A.M. Deelder

Immunodiagnosis of schistosomiasis by determination of the circulating antigens CAA and CCA, in particular in individuals with recent or light infections

In the present paper, we evaluate determination of circulating anodic (CAA) and cathodic (CCA) antigen for the diagnosis of an active Schistosoma infection in humans, in comparison to the diagnostic performance of parasitological examination and the demonstration of specific antibodies. Illustrated by three different studies, which all deal with the diagnosis of either recent or low intensity infections, we further discuss our experiences with these diagnostic methods. For the diagnosis of recent infections, specific antibody determination showed to be very sensitive, particularly in individuals originating from non-endemic areas. For the assessment of cure and for the diagnosis of active infections in endemic areas, the methods of choice are parasitological examination and CAA or CCA determination. Depending on infection levels of the target population and on logistic conditions, CAA and CCA determination may either replace parasitological examination or, in the case of light infections, may be used as a complementary diagnostic tool.

Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters

In this study the presence of two circulating schistosome derived antigens, probably both polysaccharides, was demonstrated in hamsters heavily infected with Schistosoma mansoni. One antigen was an anodic, heat-stable, high molecular weight substance; it was demonstrated in serum, adult worm antigen and in the excretory and secretory products of adult worms. The antigen was demonstrated in the epithelial cells of the schistosome gut. A second antigen, cathodic, heat-stable and a low-molecular weight substance (MW < 30,000), was demonstrated in hamster serum, hamster urine, adult worm antigen, and in the excretory and secretory products of adult worms. Two additional schistosome derived antigens, both heat-labile, were demonstrated in hamster urine.

Schistosoma mansoni: characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse, hamster, and human infections.

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.

Detection of circulating anodic antigen by ELISA for seroepidemiology of schistosomiasis mansoni

Sera of individuals from Burundi excreting eggs of Schistosoma mansoni (prevalence 35%; 178 subjects) and of similar individuals from Maniema, Zaire (prevalence 95%; 99 subjects), and of 159 Dutch and 81 Zairean non-infected controls, were screened by enzyme-linked immunosorbent assay for the presence of schistosome circulating anodic antigen (CAA). No false positive results were obtained. The sensitivity of the test was 75% in Burundi and 93% in Zaire, a significant difference (P less than 0.05). However, in matched egg output classes the test results did not differ significantly; 60% and 67%, respectively, of those excreting 1-100 eggs per gram of faeces (epg), 86% and 100% of those excreting 101-400 epg, and 100% of those excreting over 400 epg were detected. The efficiency of the assay was 91% in Burundi and 93% in Zaire. The Spearman rank coefficient of correlation between antigen titre and egg output (determined by 3 consecutive Kato egg counts) was 0.61 in Burundi and 0.82 in Zaire. The sensitivity of the test compared well with a single egg count. In addition, preliminary data showed that occasionally CAA was detectable in serum of individuals not excreting schistosome eggs. As CAA is found only in the presence of living worms, such cases reflect active infections.

Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies

We have developed an enzyme immunoassay (ELISA) for the quantification of the schistosome circulating cathodic antigen (CCA), a glycoprotein associated with the syncitium lining the gut of the parasite. A mouse monoclonal antibody of IgG3 isotype was used as coating (antigen-capture) antibody, while a biotinylated mouse monoclonal IgM was used as second (antigen-detecting) antibody. Streptavidin-alkaline phosphatase was used as enzyme label. The lower detection limit of the assay was 1.0 ng of the trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, which corresponds to approximately 0.2 ng CCA per ml. The ELISA showed a linear range from 1.0 to 62.5 ng AWA-TCA per ml. Serum and urine samples of 16 individuals infected with Schistosoma mansoni (egg counts ranging from 5 to 4820 eggs per gram of faeces) were tested in the assay. Antigen titres ranged from less than 4-8192. This assay represents a considerable advantage in diagnosis of Schistosoma infections as it allows the detection and quantification of CCA in serum and urine in even lightly infected individuals.

Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal circulating anodic antigen

We have developed a new magnetic bead antigen capture enzyme-linked immunoassay for the detection of schistosomal circulating anodic antigen. The assay utilizes IgG1 monoclonal antibody coated monodisperse magnetic beads in microtitre trays fitted to a special magnet. The total test time was found to be 1-2 h, using 0.05 mg beads per well. The lower detection level was 0.7 ng AWA-TCA per ml (approximately 0.07 ng CAA per ml). Validation by sera from uninfected and Schistosoma mansoni infected Africans and Norwegians resulted in an assay specificity of 100% and sensitivity was close to 90% for cases excreting more than 100 eggs per gram faeces. At such clinically relevant levels the inter-assay CV was below 10% and photometric absorbance correlated to antigen levels was nearly linear. There was a significant correlation between the magnetic bead EIA absorbance values and the titres obtained using the previously established ELISA. The new bead assay, however, was easier and less laborious because TCA pretreatment and the titration of positive results were unnecessary.

