N. De Jonge

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.

A simple and rapid treatment (trichloroacetic acid precipitation) of serum samples to prevent non-specific reactions in the immunoassay of a proteoglycan

In our laboratory serum components interfering in the immunoassay of the schistosome proteoglycan circulating anodic antigen (CAA) in serum necessitated us to develop a simple technique by which the non-specific reaction of negative control sera could be prevented. Trichloroacetic acid was added to serum samples to precipitate interfering (glyco-)proteins. After centrifugation, the supernatant was neutralized and used directly either in the ELISA or in the indirect haemagglutination. This method gave satisfactory results, i.e., negative control sera did no longer give false positive reactions, while the titre of the positive controls remained unaffected. This method could also be successfully applied for the pretreatment of urine samples.

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.

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)

Quantitative determination of circulating anodic and cathodic antigens in serum and urine of individuals infected with Schistosoma intercalatum

Circulating anodic and cathodic Schistosoma antigens (CAA and CCA) have been determined by enzyme immunoassays in serum and urine of 60 individuals infected with S. intercalatum in Equatorial Guinea. The median egg output was 29 eggs/g of faeces (range 3-840). The egg output strongly correlated with concentrations of serum CAA (p = 0.47) and urine CAA (p = 0.42) (P < 0.001 for both); the later 2 quantities were also correlated with each other (p = 0.44, P < 0.001). All except 3 infected individuals had detectable amounts of serum CAA and/or urine CCA, a sensitivity of 95% for these 2 tests combined. Urine CAA was detected in 43% of patients. Serum CCA was detected in all infected individuals; however, no significant correlation was obtained between serum CCA levels and egg output in the stools of individual patients. This is the first study to demonstrate CCA in specimens of patients infected with S. intercalatum. The detection of CCA in urine is a new, non-invasive diagnostic method for S. intercalatum infection.

Schistosome circulating anodic antigen in serum of individuals infected with Schistosoma japonicum from The Philippines before and after chemotherapy with praziquantel

The presence of the schistosome circulating anodic antigen (CAA) in serum of patients infected with Schistosoma japonicum from The Philippines has been investigated using an enzyme-linked immunosorbent assay (ELISA). Serum samples were tested from 48 patients who excreted S. japonicum eggs, 9 individuals with a negative stool examination, and 20 controls with both a negative stool and a negative circumoval precipitin test. No false positive result was detected for the unequivocally negative controls. CAA could be demonstrated in 72.9% of the egg-excreting patients. A positive correlation between parasite burden (eggs per gram of faeces) and antigen level (CAA titre) was found (Spearmans rho = 0.48, P < 0.001, n = 48). Four of 18 sera from the egg-negative individuals were positive in the ELISA. In view of the fact that anti-worm antibodies were also detected in these 4 sera, those reactions suggest active infection not detected by stool examination. In serum from patients treated with praziquantel, a significant drop in CAA titre was seen within 5 d after treatment (Wilcoxons chi T = -2.23, P = 0.0258, n = 21). In conclusion, the detection of CAA by ELISA in S. japonicum infection can give valuable information in both individual diagnosis and therapeutic drug monitoring, as well as in epidemiological studies or disease control programmes.