636 TRANSACTIONS OPTHEROWI. SOCIETYOFTROPICAL. MEDICINEANDHYGIENE,
VOL.
73, No.
6, 1979
Application of the enzyme linked immunosorbent assay (ELISA) for the serodiagnosis of Schistosoma mansoni infections in St. Lucia MOIRA L. MCLAREN’, E. G. LONGS, R. W. GOODGAME~ AND JANE E. LILLYWHITE~ 1 Ross Institute, London School of Hygiene & Tropical Medicine, Keppel St., London WCIE 7HT 2 Research & Control Department, P.O. Box 83, Castries, St. Lucia 3 Massachusetts General Hospital, Massachusetts, USA
Summary As part of our search for a serodiagnostic assay to replace the expensive and tedious stool examination in the diagnosis of Schistosoma mansoni infection, we have studied the sensitivity, specificity and quantitative features of an enzyme linked immunoassay (ELISA) using crude S. mansoni egg antigen preparation. Results of studies carried out in both London and St. Lucia indicate that the assay can give useful serodiagnostic information ranging from 82 to 99 *5 % sensitivity, depending on level of infection intensity and method of blood collection and 100 71 specificity in St. Lucian/St. Vincent populations. The St. Lucia study also showed that the assay could be operated in a qualitative form in an endemic area. Introduction A scheme to control transmission of Schistosoma mansoni infection in St. Lucia, using a combination of mollusciciding, chemotherapy and improved water supplies has resulted in prevalence rates being reduced to below 59/o in several areas. Diagnosis of infection by stool examination alone becomes increasingly difficult in areas of low transmission and low infection intensity and there is an serodiagnostic ncreasing need for improved methods (JORDAN, 1977). The enzyme linked immunosorbent assay (ELISA), which has had wide application in the serodiagnosis of parasitic infections (VOLLER et al., 1976), has shown promise for the detection of antibodies in S. mansoni infections when tested on large numbers of sera from infected people (MCLAREN et al., 1978). The assay has not, however, been used for screening populations with very low infection intensities, as in St. Lucia, and has not been operated under conditions experienced in an endemic area. The use in the assay of sera eluted from filter paper blood spots is an&her important aspect which needs more rigorous evaluation. This method of blood collection will probably be used for routine surveys in St. Lucia, as it provides a convenient means of transporting test material from the field (LONG et al., in press). ’ This report de&rib& two studies carried out in London and St. Lucia to assess: (i) sensitivity of the assay in detecting antibodies in .a wide range of infection intensities from St. Lucian patients using serum collected by venepuncture and as filter paper
blood spots; (ii) specificity of the assay when tested with uninfected controls from St. Vincent; (iii) quantitative features of the assay in relation to infection intensity. Materials and Methods Study l-Ross Institute, London Serum samples Sera from 213 male and female patients infected with S. mansoni, whose ages ranged from under six years to over 50 were examined. Each serum sample had been drawn by venepuncture before treatment and at the same time the intensity of infection was determined by taking the arithmetic mean of three stool samples quantitatively analysed by the filtration staining technique (BELL, 1963). The samples were frozen at - 20°C until the present study was done, Intensities of infection varied from 10 to 200 eggs per gram of faeces (e.p.g.), with 71 patients excreting fewer than 25 e.p.g. No patients had hepatosplenic schistosomiasis. To assess test specificity, sera from 100 schoolchildren aged six-to 10 years were collected on filter paper from a single finger stab wound following the method of BRUCE-CHWATT et al. (1973). The samples consisted of 94 St. Vincentian control sera intersnersed with six St. Lucian positives. The St. Vinceitians all had a single stool specimen examined for S. mansoni and othe; intestinal helminths. None was nositive for S. mansoni. 49% had Trichuris. 38 “A had Ascaris and 14 Y, had hookworm infections: Study 2-Research & Control Department, St. Lucia 326 serum samples obtained in 1978 from St. Lucian patients were examined. These were collected as drops of blood on filter paper. All patients had been regularly examined for ova by the Bell filtration technique during the period 1968-77 and, on the basis of parasitological diagnosis were divided into four groups: (i) individuals repeatedly positive by stool examination; (ii) individuals positive once by stool examination; (iii) individuals consistently negative by stool examination; (iv) individuals with recently acquired infections ( f one year). Enzyme Assay In both studies the assay was performed as previously described (MCLAREN et al., 1978). All tests were carried out using a soluble egg antigen at an optimum concentration of 2.5 pg/ml protein. All serum samples were screened initially at a
M. L. MCLAREN
637
et cd.
