Acta Tropica, 48(1991)25-35
25
Elsevier ACTROP 00079
Mixed Sch&tosoma haematobium and S. mansoni infection: Effect of different treatments on the serum level of circulating anodic antigen (CAA) N . de Jonge 1, G. S c h o m m e r 2, H. Feldmeier 2, F.W. Krijger 1, A . A . D a f a l l a 3, U. Bienzle 2 a n d A . M . D e e l d e r 1 I Laboratorv of Parasitology, Medical Faculty, University of Leiden, Leiden, The Netherlands, 2State Institute for Tropical Medicine Berlin, Berlin, Germany, and 3National Council for Research, Khartoum, Sudan (Received 21 September 1989; accepted 21 January 1990)
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. Key words: Trematode; Schistosomiasis haematobium; Schistosomiasis mansoni; Immunodiagnosis; ELISA; Monoclonal antibody; Circulating antigens; Chemotherapy; Praziquantel; Oxamniquine; Metrifonate
Introduction In schistosomiasis the effect of drug treatment is usually assessed by looking for schistosome ova in stool, urine or biopsy specimens. Precise assessment of chemotherapy, though, needs quantitative parasitological techniques, which are timeconsuming, especially when egg excretion is at a low level. However, recently, it has been shown that active schistosome infections may quantitatively be assessed by the determination of circulating schistosome antigens (Abdel-Hafez et al., 1983; Feldmeier et al., 1986; De Jonge et al., 1988, 1989a), and that the level of circulating antigen shows less day-to-day fluctuation than egg counts (De Jonge et al., 1989a). One of the major antigens which can be detected in the circulation of schistosomiaCorrespondence address." N. de Jonge, Laboratory of Parasitology, Medical Faculty, University of Leiden, P.O. Box 9605, 2300 RC Leiden, The Netherlands. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
26 sis patients is the schistosome gut associated circulating anodic antigen (CAA), a 70 kDa proteoglycan (Berggren and Weller, 1967; Gold et al., 1969; Nash, 1974; Nash et al., 1974, 1977; Deelder et al., 1976, 1980; Kestens et al., 1988). This antigen could be detected in the serum of patients infected with S. mansoni (Feldmeier et al., 1986; De Jonge et al., 1988), S. intercalatum (De Jonge et al., 1989b), S. haematobium (Feldmeier et al., 1986; De Jonge et al., 1989a) and S.japonicum (De Jonge et al., unpublished data), and its monitoring is a reliable method to assess the efficacy of chemotherapy. Recently, we described a very rapid decrease in CAA titres after treatment with praziquantel in S. mansoni infections (De Jonge et al., 1989a). As infections with schistosomes can be treated with a variety of drugs, it is of interest whether the efficacy of these drugs can be assessed by quantitatively monitoring antigenemia over a prolonged period of time. We therefore submitted patients with a mixed S. haematobium/S, mansoni infection to differential chemotherapy with praziquantel, oxamniquine, or metrifonate, and assessed natural fluctuation of egg excretion and antigenemia over a period of five months in a group of untreated controls. Materials and Methods
Stud). population
We studied the sera of 160 patients from the Gezira irrigated area in Central Sudan. Epidemiological, clinical and immunological characteristics of these patients have been previously described (Doehring et at., 1985; Feldmeier et al. 1985, 1986). The patients were male, 6 11 years old, and pupils in a primary school. Informed consent for participation in the study was obtained in the arabic language from the parents. The patients were randomly divided into four groups: 38 patients (Group 1) received metrifonate (two times 10 mg/kg bodyweight two weeks apart), 53 patients (Group 2) received oxamniquine (single oral dose 60 mg/kg) and 48 patients (Group 3) received praziquantel (single oral dose 40 mg/kg); a control group of 21 patients (Group 4) received a multivitamin preparation. At the end of the study, all patients were treated with praziquantel. Parasitological examination
Both stool and urine examinations were performed before treatment as well as one month and five months after initial treatment, each time on three consecutive days. Urine specimens were examined by the filtration trypan blue staining technique (Feldmeier et al., 1979). Day-to-day variation in urine egg output was minimized by giving all patients a 250 ml soda drink 45 rain prior to urine collection; this soda drink was given at 11:00 h (Doehring et al., 1983). If the egg concentration was less then 10 eggs per 10 ml of urine, the whole volume (up to 350 ml) was filtered. Faecal egg excretion was quantified by a modified Kato thick smear technique (Teesdale and Amin, 1976); five thick Kato smears of 40 mg were examined on each day. Blood collection and pretreatment of sera
Blood samples were taken by venapuncture. After clot retraction, the samples were centrifuged and the serum was carefully removed. Serum samples were transported
27 frozen to Germany and stored at - 7 0 ° C until use. The serum samples were pretreated with trichloroacetic acid (TCA) before testing to remove interfering proteins and to dissociate immune complexes as described by De Jonge et al. (1987).
