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The present status of serodiagnosis and seroepidemiology of schistosomiasis
DIAGNMICROBIOLINFECTDIS 1987;7:93-105
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REVIEW ARTICLE
The Present Status of Serodiagnosis and Seroepidemiology of Schistosomiasis Shirley E. Maddison
The literature of the past 4-5 yr on serodiagnosis and seroepidemiology of schistosomiasis is reviewed. A variety of assays with different antigens are being used for serodiagnasis. Several purified antigens appear to be sensitive and specific, but have little if any capability of indicating duration of infection, parasite burden, or effect of chemotherapy. The results of long-term posttherapy field studies indicate that serology has a role in monitoring control programs. Standardized serologic assays and the need for International Standard Reference Sera are emphasized. A standardized enzyme-linked immunosorbent assay based an the Falcon Assay Screening Test ®system (FAST-ELISA), and involving a standard reference serum pool, is suitable for both serodiagnosis and field studies. Measurement of circulating antigens as a parameter of active infection is considered to have increased potential, compared with antibody measurement, in management of clinical disease and in control programs. Recombinant DNA technology may be useful for producing standard antigens for use in assays measuring antibody or circulating antigen. Time-resolved immunofluorescence involving europium-labeled conjugates may provide the increased assay sensitivity needed for measurement of circulating antigen. INTRODUCTION In spite of the fact that safe, effective drugs against schistosomiasis in h u m a n s have been available for the past 5 yr, transmission continues in parts of the tropics e n d e m i c for the disease. Identification of schistosome eggs in stool, urine, or tissue constitutes definitive diagnosis. Detecting antibodies to schistosomiasis, however, is valuable in the United States and Europe as an aid to diagnosis in immigrants or in expatriates returning from an e n d e m i c area. In addition, positive serology may be an important lead in diagnosing patients w i t h s y m p t o m s c o m p a t i b l e w i t h ectopic schistosomiasis who are not passing eggs in feces or urine. This review e n c o m p a s s e s the schistosomiasis literature for the past 5 yr. Developments in serodiagnosis, seroepidemiology, n e w serodiagnostic antigens, standardization with emphasis on a rapid enzyme-linked immunosorbent assay (ELISA) known as the FAST-ELISA, detection of antigenemia, and advances in recombinant DNA technology will be discussed. SERODIAGNOSIS
The recent literature i n c l u d e s relatively few reviews of routine serodiagnosis of parasitic infections in general, and schistosomiasis in particular (Nash, 1982; Barakat et al., 1983; Walls and Wilson, 1983; Higashi, 1984; Bruckner, 1985; de Savigny and From the Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Human Services, Atlanta, GA. Address reprint requests to: Shirley E. Maddison, PhD, Division of Parasitic Diseases, Centers for Disease Control, Atlanta, GA 30333. Received January 20, 1987; revised and accepted March 18, 1987.