Table I-Sensitivity and specificity levels in a group of S. mansoni-infected patients, using serum collected by venepuncture, and a control group of uninfected children from St. Vincent
Serum samples
No. tested
St. Lucia
213
St. Vincent
Table
5; Seropositive/Mean anti-egg total (-W 99.510.79
94
II-Anti-egg
total
and IgG levels
Egg count (em)
0
jO.08 + 0.01
and relationship
to infection
Anti-egg MeanE400
No. tested
IO-25 26-200 200
i 0.02
71 114 28
OD & SE. anti-egg IgG (E490) 98.610.68 0
31 0.03
jo.09 + 0.01
intensity Anti-egg IgG Mean E490 i S.E.
total & S.E.
0.7410.03 0.77 10.02 0.83 10.04
“0.58 kO.03 0.72&0*02 *0.78 kO.03
* Mean ODs of the high and low intensity groups different from each other by unpaired t-test, p c 0.001. Table III-Sensitivity filter paper blood Serum samples
of ELISA spots
No. tested
in detecting
antibodies
Mean frequency stool examin.
in two groups
of infected
patients
using
?b Seropositivej Mean E400 i S.E.
Mean egg count (epg)
Group 1
157
6.4
24.2
*93/0.38 ho.01
Group 2
180
6.4
15.6
*82/0.35 10.02
* Includes samples with values of between 0 * 18-0.25 Table IV-Serological infections Serum samples
reactions
in a group
parasitologically
No. tested
Mean frequency stool examin.
Group 3
53
6.7
Group 4
36
7.0
** Re-examination
and in a group
Mean egg count (epg)
with recent
“a Seropositive
0
““42
-
of seropositive reactors by stool examination
dilution of 1:300, and subsequently some blood eluates were rescreened at a lower dilution of 1:150. The amount of specific bound antibody was measured with both a phosphatase labelled antihuman total and IgG conjugate, and a peroxidase labelled anti-human IgG conjugate. Paranitrophenyl phosphate was used as substrate and 3M NaOH as inhibitor in the phosphatase system, and orthonhenylene diamine-H,0,/8N HeSO, as substrate and inhibitor in the peroxidase system. The amount of bound antibody was assayed photometrically at 400 nm for phosphatase and 490 nm for peroxidase, bv measuring the product of enzyme substrate breakdown when the reference positive serum reached an OD level of 0.75
negative,
97
revealed 48 9, to be infected
Samples with OD levels of z 0 ~25 were considered positive, and those with readings of 0 * 18 to 0.25 as borderline positives. Where possible these samples were rescreened at a lower serum dilution of 1: 150 to increase the level of reactivity. All tests were performed without knowledge of the infection status of the patient. Results Study 1 Table specificity collected labelled antibody
I shows the levels of sensitivity and of the assay, obtained with serum samples by venepuncture. With a phosphatase anti-globulin which measures the total response only one adult St. Lucian serum
638
APPLICATION OF ELISA FOR S. mUnSOniSERODIAGNOSISIN ST. LUCIA
was negative. With the peroxidase anti-IgG three false negative reactions occurred in adults; these were in individuals excreting 10 e.p.g., the minimum detected by the filtration staining technique. Of the 100 blood spots examined blind for test specificity, no cross reactivity was observed with the 94 St. Vincent control samples and all had OD levels of below 0.18 when screened at dilutions of 1:300 and 1: 150. Five of the six interspersed St. Lucian positives reacted in the test and subsequent parasitological re-examination of the one seronegative revealed this individual to be uninfected. Table II shows the quantitative aspects of the assay, When grouped by intensity of infection a significant difference exists between the mean OD from low to high intensity of infection when using the peroxidase labelled anti-IgG system. No such correlation exists with the phosphatase total antiglobulin system. Regression analysis by the least squares fit method for the anti-IgG system yields a correlation coefficient of 0.183, p < 0.05.
