Serum circulating egg antigen levels in two areas endemic for Schistosoma mansoni

TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(1998)92,350-354

Serum circulating mansoni

egg antigen

levels in two areas endemic

for Schistosoma

H. A. M. Nibbeling*, L. Van Lieshout, K. Polman, F. F. Stelma, A. M. Polderman and A. M. Deelder Department of Parasitology, Leiden University Medical Centre, l? 0. Box 96052300 RC Leiden, The Netherlands Abstract A monoclonal antibody-based enzyme-linked immunosorbent assay detectingSchistosoma rnansoni circulating soluble egg antigen (CSEA) was applied in epidemiological studies. The serum CSEA levels were determined for 2 populations with a high prevalence (>95%) and high intensity of infection as determined by faecal egg counts. In one population (Maniema, Zaire) transmission had been occurring for several decades, while in the other population (Ndombo, Senegal) transmission had started only recently. CSEA could be detected in 88% and 70% of the serum samples from Maniema and Ndombo, respectively. The sensitivity of the CSEA assay increased with rising egg count.The age-related CSEA profiles of the Maniema population followed a pattern similar to that of egg counts and of the adult worm antigen CAA (circulating anodic antigen). However, the recently infected Ndombo population showed a clearly different profile: while the CSEA prevalence reached a peak in children and adolescents, the mean CSEA levels did not vary significantly in the different age groups. CSEA levels were significantly lower in Ndombo than in Maniema. As egg antigens in serum are thought to be in part, or even primarily, derived from eggs in the tissues, these findings indicate a relatively smaller tissue egg load in Ndombo than in Maniema. Keywords: schistosomiasis, Schtitosomamansoni,circulating soluble egg antigen, epidemiology, Zaire, Senegal Introduction The diagnosis of Schistosoma mansoni infection is primarily based on detection of eggs in stool. However, the sensitivity of the currently applied modified Kato thick smear technique is low in lightly infected populations and is thought to result in an underestimation of prevalence (DE VLAS & GRYSEELS, 1992). As an alternative quantitative method, many efforts have been made to develop assays for detection of schistosome-derived circulating antigens. The best studied antigens are adult worm antigens, especially circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), for which enzyme-linked immunosorbent assays (ELISAS) havebeendeveloped (DEELDER~~~Z., ~~S~;DEJONGE et al., 1990) which allow their detection in serum and urine of schistosomiasis patients. The presence of CAA and CCA indicates an active infection (DE TONGE et al.. 1989; VANLIESHOUT et al., 1991, 1993), aid in animal models antigen levels are strongly correlated with worm burden (AGNEW et al., 1995). Epidemiological studies, involving measurement of egg output and CAA levels, have been conducted in areas with a different S. mansoni transmission history. In one population (Maniema, Zaire), transmission had been occurring for several decades, while in a second population (Ndombo, Senegal) transmission had started only recently. Both areas had high transmission levels of S. mansolzi, with egg counts ranging from low to extremelvhigh levels (POLDERMAN etal.. 1985a; STELMA et al., -1993). Serum CAA levels were age-rdlated, resembling the characteristic age-related egg count pattern in both populations, but were significantly lower in Ndombo than in Maniema (POLMAN et al., 1995; VAN L1~~~0~~etaZ.,1995,1998).As CAAlevelsinhumans are also assumed to reflect worm burden, this finding was interpreted as indicating higher parasite fecundity in the recently exposed population of Ndombo (VAN LIESHOUT~Z~~., 1998). The induction of granulomas around eggs retained in the tissues is recognized to be the main cause of pathology in schistosomiasis. However, detection of adult worm antigens and counting excreted eggs in faeces do not necessarily provide accurate information about tissue egg loads. Assessment of the levels of antigens secreted by eggs into the circulation could provide more information about the proportion of eggs retained in the *Author for correspondence; phone +31 71 527 6859, fax +31 71 527 6850.