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Speiser, 1985; Maddison, 1986). Bruckner, in a discussion of the immunodiagnosis of parasitic infections commonly found in U.S. children did not include schistosomiasis, because transmission does not occur in the continental United States but is limited to the island of Puerto Rico. Bruckner pointed out that because of the complex life cycles of many parasitic organisms, few serologic tests for parasitic infections could be accurately used for their predictive values. A variety of assays are used for the serodiagnosis of schistosomiasis, but most laboratories report test results as positive or negative; few have the capability of furnishing the physician with information relative to the activity status of the infection. Speiser (1985) in Switzerland used a rapid multiantigen ELISA for antibody screening and relied on the differential level of response to the battery of crude antigens to presumptively diagnose the causative organisms. He advocated, however, the development and use of purified specific antigens. At the Centers for Disease Control (CDC), where reference diagnosis has been performed for years, serum specimens were tested by indirect immunofluorescence (IIF) (Wilson et al., 1974). The assay, which used frozen sections of adult worms of Schistosoma mansoni and anti-IgG heavy and light chain conjugate, was positive in 85% of parasitologically proven cases (Walls and Wilson, 1983). No correlation was found, however, between titer and onset of disease or severity of symptoms. Nash (1982) identified specific fluorescence of the gut epithelial cells of adult schistosome worms (GASP antigen). U.S. expatriates infected within the previous 4 yr all had increased IgM and IgG reactivity to GASP compared with the serum reactivity of chronically infected patients from Puerto Rico and Brazil. Even in very light, recent infections, anti-GASP response was detectable. In contrast, when Nash (1982) fractionated the proteoglycan antigens of adult worms by diethylaminoethyl (DEAE) ionexchange chromatography, he found that antibody response in radioimmunoassay to a phenol sulfuric test active peak (PSAP) was dependent on intensity of infection as determined by fecal egg counts. In addition, acute versus chronic infections in the PSAP assay were differentiated by the predominant IgM antibody response in acute infections. Norden and Strand (1985), in an experimental study, used radioimmunoprecipitation of adult male worm glycoproteins to differentiate acute and chronic infections. Sera from acute infections precipitated glycoproteins of 55, 52, and 35 kD: sara from chronically infected patients precipitated all major glycoproteins ranging from 400 to 12 kD. Infections of Dutch and U.S. tourists in Ethiopia have allowed serologic studies of early infections within 8 wk-1 yr of exposure to S. mansoni (Deelder and Kornelis, 1981; Maddison and Tsang, 1983). In the study of the Dutch tourists, all individuals who had been in contact with water harboring cercariae had demonstrable antibody, whether or not they were passing schistosome eggs or had clinical symptoms. Highest antibody titers were observed with gut-associated polysaccharide antigens, both in IIF and ELISA. Maddison and Tsang (1983) observed moderate levels of reactivity with a standardized plate ELISA (Maddison et al., 1985) in the sera of the U.S. tourists. All the tourists had low fecal egg counts. The sera were obtained within 6 wk-2 mo of exposure. S. mansani adult microsomal antigen (MAMA)(Tsang et al., 1983a) was used in this study. SEROEPIDEMIOLOGY Seroepidemiology of schistosomiasis has not been an integral component of control programs in endemic areas, with the possible exception of the People's Republic of China. This may reflect the insensitivity and nonspecificity of crude schistosomal
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antigens and the insensitivity and cumbersomeness of assays available. Thus, at the present time the information provided by seroepidemiologic studies is essentially limited to determining the prevalence of past and present infection, but not disease, in an endemic area. Mott (1984) concluded from the evaluation initiated by the World Health Organization (WHO) of S. japonicum egg and adult worm antigens that immunodiagnosis may play an important role in the monitoring and surveillance phases of control programs for schistosomiasis japonica. The WHO Expert Committee on schistosomiasis (Technical Report Series 728, 1985) suggested that serologic tests for S. jQponicum infections be developed further because of their potential usefulness. Nontheless, recent field studies have made significant contributions to the epidemiology of schistosomiasis in endemic areas in both Southeast Asia and Africa. A retrospective study using ELISA and the circumoval precipitin test (COPT) to examine sera collected between 1974 and 1975 suggested that S. japonicum-like infections may be more prevalent in some areas of Malaysia than was previously thought (Anuar et al., 1984). Yogore et al. (1983) reported that ELISA using a crude egg antigen of S. japonicum was considerably more sensitive than a single stool examination using a modified merthiolate-formaldehyde procedure in a study in the Philippines. Serology was performed on plasma samples obtained from whole blood collected in heparinized capillary tubes. This