Study 2 Table III shows the levels of sensitivity of the assay obtained with blood eluates in two groups of infected patients. In Group 1, those positive parasitologically on more than one occasion, the seronegatives occurred in individuals excreting 10 e.p.g. A greater number of false negatives occurred in Group 2 and again these were generally in individuals with minimal infection intensities. Table IV shows the results obtained in Grouts 3 and 4. 42 ‘:A of individuals in Group 3, who were consistently ova-negative, reacted in ELISA. Repeat stool examination of the seropositives revealed 487; of them to be infected when three papers were re-examined by the filtration technique. This group comprised two distinct age categories, three to 18 and 40 to 73. The seronegatives showed a bimodal distribution, but all but one of the seropositives occurred in the three to 18 age group. Group 4 comprised individuals with recently acquired infections, both new cases and reinfections. Antibodies were detected in all but one individual who, when re-examined parasitologically, was found to be negative on four separate examinations. Hence the test was 100 u; in detecting antibodies in recent infections. Discussion These studies were performed as part of a search for a sensitive and specific serological test for S. mansoni infection which could be used in St. Lucia to supplement or replace stool examination in schistosomiasis control schemes. Such a test must be valid in all age groups and in very low intensity infections. The results from the tests carried out in both studies one and two showed a range of sensitivity levels in the assay. Using serum samples collected by venepuncture, test sensitivity was almost 100”; and the few false negative reactions that occurred were all in patients with a minimum egg count of 10 e.p.g. The study in St. Lucia was based entirely on blood samples, collected on filter paper, from patients with extremely low infection levels. Test
sensitivity in this population ranged from 82 to 9376. Antibody levels were also lower, as reflected by lower absorbance readings compared to those obtained in the earlier study, and there were also a greater number of borderline positive reactions. Using a lower serum dilution of I:150 for test sera as in a previous study (MCLAREN et al., 1978) increased the level of antibody reactivity. A comparative study of matched serum samples and blood eluates from infected Sudanese patients did not reveal any quantitative difference in antibody response in the assay (MCLAREN, unpublished data) however, the sera examined were from heavily infected patients with high antibody levels. In cases where antibody levels are very low it may be less stable when absorbed on to paper. Most of the false negative reactions occurred in individuals from Group 2 who had been diagnosed as ova-positive on only one occasion and who had very low egg counts (group mean 15.6 e.p.g.). The possibility that some of these individuals were stool false positives cannot be discounted, particularly as swapping stool samples has been known to occur on a small scale in some villages and there is also a small, but possible, risk of contamination of the apparatus used in the Bell filtration technique. The assay also detected antibodies in a group of individuals who were found to be consistently negative by routine stool examination. All but one of the seropositives occurred in the three to 18 age group, and 48”, of these were confirmed as stool positive when they were examined parasitologically on the basis of three stained papers, instead of one which is the standard for routine examination. It is hiehlv likelv that all the serooositive individuals in thi three to 18 age group were infected and had been missed on stool examination because they had minimal infection intensities, below the level of consistent detection (10 e.p.g.) by the Bell filtration technique. In addition to being able to detect antibodies in very low level infections, antibodies were also detected in a group of individuals with recent infections. This represents an important group with respect to possible future use of the test for incidence measurements. The specificity of the antibody response was tested using sera from St. Vincentian schoolchildren. St. Vincent is an island 30 miles south of St. Lucia with similar geography, peoples and culture but without S. mansoni. The suitability of this population