tissues. The development of a monoclonal antibodybased ELISA specific for S. mansoni circulating soluble egg antigens (CSEA) has been reported previously by us (NOUREL DIN et al., 1994a), and the usefulness of this CSEA assay as a diagnostic technique complementary to the measurement of adult worm antigens and egg output has been demonstrated by NOUREL DIN et al. (1994b). In the present study we compared the CSEA levels in the serum of the recently infected population of Ndombo with those of the chronically infected population of Maniema. Although many laboratories have described the serodiagnostic potential of several egg antigens, this was the first time that a specific S. mansoni egg antigen assay had been used in an epidemiological study. It was shown that measurement of circulating egg antigens may provide additional information on the dynamics of parasite populations, in relation to intensity of infection and the age of the host. Materials and Methods Study populations The first data set came from a population survey in Maniema (Kivu Province. eastern Zaire). This area has been endemic for schistdsomiasis mansoni for several decades @'OLDERMAN et al., 1985a, 1985b). Serum samples were collected from a random sample of this population. The second data set came from Ndombo, a recently exposed and heavily infected community in northern Senegal. The focus and its epidemiological details have been described elsewhere (TALLA et al., 1990; STELMA et al., 1993). The serum CAA levels of both populations h&e also been reported (POLMAN et al., 1995: VAN LIESHOUT et al.. 1995). For the current study, CSEA levels were de&nine2 in serum samples from 498 individuals from Maniema and 179 individuals from Ndombo. Parasitological examination Faecal egg counts, expressed as eggs per gram of faeces, were based on 2 stool examinations with an interval of a few weeks, each consisting of duplicate 25 mg Kato slides (KATZ etal., 1972; FOLDERMAN etal., 1985b). Determination of circuculatingsoluble eg,, ant&en The anti-egg monoclonal anti&dies- (mabs) 1145Bl-A (immunoglobulin [Ig] Gl) and 114-4D12-A (IgG3) were prepared, characterized and purified as described elsewhere (NOUREL DIN et al., 1994a). The mabs were biotinylated with sulphosuccinimidyl-6-

CIRCULATING EGG ANTIGEN IN SCHISTOSOMIASISMANSONI (biotinamido) hexanoate (Immunopure@, NHS-LC-Biotin, Pierce), according to the manufacturer’s instructions. The ELISA for quantitative determination of CSEA was performed as described previously (NOUREL DIN et al., 1994b), with some modifications. Briefly, polystyrene microtitration plates (Maxisorp@, Nunc, Denmark) were coated with a mixture of the mabs 1145Bl-A and 114-4D 12-A and blocked with bovine serum albumin. Serum samples were pretreated with trichloroacetic acid (TCA) to remove interfering proteins and to dissociate immune complexes (DE JONGE et al., 1987) and subsequently tested in a two-fold dilution series starting with an initial dilution of 1:4. A mixture of biotinylated mabs I14-5Bl-A and 114-4D12-A was used as the detection antibody, visualized with streptavidin-alkaline phosphatase (Zymed, USA). After colour development, using 4-nitrophenyl phosphate as a substrate, the absorbance was measured at 405 nm. The CSEA concentrations in the serum samples were calculated from a standard dilution curve of the TCA soluble fraction of soluble egg antigen (SEA-TCA) and expressed as ng/mL. The lower detection limit of the CSEA assay was approximately 0.4 ng SEA-TCA/mL. Samples were considered positive if the absorbances were higher than the background (mean of 10 buffer controls plus 2 SD). The specificity of the assay was lOO%, as determined by testing sera of 72 healthy Dutch individuals. Analysis

of results

Since the egg counts and serum antigen level distributions were skewed, data were normalized by loglo transformation of all positive values. Data were expressed as ranges and geometric means with their 95% confidence intervals (95% CI) of the positive values. x2 and t tests were used to assess differences between the 2 study populations. Pearson product-moment correlation was initially used to estimate the association between circulating antigen levels and egg counts. Each population was divided into 8 age categories and the variation of intensity of infection with age was assessed by oneway analysis of variance (F test). The data were further examined by multiple regression analysis, with CSEA levels as the dependent variable. Individuals with negative egg counts and determinations of both antigens were considered as non-infected and omitted from the analysis (16 persons in Maniema and one in Ndombo). Individuals with positive results for at least one of these variables were included, and a value equal to half the detection limit of the assay concerned was assigned to any negative results (VAN LIESHOUT et al., 1995). The egg counts and CAA levels were analysed by several models after allowing for age group and sex. Results The characteristics of the populations studied in Maniema and Ndombo are summarized in Table 1. The 2 groups had similar prevalences and egg counts, al-

