Yacoub AA, Lillywhite J. The effect of chemotherapy on the serological response of patients with schistosoma haematobium infection using the Enzyme linked immunosorbent assay. Journal of Faculty of Medicine-Baghdad 1985;27(3):19-29.

1. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
29
THE EFFECT OF CHEMOTHERAPY ON THE SEROLOGICAL RESPONS OF
PATIENTS WITH SCHISTOSOMA HAEMATOBIUM INFECTION USING THE
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Alim A.H. Yacoub and Jane E. Lillywhite
Department of Tropical Hygiene, London School of Hygiene & Tropical Medicine, Keppel
Street, London WCIE 7HT.
‫بال‬ ‫العالج‬ ‫تأثير‬
‫ع‬
‫قا‬
‫ق‬
‫اختباراالليزا‬ ‫باستعمال‬ ‫البولية‬ ‫بالبلهارزيا‬ ‫مصابين‬ ‫لمرضى‬ ‫المصلية‬ ‫االستجابة‬ ‫على‬ ‫ير‬
‫الخالصة‬
‫اجر‬
‫ي‬
‫من‬ ‫دم‬ ‫نماذج‬ ‫على‬ ‫األليزا‬ ‫اختبار‬
٢٣
‫وجدوا‬ ‫طفال‬
‫بالبلها‬ ‫مصابين‬
‫رزيا‬
‫أجر‬ .‫البولية‬
‫ي‬
‫بعد‬ ‫و‬ ‫العالج‬ ‫قبل‬ ‫االختبار‬
‫ه‬
‫بثالثة‬
‫اشه‬
.‫ر‬
‫العال‬ ‫لغرض‬ ‫االترينول‬ ‫عقار‬ ‫استعمل‬
‫االدرار‬ ‫فحص‬ ‫نتيجة‬ ‫كانت‬ . ‫ج‬
‫سالبة‬
‫من‬ ‫اشهر‬ ‫ثالثة‬ ‫بعد‬ ‫المصابين‬ ‫االطفال‬ ‫لكل‬
‫نسبة‬ ‫في‬ ‫ملحوظا‬ ‫انخفاضا‬ ‫هناك‬ ‫ان‬ ‫وجد‬ .‫العالج‬
‫ان‬ ‫ضد‬ ‫المتكونة‬ ‫المضادة‬ ‫االجسام‬
‫ت‬
‫تغير‬ ‫أي‬ ‫يالحظ‬ ‫لم‬ ‫ولكنه‬ ‫الديدان‬ ‫جيات‬ ‫ي‬
‫في‬
‫د‬ ‫مع‬ ‫عليها‬ ‫حصلنا‬ ‫التي‬ ‫النتائج‬ ‫قورنت‬ . ‫نفسها‬ ‫الديدان‬ ‫جينات‬ ‫انتي‬ ‫ضد‬ ‫المتكونة‬ ‫المضادة‬ ‫االجسام‬ ‫نسبة‬
‫ونوقشت‬ ‫مماثلة‬ ‫راسات‬
‫اهمي‬ ‫ضوء‬ ‫في‬
‫ت‬
‫الو‬ ‫للدراسات‬ ‫ها‬
‫ب‬
‫ائية‬
.‫البلهارزيا‬ ‫لمرض‬
SUMMARY
The enzyme linked immunosorbent assay (ELISA) was performed on blood samples obtained from
23 children found infected with Schistosoma haematobium before and three months after treatment
with hycanthone. All of the children were negative by urine examination three months after
treatment. Egg and worm antigens of S. mansoni were used in the assay. There was statistically
significant decline in the level of antibodies against egg antigens but there was no difference in
the level of antibodies against worm antigens. The comparability of the results with similar studies
and their relevance to the interpretation of serological data obtained from epidemiological studies
are discussed.

2. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
30
INTRODUCTION
One of the potential applications of serological tests in schistosomiasis is to evaluate the
effectiveness of chemotherapy whether at an individual level, to test for a complete cure, or at a
community level, to monitor a control programme. This requires an understanding of the pattern
of antibody to treatment.
Earlier studies, conducted to find out the impact of chemotherapy on the level of antibodies among
cases of schistosomiasis, applied such serological tests as the circumoval precipitin test,
immunofluorescence, haemagglutination, double gel diffusion, complement fixation and skin tests
(da Silva et al. 1975, da Silva et al. 1976, Hoshino et al. 1970, Rifaat et al. 1969 and Umaly et al.
1974)1-5
. Various antigens were used in these tests such as adult extracts of Fasciola gigantica, S.
mansoni and S. japonicum, erythrocytes sensitised with worm extracts, worm particles, and
schistosomes eggs.
The recent development of the enzyme linked immunosorbent assay (ELISA) and its evaluation
as a screening tool in epidemiological studies, using more purified antigens (Janitschke et al. 1981,
McLaren et al. 1987, McLaren et al. 1979 and Schinske et al. 1979)6-9
, raised the need to examine
the relation between chemotherapy and sero - reactivity using this test.
Mott and Dixon (1982)10
and Butterworth et al. (1984)11
have recently used the ELISA to measure
pre- and posttreatment antibody levels of patients infected with S. mansoni.
The present study reports the results of the ELISA before and after treatment of children found
infected with S. haematobium.
MATERIALS AND METHODS
The study was carried out in Basrah governorate, southern Iraq. The area is known to contain
foci endemic for S. haematobium. A control programme based on selective chemotherapy is being
implemented by the Endemic Diseases Centre of Basrah.
A survey was carried out among primary school children from October 1983 to March 1984.
The initial screening was done by urine examination using the sedimentation method. Twenty-
three children found infected with S. haematobium were treated with hycanthone 2 mg per kg body

3. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
31
weight intramuscularly. Blood and urine samples were obtained before and three months after
treatment. Samples were also collected from ten uninfected children at the same time.
In addition, we were able to identify seven children who were found infected and treated
during the survey which was carried out a year prior to the present study. Blood and urine samples
were collected from them as well.
The pre- and post-treatment urine samples were examined by the Nucle- pore filtration method
as described by Peters et al. (1976)12
. Five millilitres of urine were extracted from each sample
using plastic disposable syringes, and injected through filter holders containing Nuclepore filters
of 12 microns pore size and 13 mm diameter.
Finger prick blood samples were absorbed onto Whatman number 3 filter paper, allowed to dry
and kept in plastic bags at -20°C to be tested later by the ELISA.
The enzyme linked immunosorbent assay
The dried blood spots on the filter paper were cut using a punch calibrated to produce 8
microlitres blood samples. Each spot was eluted in 200 microlitres of incubation buffer made of
one litre of Phosphate Citrate buffer and 0.45 mls of Tween 20.
The test was based on that described by McLaren et al7
. (1978) with some modifications.
Soluble egg antigen (SEA), Excretory Secretory (ES) and Freeze Thaw (FT) worm antigens of S.
mansoni were used at concentrations of 2, 200 and 2.5 microgrammes per ml respectively. FT
antigen was prepared following the method of Ramalho - Pinto et al. (1978)7
. SEA and ES antigens
were prepared according to the methods described by Boros and Warren (1970)14
and Deedler et
al. (1976)15
respectively.
S. mansoni antigens were used because standardized S. haematobium antigens were not
available due to difficulties in maintaining S. haematobium within the laboratory.
The test samples, the reference positive and the negative sera were used at 1 in 300 dilutions.
Anti-human IgG labelled peroxidase conjugate (Dakopatts) was used at a dilution of 1 in 4000.
Ortho-phenylene diamine was used as a substrate. The reaction was stopped with 2.5 M sulphuric
acid. The absorbance values were read at an extinction point of 492 nm when the reference positive
read 1. This was selected rather than 0.75 used by McLaren et al. (1978) since greater sensitivity

4. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
32
was needed to detect S. haematobium infection when using S. mansoni antigens. A cut off point
of 0.4 was selected to discriminate between positives and negatives.
STATISTICAL ANALYSES
The ELISA readings were transformed into their log values. The geometric means, 95%
confidence intervals and tests of significance were calculated from the transformed data.
A paired test was performed to assess the statistical significance of the change in the levels of
antibodies to different antigens before and after treatment.
RESULTS
The geometric mean of the egg output of the infected cases before treatment was 13.6 eggs/5
ml (range 1 to 400 and the 95% confidence interval= 7.5-24.6 eggs/5 mis). All of the treated
children were found to be negative by urine examination 3 months later (only dead ova were
identified in a few cases).
The distribution of the ELISA readings before and after treatment, using SEA and FT worm
antigen is shown in Figure 1. The distribution of the absorbance values using ES antigen is not
shown since it is very similar to that of the FT antigen. Using SEA, 19 out of 23 had absorbance
values above 0.4 (82.6%) before treatment. Six of these 19 (31.6%) had values below 0.4 three
months following treatment.

5. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
33
Fig.1.The distribution of absorbance value of individuals infected with S. haematobium before and
after treatment, using soluble egg and freeze thaw antigens.
Table 1 shows the geometric means and 95% confidence intervals of the absorbance values,
using SEA, for the infected children before and after treatment. The reduction in the level of
antibodies against egg antigens. The reduction in the level of antibodies against egg antigens
following treatment was found to be statistically significant by the paired t test (t=7.094 degrees
of freedom 22 P<0.001).
Table 1. the geometric means (E 492 nm) and 91% confidence intervals using SEA before and
after treatment of infected and uninfected children.
Geometric mean (95%) confidence interval)
Group Number Before treatment After treatment t p
Infected children 23 0.55 (0.44-0.69) 0.42 (0.34-0.52) 7.094 0.001
Uninfected children 10 0.25 (0.18-0.35) 0.21 (0.19-0.23) 1.343 NS
For the uninfected children there was no significant difference in the ELISA readings before
and after treatment over the same period of time (t = 1.34 df = 9 NS). The mean value of this group
is significantly lower than those of the infected children whether before or after treatment (0.25
compared to 0.55 before treatment and 0.21 compared to 0.42 after treatment).

6. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
34
The results of testing the samples from the infected children against worm antigens are shown
in table 2. The difference in the level of antibodies before and after treatment was not found to be
statistically significant for both types of worm antigens (t=0.248 for FT antigen and t=0.738 for
ES antigen NS).
The seven children, who were treated one year prior to the present study, were all negative by
urine examination. The geometric mean of the absorbance values was 0.59 (95% confidence
interval 0.37-0.94). Six of them had values above 0.4.
Table 2. The geometric mean (E 492 nm) and 95% confidence intervals of samples from infected
children before and after treatment using FT and ES worm antigens.
Geometric mean (95% confidence interval)
Antigen Before treatment After treatment t p
FT 0.36 (0.30-0.44) 0.37 (0.30-0.44) -0.248 NS
ES 0.38 (0.31-0.47) 0.42 (0.32-0.55) -0.738 NS
DISCUSSION
The sensitivity of the ELISA in detecting S. haematobium infection using either egg or worm
antigens of S. mansoni was reported to range between 80% and 86% (Janitschke et al. 1981,6
McLaren et al. 19787
and Schinski et al. 19769
). Ismail (1980)16
tested sera, by the ELISA, from
Egyptian children and adults who were found infected with S. haematobium. Egg, worm and
cercarial antigens of both S. haematobium and S. mansoni antigens were used. The S. mansoni
antigens were found to be as reactive as the S. haematobium antigens against the same set of sera.
S. mansoni egg antigen was found more reactive than either worm or cercaial antigens. In this
study, 19 out of the 23 (83%) infected children could be considered as serologically positive while
all the uninfected children had absorbance values below the selected 0.4 cut-off point.
Thus it seems that the ELISA, using S. mansoni antigen, is of appropriate validity for the
purpose of the interpretation of the results obtained in this study.
The significant reduction in the level of antibodies to egg antigen following chemotherapy, as
reported here, is in agreement with the results obtained from testing, by the (ELISA) pre- and post-
treatment sera of S. mansoni infection (Matt and Dixon 1982)10
. They, however, reported a

7. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
35
significant reduction in the level of antibodies to worm antigens as well. This is probably due to
following up their cases six rather than three months after treatment.
On the other hand, Butterworth et al. (1984)11
measured the level of antibodies, to egg and
worm antigens, of S. mansoni cases using the ELISA test before and 5 weeks after treatment. There
was a detectable rise in the level of antibodies to worm antigens but there was no variation in the
level of antibodies to egg antigens.
A rise in the level of antibodies to worm antigens within four weeks of treatment of cases of
schistosomiasis using other serological tests has been reported by da Silva et al. (1975)1
, da Silva
et al. (1976)2
and Hoshino et al. (1970)3
. Such a rise has also been reported by Umaly et al.5
(1974).
However, the latter reported their findings in terms of the distribution of the positives before and
after treatment rather than using the actual level of antibodies in assessing the time impact of
chemotherapy.
A reduction in the level of antibodies to egg antigens following treatment of cases of
schistosomiasis was also reported by Rifaal et al. (1969)4
.
Thus, a provisional time sequence pattern of the serological response following chemotherapy
of cases of schistosomiasis could be described based on the results of the present study and those
already reviewed. Antibodies to egg antigens start to decline after approximately a month of
commencing treatment and continue such decline unless there is a further exposure to infection.
Antibodies to worm antigens, on the other hand, show a rise within the first four weeks of
chemotherapy and start to decline thereafter to reach their pretreatment level approximately three
months later and decline further afterwards.
In this study the rate of sero-reversion among infected cases was found to be 311. During the
period of the follow up there was no possibility of reinfection since water contact was minimal
because of the cold weather and the fact that Bulinus snails (the intermediate hosts) have never
been detected at this time of the year by the staff of the Endemic Diseases Centre (personal
communication).
However, these reverted cases might convert serologically on further exposure irrespective of
whether such exposure leads to patient infection. This was apparent in the case of the seven
children who were found infected and treated the year before the present survey. Their level of
antibodies (as measured by the geometric mean of the absorbance values) was found to be higher

8. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
36
than that of the children with current infection. This might suggest that infected children become
sensitized to schistosomal antigens and that on further exposure they start producing antibodies at
an accelerated rate even if they do not get a patent infection. This hypothesis needs to be tested
further by serologically monitoring re-exposure to infection in a larger population for at least a
year. The results obtained here illustrate a problem associated with conversion following re-
exposure when chemotherapy has been the sole means of control. It follows that serology would
be most useful in assessing control programmes based on chemotherapy in conjunction with
molluscicides.
ACKNOWLEDGEMENTS
We would like to express our gratitude to Dr. C.C. Draper. Director of the Sero-epidemiology
Laboratory, London School of Hygiene and Tropical Medicine and the staff of the Endemic
Diseases Centre, Basrah for their help during the present study.
REFERENCES
1. Da Silva, L.C., Hoshino-Shimizu, S., Kanamura, H., Strassmann, P.G., Camargo, M.E., Júnior,
H.S., Lopes, J.D., Chamone, D.A.F., Raia, S. and Da Silva, G.R., 1975. Serum antibody changes
after chemotherapy of patients with schistosomiasis mansoni. A statistical analysis. Revista do
Instituto de Medicina Tropical de São Paulo, 17(6), pp.344-349.
2. da Silva, LC., Hoshino Shimizu, S., Kanamura, H., Strassman, P.G., Camargo, M.E., Sette, H.
JR., Lopes, J.D., Chamone, D.A.F., Raia, S., & da Silva, G.R. (1976). Serum antibody changes
after repeated chemotherapeutic series in parasitologically cured patients with S. mansoni. Revista
do Instituto de Medicina Tropical de Sao Paulo, 18, 206-210.
3. Hoshino, S., Camargo. M. & da Silva, L. C. (1970). A comparison between the
haemagglutination test with formalin treated erythrocytes and the immunofluorescent test with
worm particles, for schistosomiasis. Revista do Instituto de Medicina Tropical de Sao Paulo, 12,
136-143.

9. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
37
4. Rifaat, M.A., Ismail, I., El-Mahallawy, M.N., Awaad, S. & Essawy, M. (1969), A comparative
study of some immunological tests for schistosomiais before and after treatment. Transactions of
the Royal Society of Tropical Medicine and Hygiene, 63, 338-342.
5. Umaly, R.C., Delerich, S. & Haas, J. (1974). Immunological tests for bilhariziasis.
Tropenmedizin und Parasitologie, 25, 422-432.
6. Janitschke, K., El-Kalouby, A.H., Braun Munzinger, R.A., El-Baz, R.A. & Mahmoud, M.
(1981). Evaluation of the ELISA test as an epidemiological tool in schistosomiasis. The Journal
of Tropical Medicine and Hygiene, 84, 147-154.
7. McLaren M., Draper, C.C., Roberts, J.M., Minter - Goedbloed, E.F., Ligthart, G.C., Taesdale,
G.H., Amin, M.A., Omer, A. H.S., Bartlett, A. & Voller, A. (1978). Studies on the enzyme linked
immunosorbent assay (ELISA) test for S mansoni infections. Annals of Tropical Medicine and
Parasitologu, 72, 243-253.
8. McLaren, M.L., Long, E.G., Goodgame, R.W. & Lillywhite, J.E. (1979) Application of the
enzyme linked immunosorbent assay (ELISA) for the serodiagnosis of S. mansoni infection in St.
Lucia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 73, 636-639.
9. Schinski, V. D., Cutter, W.C. & Hurrel, K. D. (1979). Enzyme and 1125
-label- led anti-
immunoglobulin-assay in the immunodiagnosis of schistosomiasis. American Journal of Tropical
Medicine and Hygiene, 25, 824-831.
10. Mott, K.E. & Dixon, H. (1982). Collaborative study on antigens for immunodiagnosis of
schistosomiasis. Bulletin of the World Health Organization, 60, 729-753.
11. Butterworth, A.E., Dalton, P.R., Dunne, D.W., Mugambi, M, Duma, JH., Richardson, B.A.,
Siongok, T.K. & Sturrock, R.F. (1984) Immunity after treatment of human Schistosomiasis
mansoni. 1. Study design, pretreatment observation and the results of treatment. Transactions of
the Royal Society of Tropical Medicine and Hygiene, 78, 108-123.
12. Peters, P.A., Warren, K.S. & Mahmoud, A.F. (1979) Rapid, accurate quantification of
schistosome eggs via Nuclepore filters. Journal of Parasitology, 62, 154-155.
13. Ramalho Pinto, F. J., Goldring, O.L. Smithers, S.R. & Playfair, J.H.L. (1976). T-cell helper
response to antigens of S. mansoni in CBA mice Clinical and Experimental Immunology, 26, 327-
333.

10. J. Fac. Med. Baghdad 1985 Vol. 27 No. 3
38
14. Boros, D.L. & Warren, K.S. (1970). Delayed hypersensitivity type granuloma formation and
dermal reaction Induced and elicited by a soluble factor isolated from S. mansoni eggs. Journal of
Experimental Medicine, 132, 488-507.
15. Deedler, A.M., Klappe, H.T.M., Van den Aardweg, G.J.M.J. & Van Meer- beke, E.H.E.M.
(1976) S. mansoni: Demonstration of two circulating antigens in infected hansters. Experimental
Parasitology, 40, 189-197.
16. Ismail M.M. (1980) Observations on some serological tests in Schistosoma haematobium in
man and infected animals. PhD Thesis. University of London. 105-122.