as a control has been discussed (WARREN et al., 1973). All of the sera from this population were negative when both phosphatase and peroxidase conjugates were used in the assay. At serum dilutions of both 1:300 and 1: 150 all sera gave OD levels of below the borderline positive level of 0.18. A study of the quantitative aspects of the assay showed the absorbance in the anti-egg IgG system to be crudely quantitative. The results given here show that higher egg counts tend to give higher OD levels, a finding not observed when the total antibody response is measured. Studies carried out on Sudanese children with wide-ranging S. mansoni infection intensities have revealed similar findings (unpublished data). This suggests that the IgG
M. L. MCLAREN
component of the anti-SEA response may be reacting with fewer, but more specific, antigens than the broad total antibody response. This finding is not however of good enough fit to be of any practical value at this point. Using a more purified antigen may increase the quantitative aspects of this test. These results compare favourably with similar studies carried out by LONG et al., on the use of the qualitative MSAl-radioimmunoassay for serodiagnosis of S. mansoni infection in St. Lucian populations. Acknowledgements We would like to thank Dr. P. Jordan and Dr. C. C. Draper for helpful advice and encouragement. This investigation received financial support from the UNDPWorld Bank/WHO Soecial Programme for Research and Training in Tropical Diseases. and the Edna McConnell Clark Foundation. References Bell, D. R. (1963). A new method S. mansoni eggs in faeces. Bulletin Health
Organization,
for counting of the World
29, 525-530.
et cd.
639
control.
American Journal and Hygiene, 26, 877-886.
of
Tropical
Medicine
Long, E. G., Pelley, R. P. & Hunt, W. (1979). Radioimmunoassay for the detection of Schistosoma mansoni infection using blood spots on filter paper. Bulletin of the World Health Organization, (in press). McLaren, M. L., Draper, C. C., Roberts, J. M., Minter-Goedbloed, E., Ligthart, G. S., Teesdale, C. H., Amin, M. A., Omer, A. H. S., Bartlett, A. & Voller, A. (1978). Studies on the enzyme linked immunosorbent assay (ELISA) test for Schistosoma mansoni infections. knnals df Tropical Medicine and Parasitolopv.Y.2, 72. 243-253. Voller, A., Bartlett, A. & Bidwell, D. E. (1976). Enzyme immunoassays for parasitic diseases. Transactions of the Royal Society Medicine and HJjgiene, 70, 98-106.
of
Tropical
Warren, K. S. Kellermeyer, R. W., Jordan, P., Littell, A. S., Cook, J. A. & Kagani, G. (1973). Immunologic diagnosis of schistosomiasis. 1. A controlled study of intradermal (immediate and delayed) and serologic tests in St. Lucians mansoni and uninfected infected with Schistos&a St. Vincentians. American Journal of Tropical Medicine and HJjgiene, 22, 189-198.
Bruce-Chwatt, L. J., Draper, C. C. & Konfortion, evidence of P. (1973). Seroepidemiological eradication of malaria from Mauritius. Lancer, iii, 547-55 1. Jordan, P. (1977). Schistosomiasis-Research to
Accepted
Admission to the Fellowship of the Society All registered medical and veterinary practitioners and others interested in scientific pursuits relating whose qualifications are to tropical medicine, deemed satisfactory by the Council, are eligible for election as Fellows of the Society. The annual subscription payable by Fellows is E15-00 which becomes due in advance on the 1st April of each year. The Transactions and the current Year Book of the Society are posted regularly to every Fellow whose subscription is not in arrear. Further information may be obtained from the Hon. Secretaries, Manson House, 26 Portland Place. London WlN 4EY. or from the Local . Secretary of the district.
CORPORATE MEMBERSHIP WITH THE ROYAL COMMONWEALTH SOCIETY, 18 NORTHUMBERLAND AVENUE, LONDON, WC2 5BJ The Society has taken up Corporate Membership with the above Society, which has premises just off Trafalgar Square. Overseas Fellows can now use the facilities of this Society, when they come to London, for a period of 21 days in any three months provided that all booking is done through the Secretary at Manson House. Fellows who wish to use these facilities must give the Secretary adequate notice of their requirements particularly during the summer when, at least two months’ notice will be required. Details of the facilities available will be sent to Fellows on request.
for
publication
30th January,
1979.