Determination of circulating anodic antigen CAA was quantified by an enzyme-linked immunosorbent assay (ELISA) (Deelder et al., 1989a). Briefly, microtitration plates were coated with mouse ascitic fluid, containing ~-CAA monoclonal antibody 120-1B10-A (IgG1), by incubation overnight at room temperature. The plates were then washed and blocked with bovine serum albumin. After incubation for 1 h at 37°C, the plates were washed again and stored dry at - 7 0 ° C until use. Because dose-response curves of individual sera were incongruous in their linear range, it was not possible to use the absorbance of a single serum dilution as an indicator of antigen concentration. Therefore TCA pre-treated serum samples were tested in a two-fold dilution series in assay buffer (0.035 M PBS, 0.02% Tween-20, 0.5% PEG-1000, pH 7.8). 75 gl sample was added to each well. After 1 h incubation at 37°C, the plates were washed and 75 gl alkaline phosphatase conjugated Protein-A purified monoclonal antibody 120-1BI0-A was added to each well. Following incubation for 1 h at 37°C and a final washing procedure, 75 lal p-nitrophenyl phosphate substrate solution was added to each well. After incubation at 4°C overnight, the absorbance was read at 405 nm, using a Biotek EL-310 EIA-reader. The reciprocal value of the last sample dilution resulting in an absorbance above background level (mean of twelve replicated buffer assays plus two times the standard deviation) was taken as the titre. Titres /> 4 were considered positive. Pooled sera of healthy Dutch blood donors or healthy German volunteers were included as negative control samples in each run. No false positive results were obtained for these controls. The ELISA was found earlier to be entirely specific. We have tested several hundreds of negative control sera, and false-positive results were never obtained (Deelder et al., 1989a; De Jonge et al., 1988, 1989a, b). The detection limit of the ELISA with the conjugate batch used was 0.8 ng/ml of the TCA soluble fraction of adult S. mansoni worm antigen.
Statistical methods Neither egg counts, nor CAA-titres were normally distributed. Therefore, nonparametric statistical methods were used to evaluate the results. Data were characterized by minimum- and maximum-value, median and 90-percentile value. Accordingly, Kendall's coefficient of concordance, Spearman's rank coefficient of correlation, Wilcoxon's matched pairs signed ranks test and Mann-Whitney test were applied where appropriate. These tests were run on an IBM/XT compatible computer using the SPSS/PC+ statistical package (SPSS, Chicago, IL). Results
Parasitological examinations Within the total study population egg excretion in stool and urine varied considerably and showed a typical binomial distribution with few individuals excreting very
28
high numbers of eggs. Faecal egg counts ranged from 37 to 6563 eggs per gram faeces (epg), median 691 epg, 90-percentile 1954.3 epg. S. haematobium egg count in urine ranged from 1 to 9009 eggs per 10ml (epl0ml) (median 68.2, 90-percentile 1511 epl0ml). There was no significant difference in S. haematobium or S. mansoni egg counts between the four treatment groups (Table 1). The numbers of patients excreting eggs of either schistosome species is given in Table 2. TABLE 1 Effect of differential chemotherapy on egg excretion in stool and urine Treatment
Time a
group
Egg excretion S. haematobium (eggs per I0 ml urine) b
S. mansoni (eggs per gram faeces) b
min
med
90-per
max
rain
med
90-per
max
Metrifonate ( n = 38)
0 1 5
0 0 0
95 l 1
1695 29 14
4530 161 351
66 20 0
557 740 623
2325 1873 2722
6563 5620 5460
Oxamniquine ( n = 53)
0 1 5
0 0 0
49 44 46
965 826 661
4067 1426 1343
37 0 0
700 0 3
1672 61 80
4070 440 230
Praziquantel (n=48)
0 ! 5
! 0 0
66 1 0
1889 31 6
9009 139 113
61 0 0
755 0 0
1764 10 10
2430 330 55
Placebo (n = 2l)
0 1 5
1 0 33
124 58 95
1112 2174 836
1722 3560 2710
97 120 0
680 381 635
3376 2561 3060
4629 2835 4360
pre-treatment, 1 = one m o n t h after treatment, 5 = five m o n t h s after treatment. bValues rounded to integers. M i n i m u m S. haematobium egg excretion was 0.3 egg per 10 ml for metrifonate treated group and 0.1 egg per 10 ml for oxamniquine treated group. a0 =
TABLE 2 N u m b e r s of patients excreting eggs following treatment Treatment group
