F.W. Krijger

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.

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.

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.

The relationship between worm burden and levels of a circulating antigen (CAA) of five species of Schistosoma in mice

This study examines the ability of an assay which measures the amount of a schistosome specific antigen (CAA) in the host circulation to reliably reflect relative worm burden. Mice were infected with 5 species of schistosome with a range of infection dose. The levels of serum CAA increased during schistosome maturation. In all species tested CAA levels correlated well with adult worm burden once the parasites achieved sexual maturity and remained relatively stable during the establishment of egg production. The amount of CAA produced varied between species but within each species CAA levels were proportional to worm numbers: no density-dependent effects on CAA levels were observed even when mice carried worm burdens that were very large relative to host size. T-cell deprivation of the host had no effect on the CAA/worm burden relationship in either Schistosoma mansoni or S. haematobium infections and the CAA equilibrium was unaltered in intact mice when reduction of worm fecundity occurred. These data support the use of the CAA as an accurate and robust estimate of relative schistosome burden in man.

Levels of the schistosome circulating anodic and cathodic antigens in serum of schistosomiasis patients from Brazil

Serum levels of 2 schistosome circulating antigens, the circulating anodic antigen (CAA) and the circulating cathodic antigen (CAA), were determined in persons infected with Schistosoma mansoni in Brazil. Sensitive monoclonal antibody-based enzyme-linked immunosorbent assays were used to measure levels of the 2 antigens. The study group consisted of 38 individuals with intestinal schistosomiasis, and 20 persons with the hepatosplenic form of the disease. Age and intensity of infection were comparable for the 2 groups. CAA was detected in 65.5% of all patients sera and CCA was found in the serum of 82.8% of all patients. CAA levels correlated well with the egg output, as determined by duplicate Kato-Katz smears; CCA was significantly positively correlated with egg output in patients with intestinal schistosomiasis only. Whereas no significant difference was found between CAA titre in patients with intestinal schistosomiasis and those with the hepatosplenic form, a significantly higher CCA titre was found in patients with hepatosplenomegaly compared to patients with intestinal schistosomiasis.

Monitoring the efficacy of different doses of praziquantel by quantification of circulating antigens in serum and urine of schistosomiasis patients

We evaluated the quantitation of two schistosome circulating antigens in serum and urine as a tool for the assessment of the efficacy of praziquantel dosage regimens (40 versus 60 mg/kgbw). In addition we compared the efficacy of two different brands of praziquantel (Biltricide and Distocide), given at the same dosage (40 mg/kgbw). Thirty five Egyptian hospitalized schistosomiasis mansoni patients participated in this study. Thirteen patients (Group 1) received 60 mg/kgbw Biltricide, administered in 3 oral doses of 20 mg in one day; 22 individuals (Group 2) were treated with 40 mg/kgbw (12 Biltricide, 10 Distocide), given in one oral dose. Circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were quantitated by monoclonal antibody-based ELISAs before, and 1, 3 and 6 weeks after chemotherapy. Before treatment, all patients were positive for at least one of the circulating antigen assays. Three to six weeks after treatment significantly more patients were found to be negative in Group 1 compared to Group 2 (X2 = 7.13, P = 0.008, n = 35). Also the levels of CCA and CAA in serum and of CCA in urine were found to be significantly higher in Group 2 (Mann-Whitney U < 85, P < 0.05, n = 35). These results were confirmed by parasitological data. No differences were found between treatment with Biltricide or Distocide.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple technique to pretreat urine and serum samples for quantitation of schistosome circulating anodic and cathodic antigen