VOL.
73, No.
6, 1979
Application of the enzyme linked immunosorbent assay (ELISA) for the serodiagnosis of Schistosoma mansoni infections in St. Lucia MOIRA L. MCLAREN’, E. G. LONGS, R. W. GOODGAME~ AND JANE E. LILLYWHITE~ 1 Ross Institute, London School of Hygiene & Tropical Medicine, Keppel St., London WCIE 7HT 2 Research & Control Department, P.O. Box 83, Castries, St. Lucia 3 Massachusetts General Hospital, Massachusetts, USA
Summary As part of our search for a serodiagnostic assay to replace the expensive and tedious stool examination in the diagnosis of Schistosoma mansoni infection, we have studied the sensitivity, specificity and quantitative features of an enzyme linked immunoassay (ELISA) using crude S. mansoni egg antigen preparation. Results of studies carried out in both London and St. Lucia indicate that the assay can give useful serodiagnostic information ranging from 82 to 99 *5 % sensitivity, depending on level of infection intensity and method of blood collection and 100 71 specificity in St. Lucian/St. Vincent populations. The St. Lucia study also showed that the assay could be operated in a qualitative form in an endemic area. Introduction A scheme to control transmission of Schistosoma mansoni infection in St. Lucia, using a combination of mollusciciding, chemotherapy and improved water supplies has resulted in prevalence rates being reduced to below 59/o in several areas. Diagnosis of infection by stool examination alone becomes increasingly difficult in areas of low transmission and low infection intensity and there is an serodiagnostic ncreasing need for improved methods (JORDAN, 1977). The enzyme linked immunosorbent assay (ELISA), which has had wide application in the serodiagnosis of parasitic infections (VOLLER et al., 1976), has shown promise for the detection of antibodies in S. mansoni infections when tested on large numbers of sera from infected people (MCLAREN et al., 1978). The assay has not, however, been used for screening populations with very low infection intensities, as in St. Lucia, and has not been operated under conditions experienced in an endemic area. The use in the assay of sera eluted from filter paper blood spots is an&her important aspect which needs more rigorous evaluation. This method of blood collection will probably be used for routine surveys in St. Lucia, as it provides a convenient means of transporting test material from the field (LONG et al., in press). ’ This report de&rib& two studies carried out in London and St. Lucia to assess: (i) sensitivity of the assay in detecting antibodies in .a wide range of infection intensities from St. Lucian patients using serum collected by venepuncture and as filter paper
blood spots; (ii) specificity of the assay when tested with uninfected controls from St. Vincent; (iii) quantitative features of the assay in relation to infection intensity. Materials and Methods Study l-Ross Institute, London Serum samples Sera from 213 male and female patients infected with S. mansoni, whose ages ranged from under six years to over 50 were examined. Each serum sample had been drawn by venepuncture before treatment and at the same time the intensity of infection was determined by taking the arithmetic mean of three stool samples quantitatively analysed by the filtration staining technique (BELL, 1963). The samples were frozen at - 20°C until the present study was done, Intensities of infection varied from 10 to 200 eggs per gram of faeces (e.p.g.), with 71 patients excreting fewer than 25 e.p.g. No patients had hepatosplenic schistosomiasis. To assess test specificity, sera from 100 schoolchildren aged six-to 10 years were collected on filter paper from a single finger stab wound following the method of BRUCE-CHWATT et al. (1973). The samples consisted of 94 St. Vincentian control sera intersnersed with six St. Lucian positives. The St. Vinceitians all had a single stool specimen examined for S. mansoni and othe; intestinal helminths. None was nositive for S. mansoni. 49% had Trichuris. 38 “A had Ascaris and 14 Y, had hookworm infections: Study 2-Research & Control Department, St. Lucia 326 serum samples obtained in 1978 from St. Lucian patients were examined. These were collected as drops of blood on filter paper. All patients had been regularly examined for ova by the Bell filtration technique during the period 1968-77 and, on the basis of parasitological diagnosis were divided into four groups: (i) individuals repeatedly positive by stool examination; (ii) individuals positive once by stool examination; (iii) individuals consistently negative by stool examination; (iv) individuals with recently acquired infections ( f one year). Enzyme Assay In both studies the assay was performed as previously described (MCLAREN et al., 1978). All tests were carried out using a soluble egg antigen at an optimum concentration of 2.5 pg/ml protein. All serum samples were screened initially at a
M. L. MCLAREN
637
et cd.