Time"
S. haematobium
S. mansoni
No. patients
%
No. patients
%
Metrifonate
0 1 5
38/38 24/32 28/32
I00 75.0 87.5
38/38 32/32 31/32
100 100 96.9
Oxamniquine
0 1 5
53/53 41/47 40/44
100 87.2 90.9
53/53 21/47 22/44
100 44.7 50.0
Praziquantel
0 1 5
48/48 31/40 25/35
100 77.5 71.4
48/48 9/40 8/35
100 22.5 22.9
Placebo
0 1 5
21/21 16/18 19/19
100 88.9 100
21/2 l 18/18 18/19
100 100 94.7
a0 = pre-treatment, 1 = one m o n t h after treatment, 5 = five m o n t h s after treatment.
29 Treatment with metrifonate (Group l) reduced excretion of S. haematobium ova by almost 99%, but did not change the excretion of S. mansoni eggs. Four patients still excreted low numbers of S. haematobium eggs in urine. In Group 2, S. mansoni egg excretion fell drastically within one month after treatment with oxamniquine. The S. haematobium egg output tended to decrease one month after treatment, although the difference became only significant five months post-treatment (p < 0.01). However, no significant difference was observed in urinary egg output when compared with the placebo treated group (p = 0.29 and p = 0.27 for one and five months post-treatment results respectively). Treatment with praziquantel (Group 3) was followed by a remarkable decrease in both S. haematobium and S. mansoni egg output already one month after treatment (p
25
Elsevier ACTROP 00079
Mixed Sch&tosoma haematobium and S. mansoni infection: Effect of different treatments on the serum level of circulating anodic antigen (CAA) N . de Jonge 1, G. S c h o m m e r 2, H. Feldmeier 2, F.W. Krijger 1, A . A . D a f a l l a 3, U. Bienzle 2 a n d A . M . D e e l d e r 1 I Laboratorv of Parasitology, Medical Faculty, University of Leiden, Leiden, The Netherlands, 2State Institute for Tropical Medicine Berlin, Berlin, Germany, and 3National Council for Research, Khartoum, Sudan (Received 21 September 1989; accepted 21 January 1990)
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. Key words: Trematode; Schistosomiasis haematobium; Schistosomiasis mansoni; Immunodiagnosis; ELISA; Monoclonal antibody; Circulating antigens; Chemotherapy; Praziquantel; Oxamniquine; Metrifonate
Introduction In schistosomiasis the effect of drug treatment is usually assessed by looking for schistosome ova in stool, urine or biopsy specimens. Precise assessment of chemotherapy, though, needs quantitative parasitological techniques, which are timeconsuming, especially when egg excretion is at a low level. However, recently, it has been shown that active schistosome infections may quantitatively be assessed by the determination of circulating schistosome antigens (Abdel-Hafez et al., 1983; Feldmeier et al., 1986; De Jonge et al., 1988, 1989a), and that the level of circulating antigen shows less day-to-day fluctuation than egg counts (De Jonge et al., 1989a). One of the major antigens which can be detected in the circulation of schistosomiaCorrespondence address." N. de Jonge, Laboratory of Parasitology, Medical Faculty, University of Leiden, P.O. Box 9605, 2300 RC Leiden, The Netherlands. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
26 sis patients is the schistosome gut associated circulating anodic antigen (CAA), a 70 kDa proteoglycan (Berggren and Weller, 1967; Gold et al., 1969; Nash, 1974; Nash et al., 1974, 1977; Deelder et al., 1976, 1980; Kestens et al., 1988). This antigen could be detected in the serum of patients infected with S. mansoni (Feldmeier et al., 1986; De Jonge et al., 1988), S. intercalatum (De Jonge et al., 1989b), S. haematobium (Feldmeier et al., 1986; De Jonge et al., 1989a) and S.japonicum (De Jonge et al., unpublished data), and its monitoring is a reliable method to assess the efficacy of chemotherapy. Recently, we described a very rapid decrease in CAA titres after treatment with praziquantel in S. mansoni infections (De Jonge et al., 1989a). As infections with schistosomes can be treated with a variety of drugs, it is of interest whether the efficacy of these drugs can be assessed by quantitatively monitoring antigenemia over a prolonged period of time. We therefore submitted patients with a mixed S. haematobium/S, mansoni infection to differential chemotherapy with praziquantel, oxamniquine, or metrifonate, and assessed natural fluctuation of egg excretion and antigenemia over a period of five months in a group of untreated controls. Materials and Methods