Circulating anodic antigen levels in serum before and after chemotherapy with praziquantel in schistosomiasis mansoni.

The kinetics of serum levels of circulating anodic antigen (CAA) of Schistosoma mansoni were studied in patients with intestinal schistosomiasis before and after treatment with praziquantel. Day to day fluctuation in faecal egg excretion was compared with fluctuation in antigen level in 20 patients by serum and stool examination on 3 consecutive days before treatment. Antigen levels - calculated either as absorbance value of undiluted serum or as titre - showed less fluctuation than the number of eggs per gram of faeces determined by stool examinations based on single or duplicate 25 mg Kato smears. Compared with a placebo control group of 11 individuals, there was a significant reduction in CAA level in serum of 10 patients treated with praziquantel (40 mg/kg), 10 weeks after treatment. A similar decrease in serum CAA level was observed in a group of 46 patients treated with praziquantel, 6 weeks after treatment. In both groups, patients who remained seropositive after treatment still excreted eggs in their faeces. The kinetics of the antigen decrease were studied in more detail in 20 patients in hospital. Within 10 d after treatment with a double dose of 40 mg praziquantel per kg body weight, the antigen level fell to less than 10% of the original serum level, with a CAA half-life of approximately 2 d.

Mapping fucosylated epitopes on glycoproteins and glycolipids of Schistosoma mansoni cercariae, adult worms and eggs.

The developmental expression of the antigenic fucosylated glycan motifs Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X), and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) in Schistosoma mansoni cercariae, adult worms and eggs, was surveyed using previously defined anti-carbohydrate monoclonal antibodies (mAbs). Lewis X was found both on glycolipids and glycoproteins, yet with completely different expression patterns during the life-cycle: on glycolipids, Lewis X was mainly found in the cercarial stage, while protein-conjugated Lewis X was mainly present in the egg stage. Also protein-conjugated LDN-F and LDN-DF were most highly expressed in the egg-stage. On glycolipids LDN-DF was found in all three examined stages, whereas LDN-F containing glycolipids were restricted to adult worms and eggs. The motifs F-LDN and F-LDN-F were found both on glycoproteins and glycolipids of the cercarial and egg stage, while in the adult stage, they appeared to occur predominantly on glycolipids. Immunofluorescence assays (IFA) showed that these F-LDN and F-LDN-F containing glycolipids were localized in a yet undefined duct or excretory system of adult worms. Murine infection serum showed major reactivity with this adult worm duct-system, which could be fully inhibited by pre-incubation with keyhole limpet haemocyanin (KLH). Clearly, the use of defined mAbs provides a quick and convenient way to map expression profiles of carbohydrate epitopes.

Schistosoma mansoni: thiol proteinase properties of adult worm "hemoglobinase".

Abstract This report presents evidence that the “hemoglobinase” from adult Schistosoma mansoni, first described by Timms and Bueding and later by Senft and his collaborators, belongs to the class of thiol proteinases. Proteolytic activity is stimulated by SH-containing compounds and inhibited by N-ethylmaleimide as well as other inhibitors of thiol proteinases. The enzyme can be partially purified by affinity chromatography using a Sepharose-linked organomercurial ligand. In addition to its activity on globin and hemoglobin, the enzyme can also be assayed with Azocoll, a general protease substrate, and by the activation of inactive trypsinogen to active trypsin. Extraction of the enzyme is enhanced by the addition of the nonionic detergent Triton X-100.

Immunodiagnosis of schistosomiasis mansoni in a low endemic area in Surinam by determination of the circulating antigens CAA and CCA

We evaluated the applicability of circulating antigen detection in serum and urine for the diagnosis of Schistosoma infections in a low endemic area. In total 389 individuals from Saramacca (Surinam) participated in the survey. Stool samples were examined using the Kato method, while circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were determined by highly specific monoclonal antibody-based ELISAs. Also schistosome specific IgM antibodies were measured by the indirect immunofluorescence assay, but the diagnostic performance of this test was found to be poor in this population. S. mansoni eggs were found in 29% of the examined cases, while CAA and CCA could be demonstrated in 23% and 17% of the serum samples and in 3% and 28% of the urine samples, respectively. Forty three percent of the study population was positive in at least one of these diagnostic assays, indicating that each individual test misses a substantial part of the subjects with an active infection. In most positive cases, intensities of infection were very low. As 204 individuals participated in all screening assays, diagnostic performance of each test was evaluated in this sub-population. The highest sensitivities were achieved with the urine-CCA assay and the parasitological examination, detecting 59 and 58 out of the 107 cases with an active infection, respectively. The serum-CAA assay detected 47 positive cases. Our results demonstrate that determination of circulating antigens, especially CCA in urine and CAA in serum, provides information additional to the parasitological examination, for the assessment of prevalence and intensity of Schistosoma infection in low endemic areas.