351 Table

1. Details

Schistosoma

Maniema,

populations of endemic areas Zaire and Ndombo, Senegal

0

l-100 101-400 401-1000 1001-4000 >4000 Total

24 45 101 137 162 29 498

Percentage positive 25 67 88 E 100 88

two in

aMedian (range in parentheses). bGeometric mean (range in parentheses). CExpressedas trichloroacetic acid-soluble antigen. though the Maniema population was on average older than the population from Ndombo and comprised more males. The CAA levels were significantly higher in Maniema, as described recentlv. (VAN LIESHOUT et al., . 1998). _ CSEA was detected in 88% and 70% of the serum samnles from Maniema and Ndombo. resnectivelv (Table i). Only in the Maniema population &as this*sdnsitivity comparable to that of the CAA assay (McNemar’s test, P > 0.05). Most subjects had positive results with egg counts and both antigen determinations (420 in Maniema, 124 in Ndombo), while only a few gave negative results in all 3 determinations (16 in Maniema, one in Ndombo). The levels of CSEA were significantly higher in Maniema than in Ndombo, which is in concordance with what has already been observed for CAA. The sensitivity of the CSEA assay increased with rising intensity of infection as determined by egg counts, although the increase was much less pronounced in Ndombo than in Maniema (Table 2). For persons with egg counts of more than 4000 eggs/g, the sensitivity of the CSEA assay was 100% in both populations. However, the mean CSEA concentration of the Ndombo group was much lower than that in the Maniema population (P4000 eggs/g) was the mean CSEA concentration significantly different from that of other classes (P
Maniema No. of subjects

the

Maniema Ndombo P No. studied 179 498 Males 51% 42% co.05 Age (year~)~ 30(1-66) 19(1-77) 0,05 count (eggsig)b 653(10-13183)752(10-10328) >0.05 Circulatinganodicantigen Positive(X) 95.0 90.4 >0.05 Antigenlevel (ngimL)b 31.6(0.06-514) 5.9(0.02-339)
Table 2. Percentage of individuals with Schistosoma mansoni concentration according to egg count class in two populations

Egg count (eggsk)

of

mansoni

Ndombo Antigen concentration (ng/mL)a

No. of subjects

Percentage positive

Antigen concentration (ng/mL)a

5.3(1.5-18.5) 8.3 (5.5-12.5) 17.3(14.3-20.9) 29.6 (24.2-36.3) 52.9 (42.2-66.4) 71.3(41.1-123.7) 30.9 (27.2-35.1)

4 19 30 44 68 14 179

25 16 50 70 91 100 70

10.7 (-) 5.9(1.3-27.4) 5.3 (3.8-7.3) 6.5 (5.0-8.3) 6.4(5.2-7.9) 12.5 (8.0-19.6) 6.8 (5.9-7.8)

aGeometric mean (95% confidence interval in parentheses).

352

H. A. M.

Maniema

ETAL.

NIBBELING

Ndombo 2250

75 1500 1250 .E c

50

moo

% P

750 25

P 5 0 E .z g

500

!$

250

u

0

0

75

0

1-4

5-9

10-14

IS-19

20-29

30-39

40-49

50+

l-4

5-9

w

(63)

(6%

(46)

(W

(87)

(101)

(65)

(11)

(28) (26)

10-14

15-19

20-29

30-39

40-49

so+

(27)

(31)

(2%

(11)

(16)

Age (years)

Age (years) Egg count (eggs/g)

o-1 00

101-400

401-1000

Antigen level (ng/mL)

O-10

1 I-30

31-60

>I000 >60

Figure. Curves representing Schistosonzamansoni egg counts (A) and levels of circulating anodic antigen (B) and soluble egg antigen (C) in two communities according to age group (years). The bars indicate the prevalence and intensity of infection; numbers in parentheses are the numbers of individuals in each age group. children and adolescents (aged 5-19 years), the mean CSEA levels did not vary significantly in the different age groups (P1.9, ~125, -0.07). The CSEA levels in the Maniema population showed good correlation with both egg counts (r=O.47, ~432, PcO.01) and CAA levels (r=O.68, ~426, P
ulation the correlation between CSEA levels and the other 2 assays was much weaker, although significant, for both egg counts (r=O.26, 92=125, PcO.01) and CAA levels (~0.34, ~124, Pi 0.001). However, CAA levels and egg counts were strongly correlated with each other in this population (r=O.62, n=167, P
CIRCULATINGEGGANTIGENINSCHISTOSOMIASISMANSONI