Table I-Sensitivity and specificity levels in a group of S. mansoni-infected patients, using serum collected by venepuncture, and a control group of uninfected children from St. Vincent
Serum samples
No. tested
St. Lucia
213
St. Vincent
Table
5; Seropositive/Mean anti-egg total (-W 99.510.79
94
II-Anti-egg
total
and IgG levels
Egg count (em)
0
jO.08 + 0.01
and relationship
to infection
Anti-egg MeanE400
No. tested
IO-25 26-200 200
i 0.02
71 114 28
OD & SE. anti-egg IgG (E490) 98.610.68 0
31 0.03
jo.09 + 0.01
intensity Anti-egg IgG Mean E490 i S.E.
total & S.E.
0.7410.03 0.77 10.02 0.83 10.04
“0.58 kO.03 0.72&0*02 *0.78 kO.03
* Mean ODs of the high and low intensity groups different from each other by unpaired t-test, p c 0.001. Table III-Sensitivity filter paper blood Serum samples
of ELISA spots
No. tested
in detecting
antibodies
Mean frequency stool examin.
in two groups
of infected
patients
using
?b Seropositivej Mean E400 i S.E.
Mean egg count (epg)
Group 1
157
6.4
24.2
*93/0.38 ho.01
Group 2
180
6.4
15.6
*82/0.35 10.02
* Includes samples with values of between 0 * 18-0.25 Table IV-Serological infections Serum samples
reactions
in a group
parasitologically
No. tested
Mean frequency stool examin.
Group 3
53
6.7
Group 4
36
7.0
** Re-examination
and in a group
Mean egg count (epg)
with recent
“a Seropositive
0
““42
-
of seropositive reactors by stool examination
dilution of 1:300, and subsequently some blood eluates were rescreened at a lower dilution of 1:150. The amount of specific bound antibody was measured with both a phosphatase labelled antihuman total and IgG conjugate, and a peroxidase labelled anti-human IgG conjugate. Paranitrophenyl phosphate was used as substrate and 3M NaOH as inhibitor in the phosphatase system, and orthonhenylene diamine-H,0,/8N HeSO, as substrate and inhibitor in the peroxidase system. The amount of bound antibody was assayed photometrically at 400 nm for phosphatase and 490 nm for peroxidase, bv measuring the product of enzyme substrate breakdown when the reference positive serum reached an OD level of 0.75
negative,
97
revealed 48 9, to be infected
Samples with OD levels of z 0 ~25 were considered positive, and those with readings of 0 * 18 to 0.25 as borderline positives. Where possible these samples were rescreened at a lower serum dilution of 1: 150 to increase the level of reactivity. All tests were performed without knowledge of the infection status of the patient. Results Study 1 Table specificity collected labelled antibody
I shows the levels of sensitivity and of the assay, obtained with serum samples by venepuncture. With a phosphatase anti-globulin which measures the total response only one adult St. Lucian serum
638
APPLICATION OF ELISA FOR S. mUnSOniSERODIAGNOSISIN ST. LUCIA
was negative. With the peroxidase anti-IgG three false negative reactions occurred in adults; these were in individuals excreting 10 e.p.g., the minimum detected by the filtration staining technique. Of the 100 blood spots examined blind for test specificity, no cross reactivity was observed with the 94 St. Vincent control samples and all had OD levels of below 0.18 when screened at dilutions of 1:300 and 1: 150. Five of the six interspersed St. Lucian positives reacted in the test and subsequent parasitological re-examination of the one seronegative revealed this individual to be uninfected. Table II shows the quantitative aspects of the assay, When grouped by intensity of infection a significant difference exists between the mean OD from low to high intensity of infection when using the peroxidase labelled anti-IgG system. No such correlation exists with the phosphatase total antiglobulin system. Regression analysis by the least squares fit method for the anti-IgG system yields a correlation coefficient of 0.183, p < 0.05.