Stud). population
We studied the sera of 160 patients from the Gezira irrigated area in Central Sudan. Epidemiological, clinical and immunological characteristics of these patients have been previously described (Doehring et at., 1985; Feldmeier et al. 1985, 1986). The patients were male, 6 11 years old, and pupils in a primary school. Informed consent for participation in the study was obtained in the arabic language from the parents. The patients were randomly divided into four groups: 38 patients (Group 1) received metrifonate (two times 10 mg/kg bodyweight two weeks apart), 53 patients (Group 2) received oxamniquine (single oral dose 60 mg/kg) and 48 patients (Group 3) received praziquantel (single oral dose 40 mg/kg); a control group of 21 patients (Group 4) received a multivitamin preparation. At the end of the study, all patients were treated with praziquantel. Parasitological examination
Both stool and urine examinations were performed before treatment as well as one month and five months after initial treatment, each time on three consecutive days. Urine specimens were examined by the filtration trypan blue staining technique (Feldmeier et al., 1979). Day-to-day variation in urine egg output was minimized by giving all patients a 250 ml soda drink 45 rain prior to urine collection; this soda drink was given at 11:00 h (Doehring et al., 1983). If the egg concentration was less then 10 eggs per 10 ml of urine, the whole volume (up to 350 ml) was filtered. Faecal egg excretion was quantified by a modified Kato thick smear technique (Teesdale and Amin, 1976); five thick Kato smears of 40 mg were examined on each day. Blood collection and pretreatment of sera
Blood samples were taken by venapuncture. After clot retraction, the samples were centrifuged and the serum was carefully removed. Serum samples were transported
27 frozen to Germany and stored at - 7 0 ° C until use. The serum samples were pretreated with trichloroacetic acid (TCA) before testing to remove interfering proteins and to dissociate immune complexes as described by De Jonge et al. (1987).
Determination of circulating anodic antigen CAA was quantified by an enzyme-linked immunosorbent assay (ELISA) (Deelder et al., 1989a). Briefly, microtitration plates were coated with mouse ascitic fluid, containing ~-CAA monoclonal antibody 120-1B10-A (IgG1), by incubation overnight at room temperature. The plates were then washed and blocked with bovine serum albumin. After incubation for 1 h at 37°C, the plates were washed again and stored dry at - 7 0 ° C until use. Because dose-response curves of individual sera were incongruous in their linear range, it was not possible to use the absorbance of a single serum dilution as an indicator of antigen concentration. Therefore TCA pre-treated serum samples were tested in a two-fold dilution series in assay buffer (0.035 M PBS, 0.02% Tween-20, 0.5% PEG-1000, pH 7.8). 75 gl sample was added to each well. After 1 h incubation at 37°C, the plates were washed and 75 gl alkaline phosphatase conjugated Protein-A purified monoclonal antibody 120-1BI0-A was added to each well. Following incubation for 1 h at 37°C and a final washing procedure, 75 lal p-nitrophenyl phosphate substrate solution was added to each well. After incubation at 4°C overnight, the absorbance was read at 405 nm, using a Biotek EL-310 EIA-reader. The reciprocal value of the last sample dilution resulting in an absorbance above background level (mean of twelve replicated buffer assays plus two times the standard deviation) was taken as the titre. Titres /> 4 were considered positive. Pooled sera of healthy Dutch blood donors or healthy German volunteers were included as negative control samples in each run. No false positive results were obtained for these controls. The ELISA was found earlier to be entirely specific. We have tested several hundreds of negative control sera, and false-positive results were never obtained (Deelder et al., 1989a; De Jonge et al., 1988, 1989a, b). The detection limit of the ELISA with the conjugate batch used was 0.8 ng/ml of the TCA soluble fraction of adult S. mansoni worm antigen.