353

Table 3. Analysis of data from 482 individuals in Maniema, Zaire. Multiple regression results models with Schistosoma mansoni circulating soluble egg antigen concentration as dependent and age group and sex as independent variable.+ Variable Model 1 Constant Age lo-19 years (O/l) Age 20-39 years (O/l) Age 240 years (O/l) Female sex (O/l) Egg count Model 2 Constant Age lo-19 years (O/l) Age 20-39 years (O/l) Age 240 years (O/l) Female sex (O/l) Circulating anodic antigen level Model 3 Constant Age lo-19 years (O/l) Age 20-39 years (O/l) Age 240 years (O/l) Female sex (O/l) Egg count Circulating anodic antigen level

of three variable

t

P

R2

-0.257 0.080 -0.07 1 -0.089 -0.054 0.594

-1.57 0.83 -0.76 -0.96 -0.92 12.19

0.116 0.408 0.448 0.336 0.360 0.000

0.278

0.463 0.025 0.004 -0.046 -0.058 0.676

6.20 0.33 0.06 -0.66 -1.29 24.16

0.000 0.738 0.953 0.510 0.198 0~000

0.574

0.041 0.019 0,022 -0.008 -0.062 0.180 0.608

0.33 0.26 0.31 -0.12 -1.42 4.21 19.00

0.743 0.795 0.756 0.906 0.158 0.000 0.000

0.590

Coefficient

aAntigen concentrations and egg counts were analysed after logra transformation. a multiple regression analysis was carried out with the els. By including egg counts in the model, only 28% of main objective of assessing which variable was the best the CSEA variation could be exnlained, while aooroximatelv 58% of the variation could be explained bv inpredictor of CSEA level. Three models, including age cluding CAA with or without egg coums. This close groups and sex and, in addition, egg counts (model 0, relationshin between CAA and CSEA suaeests that CAA (model 2), or both east counts and CAA (model 3), were constructed (Table?). By including egg counts CSEA levels are not merely a reflection oft& number in the model, while allowing for age and sex (model l), of eggs excreted in stool. only 28% of the variation in CSEA level could be exIn the recently infected population of Ndombo, the plained. Models 2 and 3 fitted the data better than modCSEA levels were much lower than in Maniema, alel 1 (R2=0.574 and 0.590, respectively), with the though the egg counts in both communities were nearly variables CAA (model 2) or both CAA and egg counts identical. While the age-prevalence pattern for CSEA (model 3) being highly significant. reached a peak in children 5-19 years of age, the levels The data set from Ndombo was analvsed bv multinle of CSEA showed no sianificant variation between the regression using the same 3 models as &e Maniema ;edifferent age groups. The finding that the correlation suits (data not shown). Although the large number of between CSEA levels and both CAA levels and egg persons negative for CSEA (30%) might have affected counts was much weaker than in the Maniema populathe outcome of the analysis, 36% to 42% of the CSEA tion is in accordance with the differences in age-intensilevels could be explained by the 3 models (R2=0.355, tv profiles. The absence of (significant) variation in 0.370 and 0.421, respectively), with egg count, CAA CSEA level in the Ndombo population might be due to level, and both together all being highly significant. the low CSEA concentrations. Althouah 70% of the serum samples were definitely positive in the CSEA assay, Discussion the measured CSEA concentrations approached the Neither detection of adult worm antigens nor countlower detection limit of the assay. ing excreted eggs in faeces provides accurate informaAntigens are secreted by eggs entrapped in tissues and tion about tissue egg loads. Assessment of the levels of by eggs finding their way to the intestinal lumen. The antigens secreted by eggs could generate more informaproportion of eggs retained in the tissues cannot be tion about the proportion of eggs retained in tissue and measured directly, but the general assumption is that thus be a marker for morbidity and for worm fecundity. only about 50% of the eggs produced by mated female Using the mab-based ELISA for detecting S. mansoni S. rnansolzi worms reach the faeces (WHO, 1985), implying that another 50% accumulate in the tissues. It is CSEA (NOUREL DIN et al., 1994a, 1994b), we analysed the kinetics of serum CSEA levels in 2 populations from clear that for a significant period (at least some weeks) antigens are excreted from these eggs, the mature miraareas with a different history of S. mans& transmission. In Maniema, where transmission has been occurring cidium being the major source of antigens (MITCHELL, 1990). Similarly, it is known that the eggs which are ulfor several decades, the CSEA assay showed good sensitivity (88%) comparable with the detection of CAA. timately excreted mature during migration to the gut. Therefore, although the excretion of antigens from these These findings are consistent with a previous study which demo&rated CSEA in 84% of 5l-serum samples 2 groups of eggs is poorly understood, it is not unrealistic to assume that eggs retained in the tissues contribute from individuals from Zaire chronicallv infected with S. munsoni (NOUREL DIN et al., 1994bj. In the present more to the CSEA level than eggs miaratine to the intesstudy, the range of CSEA concentrations was large, as tinal lumen. The high CSEA levels inMan:ema, togethwas also true for serum CAA concentrations and egg er with the good correlation with worm load, are counts. Similarly, the age-intensity profile of serum consistent with the assumption that the detected circuCSEA resembled those of egg counts and CAA levels. lating egg antigens are secreted by tissue eggs. The low The CSEA levels showed stronger correlation with the serum CSEA levels in the recently infected community worm-derived CAA levels than with egg counts, and this of Ndombo, combined with a high rate of egg excretion, finding was further analysed by linear regression modsuggests that the extent of deposition of eggs in the tis“-