approach to collecting blood was a marked improvement over the drying on and elution of whole blood from filter paper (Yogore et al., 1979). The ELISA had a sensitivity of 98% and specificity of 95%. The ELISA was also more reliable than stool examinations for determining incidence of schistosomiasis in the same population (Lewert et al., 1984). Lewert et al. strongly advocated the use of ELISA with crude egg antigen for monitoring schistosomiasis programs in the Philippines. In Upper Volta, Boudin and Chaize (1982) compared ELISA using a crude adult S. mansoni worm antigen to stool examinations for eggs of S. mansoni by the merthiolate-iodine-formalin method. Both serologic and coprologic examinations were insensitive and the ELISA also showed nonspecificity. In a study of 124 adult Kenyans with chronic splenomegaly, 100% of the 29 parasitologically proved infections with S. mansoni were positive serologically (de Cock et al., 1985). However, the crude egg antigen used in the ELISA probably resulted in a number of false positives in patients with splenomegaly in whose stools schistosome eggs were not found. Feldmeier et al. (1985a, 1985b) are conducting their immunologically oriented field study in Sudan. The sensitivity of their egg counts was increased by performing repeated modified Kato thick smear examinations for S. mansoni and repeated urine filtration for S. haematobium. Crude extracts of adult worms of S. mansoni and S. haematobium were used in ELISA for IgG class antibody responses and in a radioallergosorbent test (RAST) for IgE antibody responses, respectively. In the latter assay, the conjugate was labeled with B-galactosidase, thus, avoiding the use of radioisotopes. The 44 school children studied were infected with both S. mansoni and S. haematobium. T helper/T suppressor cell ratio and NK cell function decreased with increasing worm burden. T suppressor cells and IgE and IgG responses to crude S. mansoni and S. haematobium adult worm antigens increased. Posttherapy studies on this group of children will be of particular interest. The field study in the Machakos District of Kenya being undertaken by Butterworth et al. (1984, 1985) is also yielding valuable information. Selection of the 129 children in the study was based on their having a widely ranging intensity of infection. At one end of the spectrum, children had high intensity of infection with low water contact and were predicted to be nonimmnne. At the other end of the spectrum, children had low intensity of infection with high water contact and were predicted to be immune. Pretreatment evaluations were made with ELISA using crude adult
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worm antigen and soluble egg antigen (SEA) and the cation exchange purified CEF6 egg antigen of Dunne et al. (1981). All children in this study were positive when tested with each of the three antigens. The children were treated with oxamniquine. A rise in the levels of reactivity against the crude adult worm antigen was observed 5 wk after treatment. No significant changes were observed with the egg antigens at this time. Followup studies at 3-mo intervals over one year evaluated the level of intensity of infection and immunologic response. Water contact was monitored throughout the study. The results clearly indicated, as predicted, that the high intensity infection-low water contact children were nonimmune and, thus, susceptible to reinfection. This was manifest by return of high fecal egg counts within I yr of treatment. In contrast, the children who were predicted to be immune showed low intensity infections throughout the followup, in spite of frequent water contact with sites harboring infected snails. The investigators associated the acquisition of resistance to a function of the children's age and not to previous egg-induced pathology. The mean age of the resistant group was 2 yr higher than that of the susceptible group. Butterworth et al. (1985) did not rule out the possibility that duration of infection, rather than absolute age, had a strong bearing on the acquisition of resistance. No clear-cut correlation was observed between antibody responses and resistance in this study. Antibodies mediating eosinophil-dependent schistosomulum killing rose markedly in both groups, suggesting that both groups were being exposed to cercariae. After the sharp rise of antibody levels to adult worm antigens in this study as a result of treatment, these levels fell to pretreatment values within 6 mo in both groups, and thereafter remained constant. In contrast, responsiveness to egg antigen remained constant in the nonimmune children throughout the period of observation; the resistant children showed a progressive decrease. The ability of the resistant and nonimmune children to recognize different antigenic components was studied by immunoprecipitation of mRNA translation products (Taylor et al., 1984). A marked heterogeneity in responses to the various peptides was observed, but immune children showed a higher frequency of response to mRNA translation products of 27 and 24 kD than did the susceptible children. Continuation of this study by Butterworth et al. should yield greater insight into immune mechanisms and environmental factors that influence the course of schistosomiasis in a highly endemic area. The findings, thus, will enable more effective control strategies to be devised.
