

Summary In Office du Niger, an area endemic for both Schistosoma haematobium and S. mansoni in Mali, circulating anodic (CAA) and cathodic (CCA) antigen detection assays were performed on pre-treatment serum and urine samples from two villages, Rigandé and Siguivoucée, and compared with egg counting methods. The highest prevalence was obtained with the urine-CCA assay which also had the highest sensitivity to S. haematobium, S. mansoni or mixed infection. A single urine-CCA assay was as sensitive as repeated egg counts (one stool + two urine examinations per individual). When the different assays were tested in parallel, several combinations including assays on serum were found to be highly sensitive. As urine sampling is widely accepted, urine assays will be used for further monitoring these villages one and two years after chemotherapy.     

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.     

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.     

Circulating schistosomal antigen in diagnosis and assessment of cure in individuals infected with Schistosoma mansoni.

The effectiveness of praziquantel in treating schistosomiasis is most commonly assessed by quantitating egg production or anti-schistosome antibodies in serum. We have used a monoclonal antibody (MAb)-based antigen-capture enzyme-linked immunosorbent assay (ELISA) for the serologic diagnosis of schistosomiasis, and to monitor the efficacy of praziquantel therapy in 49 individuals with parasitologically proven schistosomiasis. The MAb used, 128C3/3/21, recognizes a repeating carbohydrate epitope expressed at all stages of parasite development, and antibodies recognizing this epitope are found in the serum of infected humans. The overall sensitivity of the ELISA was 78%, with a sensitivity of 100% for patients excreting greater than 100 eggs/g of feces and 72% for those excreting less than 100 eggs/g of feces. The positivity of the ELISA was directly related to the fecal egg counts obtained on days -3, -2, and -1 before treatment with praziquantel, but there was no correlation between antigen levels and the clinical stage of the disease. After praziquantel treatment, we observed a highly significant correlation (P less than 0.0001) between the time elapsed since treatment and the decrease in antigenemia. Furthermore, although no eggs were detected in any of the stool specimens at week 12 after treatment, the antigen was detected in 21% of the treated patients (seven of 33 ELISA-positive patients). Antigen levels decreased over the 12-week period in six of these patients, whereas the antigen level increased with time in one individual. The persistence of antigenemia suggests that these individuals are either still clearing antigen or remain infected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of a circulating antigen in human schistosomiasis japonica using a monoclonal antibody

A double antibody enzyme-linked immunosorbent assay (sandwich ELISA) was used for the detection of a circulating antigen from human schistosomiasis japonica infections. This assay involves the use of polyclonal rabbit Schistosoma japonicum soluble egg antigen (SEA) antiserum to bind circulating antigen and a monoclonal antibody (MabH4) to identify and quantify this antigen. Sera from 108 S. japonicum-infected patients (acute and chronic) were tested. Sera from 93 of 95 patients with chronic infection were positive for this antigen; sera from 12 of 13 patients with acute infections were also positive. Antigen was not detectable in control human sera. Sera from 35 chronic schistosomiasis patients were collected 6-12 months after praziquantel treatment. Circulating antigen was not detectable in the sera of 33 of these patients and was dramatically reduced in 2. This ELISA system may prove valuable in differentiating past and current infections.     

Praziquantel for treatment of schistosomiasis in patients with advanced hepatosplenomegaly

We evaluated praziquantel for therapy of active Schistosoma mansoni infection in 15 rural Egyptian males with hepatosplenic schistosomiasis. Criteria for inclusion in this study were two pre-treatment S. mansoni egg counts with a mean of greater than 100 eggs g-1 faeces and an enlarged spleen. Fourteen of 15 patients had hepatomegaly, five had ascites, and six had serum albumin below 3 g dl-1. Schistosoma haematobium infection (less than 10 eggs ml-1 urine) was present in three patients. Praziquantel was administered in a single oral dose of 30 mg kg-1 body weight. Eight of the 15 patients (53%) had mild and transient reactions in the form of fever (usually one day), gastrointestinal symptoms, headache and skin rash. Criteria for parasitological cure were the absence of live eggs in two stool samples and a negative rectal snip biopsy three months after therapy. Ten patients ceased to pass live eggs (cure rate 67%). For the five who were still passing live eggs there was a mean egg reduction of 95%. The three patients with S. haematobium demonstrated parasitological cures. We conclude that praziquantel is an effective and well tolerated drug for treatment of S. mansoni infection in patients with advanced hepatosplenic schistosomiasis, and it is the drug of choice for patients with coexisting S. haematobium infection.     

Detection, quantitation, and specificity of antiparasite IgE antibodies in human schistosomiasis mansoni

Sera from 15 patients with acute or chronic Schistosoma mansoni infection were evaluated for IgE antibodies directed against soluble cercarial, adult worm, and egg antigens. Both the antigen-induced release of histamine from passively sensitized human basophils and specific radioimmunoassays were used to detect these IgE antibodies, and determination of serum IgE levels before and after specific immunosorption permitted their quantification. While chronically infected patients made IgE antibodies to all three stages of the parasite, only egg antigens induced an appreciable IgE antibody response in acutely infected individuals. Despite the fact that patients with chronic infection had significantly greater levels of total serum IgE than patients with acute disease, the percentage of this IgE that was parasite specific was similar for both groups, ranging between 4% and 28%. An ancillary observation was the fact that soluble egg antigen can trigger basophil histamine release through IgE-dependent reactions and through "nonimmunologic" mechanisms that require further characterization.     