H. A. M. NIBBELING ET/X. sues is still low, and eggs find their way to the colon lumen relatively fast in this particular recently-infected community. This is consistent with the absence of severe pathology (STELMA et al., 1993), although on the

other hand pathology might need a longer time to develOP. However,

the differences

between

the 2 populations

could also be related to efficiency in clearing these antigens, or to differences in egg antigen secretion by different S. nzansoni strains. Indeed, several studies have described schistosome soluble egg antigens which may vary considerably in concentration in eggs from different geographical locations (HAMBURGER et al., 1982; PILLAY, 1997). In addition, little is known about the relation between host immunity and egg retentioniexcretion. The present data clearly show that, to understand the cause of the marked els between populations

difference in CSEA antigen levwith a different transmission

history, more research has to be done on fundamental questions like the dynamics of egg migration, stage- and strain-specific antigen secretion and antigen clearance, and their relation with host immunity. Acknowledgements We thank Dr S. le Cessie (Department of Medical Statistics, Leiden University) for advice on the statistical evaluation of the data. We also thank Dieuwke Kornelis for careful reading of the manuscript. This investigation was financially supported by the UNDPWorld Ban&WHO Special Programme for Research and Training in Tropical Diseases (TDR), by the Research and Development Programme ‘Life Science and Technology for Developing Countries (STD-3)’ of the European Communities, and by the Netherlands Organization for Scientific Research in the Tropics (NWO/WOTRO). References Agnew, A. M., Fulford, A. J. C., De Jonge, N., Krijger, F. W., Rodriguez-Chacon, M., Gutsmann, V & Deelder, A. M. (1995). The relationship between worm burden and levels of a circulating antigen (CAA) of five species of Schistosoma in mice. Parasitology, 111, 67-76. Deelder, A. M., De Jonge, N., Boerman, 0. C., Fill%, Y. E., Hilberath, G. W., Rotmans, J. I’., Gerritse, M. J. & Schut, D. W. 0. A. (1989). Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies. American Journal of Tropical Medicine and Hygiene, 40, 268-272. De Jonge, N., Fill%, Y. E. & Deelder, A. M. (1987). A simple and rapid treatment (trichloroacetic acid precipitation) of serum samples to prevent non-specific reactions in the immunoassay of a proteoglycan. Journal oflmmunological Methods, 99, 1955197. De Jonge, N., De Caluwe, I?, Hilberath, G. W., Krijger, F. W., Polderman, A. M. & Deelder, A. M. (1989). Circulating anodic antigen levels in serum before and after chemotherapy with praziquantel in schistosomiasis mansoni. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83, 368-372. De Jonge, N., Kremsner, I? G., Krijger, F. W., Schommer, G., Fillie, Y. E., Kornelis, D., van Zeyl, R. J. M., van Dam, G. J., Feldmeier, H. & Deelder, A. M. (1990). Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 84, 815-818. DeVlas, S. J. & Gryseels, B. (1992). Underestimation of Schistosoma mansoni prevalences. Parasitology Today, 8, 27P277. Hamburger, J., Lustigman, S., Arap Siongok, T. K., Ouma, J. H. & Mahmoud, A. A. (1982). Characterization of a purified glycoprotein from Schistosoma mansoni eggs: specificity, stability, and the involvement of carbohydrate and peptide moieties in its serologic activity. Journal of Immunology, 128, 1864-1869.