ANTIGENS FOR DETECTION OF ANTIBODIES
It is obvious from this discussion of serodiagnosis and seroepidemiology that serologic antigens and assays were individual for each study described. Sensitive, specific antigens available in costeffective quantities and standardized assays are required for accurate serodiagnosis and comparability between various field studies and use in seroepidemiology. WHO initiated antigen evaluation studies with both S. mansoni (Mott and Dixon, 1982) and S. japonicum (Mott, 1984} reagents. Numerous reports on schistosome antigen purification and characterization have appeared during the past 3 yr. Strand et al. have concentrated on the glycoproteins of both adult worms and eggs (Aronstein and Strand, 1983; Norden and Strand, 1984}. Genus-, species-, stage-, and sex-specific glycoproteins were identified. As mentioned above, acute and chronic infections could be differentiated on the basis of reactions in immunoprecipitation with antigen bands from adult worms. An immunogenic protein of 23 kD was isolated from the surface of adult worms of S. japonicum and identified by a monoclonal antibody (Cruise et al., 1983). The
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antigen was recognized by immunoprecipitation with the serum of an infected Philippine patient. This low molecular-weight component was considered to be a potentially important diagnostic antigen. Hayunge et al. (1983) also purified surface and other glycoproteins from adult worms of S. mansoni. Their antigens reacted with sera of infected humans. In studies directed to identification of surface antigens as potential vaccines, Taylor and Wells (1984) isolated the tegument membranes of schistosomula of S. mansoni. Immunoblot indicated that human sera showed considerable variation in antibody response to the 35 peptides demonstrable by twodimensional gel electrophoresis. Tracy et al. (1985) purified a glycoprotein antigen from eggs of S. japonicum by absorption to concanavalin A-sepharose 4B followed by repetitive gel filtration chromatography. Conventional plate ELISA, in an initial evaluation of this antigenic component has indicated that it is sensitive. This test detected homologous infection in patients with egg counts of <1000 eggs/g of feces. A marked degree of species specificity was evidenced by antigenic reactivity with the sera of only three of 24 patients with S. haematobium eggs ~2000/10 ml urine, and none of 20 patients infected with S. mansoni. Deelder et al. (1980a) in the Netherlands, and Nash (1982) in the U.S., each independently, isolated two very similar glycoproteins. Deelder determined that both his components could also be identified as circulating antigens; only one was found to be a circulating antigen by Nash. A useful collaborative report has been published by Nash and Deelder (1985), showing that the gut-associated proteoglycan (GASP) and circulating anodic antigen (CAA) are identical. This particular antigenic component is now to be known as GASCAP (gut-associated circulating anodic proteoglycan). In contrast, a phenol sulfuric test active peak (PSAP) from the DEAE fractionation of the proteoglycan antigens by Nash was found to be different than the cathodic circulating antigen (CCA) of Deelder. Use of CAA in an ELISA has resulted in a sensitive assay for infection with S. mQnsoni (Deelder et al., 1980b). The limited yield of antigen, however, precludes its wide-scale usage (Deelder and Kornelis, 1981). Another antigen of diagnostic promise is the CEF6 antigen with the active W1 component from eggs of S. mansoni (Dunne et al., 1981). CEF6 is isolated by carboxymethyl cellulose and shows a high degree of species specificity. As already mentioned, Butterworth et al. (1984, 1985) have included its use in their field study; however, their results to date do not show that the purified CEF6 antigen is markedly superior to the crude SEA. Microsomal antigens from adult worms were developed by Tsang et al. (1983a, 1984). The microsomal antigen from S. mansoni is known as MAMA, and the corresponding antigen of S. japonicum as JAMA. These have proved to be specific, sensitive, stable and costeffective in production. A high degree of species specificity is evident in ELISA (Tsang et al., 1983b) and immunoblot (Tsang et al., 1984). The production of the comparable JAMA from S. haematobium is in progress.