For the detection of the circulating schistosome antigens CAA (circulating anodic antigen) and CCA (circulating cathodic antigen) in serum and urine samples of Schistosoma infected individuals, pretreatment of samples with trichloroacetic acid (TCA) is a standard procedure. In the present study several methods were evaluated in order to develop a more simple and rapid technique than the--especially for pretreatment of urine samples--laborious TCA technique. Optimal results were obtained with a method in which serum or urine samples were pretreated by a heat-incubation step (70 degrees C, 30 min) in an alkaline buffer (pH 9.6). In a comparison of the new technique with the TCA pretreatment, serum and urine samples of S. mansoni infected individuals from Zaire (n = 80) and of uninfected controls from The Netherlands (n = 208) were pretreated and assayed for CAA and CCA. Both pretreatment techniques showed similar sensitivities and specificities for CAA and CCA in serum, and CCA in urine. However, for the determination of CAA in urine the new technique performed significantly better, resulting in an increase of the sensitivity from 32 to 70% (titre determination).

Mixed Schistosoma haematobium and S. mansoni infection : effect of different treatments on the serum level of circulating anodic antigen (CAA)

In this study, levels of circulating anodic antigen (CAA) in serum were investigated after differential treatment of 160 Sudanese patients with mixed Schistosoma haematobium and S. mansoni infections. The patients were randomly divided into four groups, which were treated with metrifonate (two doses of 10 mg/kg bodyweight), oxamniquine (60 mg/kg), praziquantel (40 mg/kg), or a multivitamin preparation, respectively. Serum, stool and urine samples were taken prior to treatment as well as one month and five months after chemotherapy. Before chemotherapy CAA levels were similar in the four groups. Antigenemia remained unchanged in the control group. In patients treated with praziquantel or oxamniquine the concentration of CAA decreased to a similar extent. However, whereas in the praziquantel group absence of CAA was already observed one month after treatment, clearing of CAA from the circulation seemed to take longer in patients treated with oxamniquine. Treatment with metrifonate did not result in a reduction of the CAA titres.

Presence of circulating anodic antigen in serum of Schistosoma intercalatum-infected patients from Gabon

The presence of the schistosome circulating anodic antigen (CAA) in serum of Schistosoma intercalatum-infected patients from Gabon has been investigated using an enzyme-linked immunosorbent assay (ELISA). Blood samples were collected from 10 endemic controls, 29 patients which excreted viable S. intercalatum eggs in rectal mucosa and stool, six persons in which only non-viable eggs were found in rectal biopsy specimens and one person in which besides non-viable eggs a small number of viable eggs was found in the rectal biopsy specimen. CAA, a genus-specific antigen, could be demonstrated in 58.6% of the patients with S. intercalatum eggs in their stools. In comparison to S. mansoni infections, very light infections (0.6 eggs per gram faeces) could be detected by the ELISA. A strong correlation between parasite burden (eggs per gram faeces) and antigen-level (CAA-titer) was found (Spearmans rho = 0.65). Only one positive ELISA-results was found in the group with solely non-viable eggs in rectal tissue. As no false positive results were detected for the negative controls, the present results suggest, in accordance with results earlier obtained for schistosomiasis mansoni, that only in active S. intercalatum infections is antigen demonstrable.

A model system for the study of antigen secretion by adult Schistosoma mansoni in vivo

Schistosoma mansoni (6 weeks old) were surgically transferred from donor C57BI/6 mice to the hepatic portal veins of naive recipients of the same inbred strain. Between 70% and 100% of the parasites were alive 15 days later, and egg deposition was observed after transfer of worm pairs. The physiological status of the parasites was monitored by measuring the levels of a schistosome gut antigen, circulating anodic antigen (CAA), in the serum of the recipients. When only male worms were transferred, serum CAA levels increased slowly to a peak 9 days later, which was followed by a rapid decline. When worm pairs were transferred, there was an early peak in serum CAA levels followed by a gradual decline, but these levels were always higher than those recorded after male-only transfer; in two mice the pattern was similar to that observed following receipt of male worms. More CAA and eggs were produced after transfer of paired versus separated worms. It was concluded that although worm pairs can be successfully transferred, their physiological status may be sub-optimal. In contrast, male worms survive consistently well, and their transfer to a naive recipient provides a convenient model with which to study the release of antigens by schistosomes in vivo.