Study 2 Table III shows the levels of sensitivity of the assay obtained with blood eluates in two groups of infected patients. In Group 1, those positive parasitologically on more than one occasion, the seronegatives occurred in individuals excreting 10 e.p.g. A greater number of false negatives occurred in Group 2 and again these were generally in individuals with minimal infection intensities. Table IV shows the results obtained in Grouts 3 and 4. 42 ‘:A of individuals in Group 3, who were consistently ova-negative, reacted in ELISA. Repeat stool examination of the seropositives revealed 487; of them to be infected when three papers were re-examined by the filtration technique. This group comprised two distinct age categories, three to 18 and 40 to 73. The seronegatives showed a bimodal distribution, but all but one of the seropositives occurred in the three to 18 age group. Group 4 comprised individuals with recently acquired infections, both new cases and reinfections. Antibodies were detected in all but one individual who, when re-examined parasitologically, was found to be negative on four separate examinations. Hence the test was 100 u; in detecting antibodies in recent infections. Discussion These studies were performed as part of a search for a sensitive and specific serological test for S. mansoni infection which could be used in St. Lucia to supplement or replace stool examination in schistosomiasis control schemes. Such a test must be valid in all age groups and in very low intensity infections. The results from the tests carried out in both studies one and two showed a range of sensitivity levels in the assay. Using serum samples collected by venepuncture, test sensitivity was almost 100”; and the few false negative reactions that occurred were all in patients with a minimum egg count of 10 e.p.g. The study in St. Lucia was based entirely on blood samples, collected on filter paper, from patients with extremely low infection levels. Test
sensitivity in this population ranged from 82 to 9376. Antibody levels were also lower, as reflected by lower absorbance readings compared to those obtained in the earlier study, and there were also a greater number of borderline positive reactions. Using a lower serum dilution of I:150 for test sera as in a previous study (MCLAREN et al., 1978) increased the level of antibody reactivity. A comparative study of matched serum samples and blood eluates from infected Sudanese patients did not reveal any quantitative difference in antibody response in the assay (MCLAREN, unpublished data) however, the sera examined were from heavily infected patients with high antibody levels. In cases where antibody levels are very low it may be less stable when absorbed on to paper. Most of the false negative reactions occurred in individuals from Group 2 who had been diagnosed as ova-positive on only one occasion and who had very low egg counts (group mean 15.6 e.p.g.). The possibility that some of these individuals were stool false positives cannot be discounted, particularly as swapping stool samples has been known to occur on a small scale in some villages and there is also a small, but possible, risk of contamination of the apparatus used in the Bell filtration technique. The assay also detected antibodies in a group of individuals who were found to be consistently negative by routine stool examination. All but one of the seropositives occurred in the three to 18 age group, and 48”, of these were confirmed as stool positive when they were examined parasitologically on the basis of three stained papers, instead of one which is the standard for routine examination. It is hiehlv likelv that all the serooositive individuals in thi three to 18 age group were infected and had been missed on stool examination because they had minimal infection intensities, below the level of consistent detection (10 e.p.g.) by the Bell filtration technique. In addition to being able to detect antibodies in very low level infections, antibodies were also detected in a group of individuals with recent infections. This represents an important group with respect to possible future use of the test for incidence measurements. The specificity of the antibody response was tested using sera from St. Vincentian schoolchildren. St. Vincent is an island 30 miles south of St. Lucia with similar geography, peoples and culture but without S. mansoni. The suitability of this population as a control has been discussed (WARREN et al., 1973). All of the sera from this population were negative when both phosphatase and peroxidase conjugates were used in the assay. At serum dilutions of both 1:300 and 1: 150 all sera gave OD levels of below the borderline positive level of 0.18. A study of the quantitative aspects of the assay showed the absorbance in the anti-egg IgG system to be crudely quantitative. The results given here show that higher egg counts tend to give higher OD levels, a finding not observed when the total antibody response is measured. Studies carried out on Sudanese children with wide-ranging S. mansoni infection intensities have revealed similar findings (unpublished data). This suggests that the IgG
M. L. MCLAREN
component of the anti-SEA response may be reacting with fewer, but more specific, antigens than the broad total antibody response. This finding is not however of good enough fit to be of any practical value at this point. Using a more purified antigen may increase the quantitative aspects of this test. These results compare favourably with similar studies carried out by LONG et al., on the use of the qualitative MSAl-radioimmunoassay for serodiagnosis of S. mansoni infection in St. Lucian populations. Acknowledgements We would like to thank Dr. P. Jordan and Dr. C. C. Draper for helpful advice and encouragement. This investigation received financial support from the UNDPWorld Bank/WHO Soecial Programme for Research and Training in Tropical Diseases. and the Edna McConnell Clark Foundation. References Bell, D. R. (1963). A new method S. mansoni eggs in faeces. Bulletin Health
Organization,
for counting of the World
29, 525-530.