STANDARDIZED ELISA
In addition to sensitive, specific, costeffective antigens, quantitative, standardized serologic assays are required for accurate serodiagnosis and for comparison of results between laboratories. A standardized assay for antibody measurement involves the use of a standard serum pool from which a standard curve is prepared in each run of the assay. The reactivity of the unknowns are determined from the standard curve. We evaluated MAMA in a standardized microtest plate ELISA in which enzyme
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reactivity was chemically stopped after 30 min (Maddison et al., 1985). A reference serum pool, arbitrarily designated as having 100 activity units per microliter was used to prepare a standard curve in each plate. The levels of activity of the unknown sera were determined from the standard curve on a continuously variable response scale and recorded as activity units per microliter. Although this assay proved to be sensitive and specific, it had limitations because the enzyme reaction was being measured when it was no longer kinetic. In addition, because the reactions in our standardized assay were allowed to proceed for 0.5 hr, the moderate and highly reactive sera required further dilution in order for the activity levels to lie within the range of the single-log standard curve. The disadvantages of ELISA in which the reactions are chemically stopped are as follows: 1. The activity-stopping agent may not "freeze" color development 2. End-point color intensity (absorbance) is seldom linear with respect to antibody concentration 3. Enzyme reaction is linear with respect to activity only during brief initial phase. Most reaction times chosen grossly exceed the linear phase, and the assay is not truly quantitative. In contrast, single-well ELISA based on enzyme rate kinetics yields quantitative linear measurements for antigen or antibody. The rate analyzer used in the kinetic ELISA (Tsang et al., 1983c) for monitoring of the isolation and characterization of the microsamal antigens allowed such quantitative serologic analysis. However, the rate analyzer is expensive and would not be available in regional centers in developing countries. Such centers are usually familiar with the principles of ELISA. Hancock and Tsang (1986) recently developed a standardized kinetic-dependent FIGURE 1. Lid of microtitration plate holding eight strips of polystyrene sticks with attached beads.
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FIGURE 2. (A} Standard curve for 100-1000 U of the standard serum pool. Note that two equations, one linear and the other exponential, are necessary to accurately describe the data. (B) Standard curve for 10-100 U of the standard serum pool is best described by a linear equation, y = mx + b. The coefficient of determination (r2} = 0.976. (C) Standard curve for 100-1000 U of the standard serum pool is best described by an exponential equation, y = ae bx, r2 = 0.989. For each curve, each unit value was assayed in triplicate. After Hancock and Tsang, 1986; reproduced by kind permission of the authors and the editors of Journal of Immunological Methods. plate FAST-ELISA, based on the Falcon assay screening test (FAST®)1 system using MAMA. The FAST system consists of strips of 12 polystyrene beads on short sticks. The strips snap into the lid of a microtest plate (Fig. 1). The lid with one or more strips of beads is inverted into troughs or wells of a microtitration plate. A standard reference serum pool was an integral part of the assay. To prepare the standard curve for each plate, increasing a m o u n t s of the standard reference serum pool were reacted with both MAMA and conjugate i n excess. A 2-log range of activities from 10 to 1000 u n i t s of the standard reference serum pool was measurable. To describe the data, two c u r v e s are needed. The activity of the standard serum pool from 10 to 100 u n i t s is best described by a linear equation and from 100 to 1000 by an e x p o n e n t i a l curve (Fig. 2). 1Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Dept. of Health and Human Services.