Statistical methods Neither egg counts, nor CAA-titres were normally distributed. Therefore, nonparametric statistical methods were used to evaluate the results. Data were characterized by minimum- and maximum-value, median and 90-percentile value. Accordingly, Kendall's coefficient of concordance, Spearman's rank coefficient of correlation, Wilcoxon's matched pairs signed ranks test and Mann-Whitney test were applied where appropriate. These tests were run on an IBM/XT compatible computer using the SPSS/PC+ statistical package (SPSS, Chicago, IL). Results
Parasitological examinations Within the total study population egg excretion in stool and urine varied considerably and showed a typical binomial distribution with few individuals excreting very
28
high numbers of eggs. Faecal egg counts ranged from 37 to 6563 eggs per gram faeces (epg), median 691 epg, 90-percentile 1954.3 epg. S. haematobium egg count in urine ranged from 1 to 9009 eggs per 10ml (epl0ml) (median 68.2, 90-percentile 1511 epl0ml). There was no significant difference in S. haematobium or S. mansoni egg counts between the four treatment groups (Table 1). The numbers of patients excreting eggs of either schistosome species is given in Table 2. TABLE 1 Effect of differential chemotherapy on egg excretion in stool and urine Treatment
Time a
group
Egg excretion S. haematobium (eggs per I0 ml urine) b
S. mansoni (eggs per gram faeces) b
min
med
90-per
max
rain
med
90-per
max
Metrifonate ( n = 38)
0 1 5
0 0 0
95 l 1
1695 29 14
4530 161 351
66 20 0
557 740 623
2325 1873 2722
6563 5620 5460
Oxamniquine ( n = 53)
0 1 5
0 0 0
49 44 46
965 826 661
4067 1426 1343
37 0 0
700 0 3
1672 61 80
4070 440 230
Praziquantel (n=48)
0 ! 5
! 0 0
66 1 0
1889 31 6
9009 139 113
61 0 0
755 0 0
1764 10 10
2430 330 55
Placebo (n = 2l)
0 1 5
1 0 33
124 58 95
1112 2174 836
1722 3560 2710
97 120 0
680 381 635
3376 2561 3060
4629 2835 4360
pre-treatment, 1 = one m o n t h after treatment, 5 = five m o n t h s after treatment. bValues rounded to integers. M i n i m u m S. haematobium egg excretion was 0.3 egg per 10 ml for metrifonate treated group and 0.1 egg per 10 ml for oxamniquine treated group. a0 =
TABLE 2 N u m b e r s of patients excreting eggs following treatment Treatment group
Time"
S. haematobium
S. mansoni
No. patients
%
No. patients
%
Metrifonate
0 1 5
38/38 24/32 28/32
I00 75.0 87.5
38/38 32/32 31/32
100 100 96.9
Oxamniquine
0 1 5
53/53 41/47 40/44
100 87.2 90.9
53/53 21/47 22/44
100 44.7 50.0
Praziquantel
0 1 5
48/48 31/40 25/35
100 77.5 71.4
48/48 9/40 8/35
100 22.5 22.9
Placebo
0 1 5
21/21 16/18 19/19
100 88.9 100
21/2 l 18/18 18/19
100 100 94.7
a0 = pre-treatment, 1 = one m o n t h after treatment, 5 = five m o n t h s after treatment.
29 Treatment with metrifonate (Group l) reduced excretion of S. haematobium ova by almost 99%, but did not change the excretion of S. mansoni eggs. Four patients still excreted low numbers of S. haematobium eggs in urine. In Group 2, S. mansoni egg excretion fell drastically within one month after treatment with oxamniquine. The S. haematobium egg output tended to decrease one month after treatment, although the difference became only significant five months post-treatment (p < 0.01). However, no significant difference was observed in urinary egg output when compared with the placebo treated group (p = 0.29 and p = 0.27 for one and five months post-treatment results respectively). Treatment with praziquantel (Group 3) was followed by a remarkable decrease in both S. haematobium and S. mansoni egg output already one month after treatment (p