The intensity and effects of infection with Schistosoma mansoni in a rural community in northeast Brazil.

The intensity of infection with Schistosoma mansoni and its effects were investigated in a defined population living on three contiguous fazendas (subcounties) in a nonmalarious area of northeast Brazil near Salvador, Bahia. Quantitative stool egg counts (Bell technique) were performed on 363 of 417 individuals (90%) of all ages; physical examinations were done on 294 of 357 individuals (82%) 5 years of age and older. The maximum increase in prevalence was observed between the 1- to 4- and 5- to 9-year age groups, while the maximum increase in fecal egg count occurred between 5- to 9- and 10- to 14-year age groups. Highest egg counts were observed in the 10- to 14-year age group (geometric mean of 301 eggs per ml of stool) while the maximum prevalence (100%) was in the 20- to 24-year age group. In the fazenda with the lowest quantitative egg counts the age specific prevalence rates increased more slowly than in the fazendas with higher egg counts. In the study group nearly 50% of the total fecal egg output was accounted for by 22 individuals (6%) who had a mean age of 12.6 years. Egg counts for this selected group were all over 800 eggs per ml of stool with a mean of 1,514 eggs per ml of stool. In children under 15 years of age, the frequency of hepatomegaly and splenomegaly varied directly with the egg count; further, the degree of hepatomegaly was directly correlated with increasing egg counts. No splenic enlargement was noted in children not excreting eggs. In adults, on the other hand, neither splenomegaly nor hepatomegaly could be directly related to schistosomal infection per se. In children, neither the presence of infection with S. mansoni nor its intensity was reflected by altered anthropometric measurements. In the one fazenda tested the frequency of stools positive for occult blood correlated with increasing S. mansoni egg counts.     

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.     

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.     

Safety and immunogenicity of a recombinant hepatitis B vaccine in patients infected with Schistosoma mansoni

Thirty Egyptian males, 8-31 years of age, with active Schistosoma mansoni infection and negative serologic tests for hepatitis B markers, were vaccinated with a recombinant hepatitis B vaccine (Merck's Recombivax). The vaccine was given intramuscularly in the deltoid region in three 10 micrograms doses at 0, 1, and 6 months. All patients were treated with praziquantel 2 months after the first vaccination. Sera were collected every 2 months for 12 months and tested for anti-HBs using a quantitative ELISA technique. There were no side reactions except for mild local soreness at the injection site in 3 patients. At 12 months, all subjects seroconverted (antibody levels greater than 10 mIU/mL); 24 patients (80%) developed antibody levels greater than 1,000 mIU/mL. As with a plasma-derived vaccine, antibody levels were negatively correlated with increasing spleen size. A recombinant hepatitis B vaccine was safe and immunogenic when given to patients with schistosomiasis mansoni.     

The epidemiology of a recent focus of mixed Schistosoma haematobium and Schistosoma mansoni infections around the 'Lac de Guiers' in the Senegal River Basin, Senegal

A village with mixed Schistosoma mansoni and S. haematobium infections (probably in a early endemic phase) was identified around the Lac de Guiers in the Senegal River Basin. In documenting the epidemiology of both schistosomes, we focused on prevalence and intensity of infection, transmission patterns and the impact of treatment. S. mansoni prevalences (near 100%) and egg counts (overall geometric mean eggs per gram of faeces (epg) of 589 were high in all age groups, with 35% of individuals excreting > 1000 epg, and showing a slow decline in egg output only after the age of 30 years. The overall prevalence (28%) and egg counts (2% > 50 eggs/10 ml) of S. haematobium were low, with mean counts of 6.3 eggs/10 ml. Maximal mean S. mansoni egg counts were found in 5-9 year-old boys and in 15-19 year-old girls; S. haematobium maximal counts in 1-4 year-old boys and in girls aged 5-9. Extremely high Biomphalaria pfeifferi infection ratios were recorded over the whole year. Following a single treatment, re-infection was rapid with prevalences and mean egg counts of both Schistosoma species reaching pretreatment levels within 7 months.     