et cd.
639
control.
American Journal and Hygiene, 26, 877-886.
of
Tropical
Medicine
Long, E. G., Pelley, R. P. & Hunt, W. (1979). Radioimmunoassay for the detection of Schistosoma mansoni infection using blood spots on filter paper. Bulletin of the World Health Organization, (in press). McLaren, M. L., Draper, C. C., Roberts, J. M., Minter-Goedbloed, E., Ligthart, G. S., Teesdale, C. H., Amin, M. A., Omer, A. H. S., Bartlett, A. & Voller, A. (1978). Studies on the enzyme linked immunosorbent assay (ELISA) test for Schistosoma mansoni infections. knnals df Tropical Medicine and Parasitolopv.Y.2, 72. 243-253. Voller, A., Bartlett, A. & Bidwell, D. E. (1976). Enzyme immunoassays for parasitic diseases. Transactions of the Royal Society Medicine and HJjgiene, 70, 98-106.
of
Tropical
Warren, K. S. Kellermeyer, R. W., Jordan, P., Littell, A. S., Cook, J. A. & Kagani, G. (1973). Immunologic diagnosis of schistosomiasis. 1. A controlled study of intradermal (immediate and delayed) and serologic tests in St. Lucians mansoni and uninfected infected with Schistos&a St. Vincentians. American Journal of Tropical Medicine and HJjgiene, 22, 189-198.
Bruce-Chwatt, L. J., Draper, C. C. & Konfortion, evidence of P. (1973). Seroepidemiological eradication of malaria from Mauritius. Lancer, iii, 547-55 1. Jordan, P. (1977). Schistosomiasis-Research to
Accepted
Admission to the Fellowship of the Society All registered medical and veterinary practitioners and others interested in scientific pursuits relating whose qualifications are to tropical medicine, deemed satisfactory by the Council, are eligible for election as Fellows of the Society. The annual subscription payable by Fellows is E15-00 which becomes due in advance on the 1st April of each year. The Transactions and the current Year Book of the Society are posted regularly to every Fellow whose subscription is not in arrear. Further information may be obtained from the Hon. Secretaries, Manson House, 26 Portland Place. London WlN 4EY. or from the Local . Secretary of the district.
CORPORATE MEMBERSHIP WITH THE ROYAL COMMONWEALTH SOCIETY, 18 NORTHUMBERLAND AVENUE, LONDON, WC2 5BJ The Society has taken up Corporate Membership with the above Society, which has premises just off Trafalgar Square. Overseas Fellows can now use the facilities of this Society, when they come to London, for a period of 21 days in any three months provided that all booking is done through the Secretary at Manson House. Fellows who wish to use these facilities must give the Secretary adequate notice of their requirements particularly during the summer when, at least two months’ notice will be required. Details of the facilities available will be sent to Fellows on request.
for
publication
30th January,
1979.