Katz, N., Chaves, A. & Pellegrino, J. (1972). A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Revista do Instituto de Medicina Tropical de Sdo Paulo, 14,397400. Mitchell, G. F. (1990). Immunopathology of schistosomiasis. Reviews in Medical Microbiology, 1, 101-107. Nourel Din, M. S. A., Kornelis, D., van Zeyl, R. J. M. & Deelder, A. M. (1994a). Immunologic characterization of two monoclonal antibodies reactive with repetitive carbohydrate epitopes of circulating Schistosoma mansoni egg antigen. American Journal of Tropical Medicine and Hygiene, 50, 487-498. Nourel Din, M. S. A., Nibbeling, R., Rotmans, J. I’., Polderman, A. M., Krijger, F. W. & Deelder, A. M. (1994b). Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. American Journal of Tropical Medicine and Hygiene, 50, 585-594. Pillay, A. (1997). High performance SDS-polyacrylamide gel capillary electrophoresis of Schistosoma mansoni soluble egg antigens. Medical Science Research, 25, 5 13-5 16. Folderman, A. M., Mpamila, K., Manshande, J. I? & Bouwhuis-Hoogerwerf, M. L. (1985a). Methodology and interpretasurveillance of intestinal tion of parasitological schistosomiasis in Maniema, Kivu Province, Zaire. Annales de la SociU Beige de MLdecine Tropicale, 65, 243-249. Polderman, A. M., Mpamila, K., Manshande, J. I’., Gryseels, B. &Van Schayk, 0. (1985b). Historical, geological and ecological aspects of transmission of intestinal schistosomiasis in Maniema, Kivu Province, Zaire. Annales de la Socik! Belge de Midecine Tropicale, 65, 25 l-26 1. Polman, K., Stelma, F. F., Gryseels, B.,Van Dam, G. J., Talla, I., Niang, M., Van Lieshout, L. & Deelder, A. M. (1995). Epidemiological application of circulating antigen detection in a recent Schistosoma mansoni focus in northern Senegal, American Journal of Tropical Medicine and Hygiene, 53, 152-157. Stelma, F. F.,Talla, I., Polman, K., Niang, M., Sturrock, R. F., Deelder, A. M. & Gryseels, B. (1993). Epidemiology of Schistosoma mansoni infection in a recently exposed community in northern Senegal. American Journal of Tropical Medicine and Hygiene, 49,701-706. Talla, I., Kongs, A., Verle, I?, Belot, J., Sarr, S. & Coil, A. M. (1990). Outbreak of intestinal schistosomiasis in the Senegal River Basin. Annales de la Socie’ti Beige de Mkdecine Tropicale, 70,173-180. Van Lieshout, L., De Jonge, N., Bassily, S., Mansour, M. M. & Deelder, A. M. (1991). Assessment of cure in schistosomiasis patients after chemotherapy with praziquantel by quantitation of circulating anodic antigen in urine. American Journal of Tropical Medicine and Hygiene, 44,323-328. Van Lieshout, L., De Jonge, N., Mansour, M. M., Bassily, S., Krijger, F. W. & Deelder, A. M. (1993). Circulating cathodic antigen levels in serum and urine of schistosomiasis patients before and after chemotherapy with praziquantel. Transactions of the Royal Society of Tropical Medicine and Hygiene, 87, 311-312. Van Lieshout, L., Polderman, A. M., DeVlas, S. J., De Caluwe, I’., Krijger, F. W., Gryseels, B. & Deelder, A. M. (1995). Analysis of worm burden variation in human Schistosoma mansoni infections by determination of serum levels of circulating anodic antigen and circulating cathodic antigen. Journal of Infectious Diseases, 172, 1336-1342. Van Lieshout, L., Polman? K., Gryseels, B. & Deelder, A. M. (1998). Circulating antigen levels in two areas endemic for schistosomiasis mansoni indicate differences in worm fecundity. Transactions of the Royal Society of Tropical Medicine and Hygiene, 92, 115-l 19. WHO (1985). The Control of Schistosomiasis: Report of a WHO Expert Committee. Geneva: World Health Organization, Technical Report Series, no. 728. Received 14 January uary 1998

1998; accepted for publication

28Jan-