The unreliability of the Kato-Katz technique limits its usefulness for evaluating S. mansoni infections

The Kato-Katz technique, a (semi) quantitative stool examination technique, is generally recommended for diagnosis and evaluation of Schistosoma mansoni infection by schistosome experts. However, egg counts are subject to important variability. In order to quantify the reproducibility of egg counts using the Kato-Katz technique, field data of 1255 observations on 299 subjects infected with Schistosoma mansoni were analysed. Agreement between repeated observations was assessed both categorically (kappa statistic) and continuously (analysis of variance). The day-to-day variation of egg counts was much greater than the variation due to different observers or different slides. The quantitative reproducibility was low: the weighted kappa statistic was 0.39 between specimens of different days, 0.62 between slides of the same specimen and 0.81 between observers of the same slide. Therefore the classification of individual patients into groups based on egg counts, used as a measure of morbidity, must be interpreted with great care, especially in longitudinal studies. Usefulness of the Kato-Katz technique appears limited. Its reproducibility is low. It cannot be recommended as a routine test in a primary health care setting or in a hospital laboratory because safety and detection of other parasites are better assured by other techniques. It can be used in epidemiological studies and evaluation of schistosomiasis control programmes, but here too, other techniques might be preferred.     

Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies

From a panel of mouse monoclonal antibodies reactive with a repeating epitope of the schistosome circulating anodic antigen, an IgG1 monoclonal antibody was selected. This monoclonal antibody was applied in a sandwich enzyme-linked immunosorbent assay as capture antibody and as alkaline phosphatase labeled conjugate. This assay allowed a sensitive quantitation of circulating anodic antigen in serum samples of infected individuals, detecting less than 1 ng antigen/ml serum. In Schistosoma mansoni infected individuals from Zaire, the level of antigen in serum correlated with fecal egg output. The lower detection level of the immunoassay corresponded to a level of about 10 eggs/gm feces.     

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.     

Application of immunodiagnostic assays: detection of antibodies and circulating antigens in human schistosomiasis and correlation with clinical findings.

In an initial cross-sectional survey, serum, urine, and stool samples were collected from 370 participants representing about 10% of the population (n = 4,438) in Behbeet village, 50 km south of Cairo, Egypt, an area well known to be endemic solely for Schistosoma haematobium. Diagnosis was approached in two parallel ways. The first approach, which simulated actual conditions in many endemic areas in Egypt, was based on physical examination and urine and stool microscopic analysis. The second approach was based on two advanced immunodiagnostic assay systems. One system detected antibodies to species-specific microsomal antigens, the other detected circulating schistosomal antigens. Microsomal antigens from S. haematobium and S. mansoni were used to detect antibodies in the Falcon assay screening test (FAST)-ELISA and the enzyme-linked immunoelectrotransfer blot (EITB). Circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were quantified in serum and urine samples in a sandwich ELISA using monoclonal antibodies. Parasitologically, the prevalence of S. haematobium was 7.01% in females and 25.82% in males, giving an overall prevalence of 15.8%. The combination of urine CCA and serum CAA for detecting circulating antigens and the combination of the S. haematobium adult worm microsomal antigens (HAMA) FAST-ELISA and the HAMA EITB for detecting antibodies significantly improved the sensitivity of detecting S. haematobium circulating antigens and antibodies. Also, including a medical examination as an integral part of field studies and correlating immunodiagnostic results with other clinical and investigational data allowed us to calculate an accurate estimation of S. haematobium prevalence in this area of low endemicity.     

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.     

Presence of the schistosome circulating anodic antigen (CAA) in urine of patients with Schistosoma mansoni or S. haematobium infections

We investigated the presence of the circulating anodic antigen (CAA) in the urine of schistosomiasis patients. This genus specific antigen was hitherto demonstrated only in the serum of schistosomiasis patients. The urine of 80 patients with Schistosoma mansoni infections, 33 patients with S. haematobium infections, and 2 patients with mixed S. haematobium and S. mansoni infections were screened by a quantitative enzyme-linked immunosorbent assay (ELISA). CAA was demonstrated in 81% of those with intestinal schistosomiasis and in 97% of those with urinary schistosomiasis. CAA titers were less than 1:0.2-1:51.2. Results were compared with circulating cathodic antigen (CCA) titers in urine obtained in an indirect hemagglutination assay (IHA). CCA was generally not detectable in the urine of patients with S. haematobium infection, but was demonstrated in the urine of 85% of the patients with S. mansoni infection. Both CAA titers and CCA titers correlated positively with the number of S. mansoni eggs excreted in the feces, but CAA titers did not show a significant correlation with the number of S. haematobium eggs in urine. Both antigen titers showed a moderate correlation with the serum CAA level in schistosomiasis mansoni. The discovery of CAA in the urine of the majority of schistosomiasis patients tested suggests the use of urine samples for non-invasive immunodiagnosis of the disease.     

Immunodiagnosis of infection with Schistosoma mansoni: enzyme-linked immunosorbent assay for detection of antibody to circulating antigen.

A circulating antigen, a negatively charged polysaccharide from the trematode Schistosoma mansoni, was noncovalently bound to the surface of poly(L-lysine)-coated wells in polystyrene trays, which were then used in a micro-enzyme-linked immunosorbent assay (ELISA) test. The method provides an immunodiagnostic test for schistosomiasis of exceptional sensitivity with a high degree of specificity. Comparison of Bell egg counts and ELISA titers revealed a good correlation (r congruent to 0.80) in young individuals with low to moderate worm burdens, but this relationship was less marked in older individuals or those with high egg counts.