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The run-to-run variation of the reactivity of a serum was evaluated by repeatedly testing a serum in triplicate over a 1-mo period. Although the CV of the mean daily absorbance was 20.3%, the CV of the activity units per microliter as determined from the standard curve was only 13.6%; thus, the use of a standard curve reduces the run-to-run variation significantly. Figure 3 s h o w s the results of testing 234 sera. The previously recognized high degree of species-specificity of MAMA (Tsang et al., 1984) is reflected in the l o w level of reactivity of sera from S. japonicum-infected patients compared with that of sera from S. mansoni patients. The diagnostically significant level of reactivity was determined statistically by using the J index (Youden, 1950; Ruiz-Tiben et al., 1979). The data from the h o m o l o g o u s S. mansoni infections were used for the true positives: the normal CDC controls and the patients with other helminthic or liver infection were considered to be true negatives. Both the sensitivity and specificity of the assay are 99%. The FAST-ELISA with MAMA for infection with S. mansoni has been incorporated into our reference diagnostic laboratory at CDC. This assay does not give insight into duration of infection or intensity of infection. Immunoblots with extracts of the respective adults w o r m s are used for suspect S. haematobium and S. japonicum infections. When suitable antigens are available, w e will apply FAST-ELISA to other FIGURE 3. Results from assay of sera from 234 individuals. The groups of sera are identified on the x-axis. The numbers immediately above the x-axis are the number of individuals in the corresponding group that is immediately below the x-axis. On the y-axis is the FAST-ELISA result, expressed in U/p.l. Note that the scale in not continuous. All results on and above the line at 10 U/p.1 are positive, all results below are negative. After Hancock and Tsang, 1986; reproduced by kind permission of the authors and the editors of Journal of Immunological
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helminthic systems. Because each strip of 12 beads is independent, more than one type of antigen strip can be placed in a single lid. A preliminary field study has been made with the FAST-ELISA for schistosomiasis in Cameroon. The only equipment used was two Eppendorf pipettes and a garden spray for washing the beads. The assay appears to be surprisingly rugged, but minor difficulties were encountered with the stability of the hydrogen peroxide. This problem has been resolved by use of a very stable TMB Microwell Peroxidase Substrate System (Kirkegaard and Perry Laboratories, Inc. Gaithersburg, MD). In this study blood collected into phosphate buffered saline (PBS) containing 0.2% sodium azide as a preservative (A. Sulzer, personal communication) was suitable for transport and subsequent testing. A volume-marked capillary tube was used to collect 0.1 ml of whole blood from a finger-stick into 1.9 ml of PBS. When dilution was taken into account no significant difference was observed between the levels of reactivity of serum and the blood in PBS. This method of collecting and processing blood from finger-sticks is vastly superior to filter paper. DETECTION OF ANTIGENEMIA Antibody detection for routine serodiagnosis of schistosomiasis currently provides little, if any, information about the course or intensity of infection, or the effect of chemotherapy because antibodies persist in chronic infections and are slow to indicate significant change after chemotherapy. Detection and quantitation of worm products--surface glycoproteins and proteins that have been sloughed off, as well as secretory/excretory products--in serum or urine would identify active infection and give insight into the effect of chemotherapy. In contrast to some mycotic, viral, and bacterial diseases, however, antigen detection in parasitic disease in general, and schistosomiasis in particular, has not yet become a diagnostic procedure. French investigators with workers in Burundi (Carlier et al., 1980) and Brazil (Santoro et al., 1981) identified circulating antigen and circulating immune complexes in patients' sera. The amounts of the schistosome products were small, however, and necessitated concentration of serum and/or use of radioisotopic assays. Decider et al. (1980a) demonstrated circulating S. mansoni polysaccharide antigens (CAA and CCA) in heavily infected experimental animals. Qian and Decider (1983) quantitated CAA by ELISA in the serum of rabbits experimentally infected with S. japonicum. Circulating antigens in mice harboring 100 worms were detectable as early as I wk after exposure (Hayunga et al., 1986). This was achieved in a competitive-inhibition ELISA using a purified cercarial antigen of apparent molecular weight of 41 kD. CAA and CCA recently have been studied in mixed infection S. mansoni and S. haematobium patients (Feldmeier et al., 1986). A radioimmunometric assay could detect 150 ng/ml of CCA. The monoclonal antibody used appeared to be speciesspecific, because a correlation was observed between the amount of CCA and number of S. mansoni eggs in the stool, but not the number of S. haematobium eggs in urine. The circulating antigen assay was not as sensitive as the detection of circulating antibody by ELISA. Because of the short half-life of radioisotopes and the hazards associated with their use, alternates to isotopic assays are being sought. The development of a sensitive ELISA for measuring circulating antigens and specific immune complexes in schistosomiasis, however, will probably necessitate demanding steps, such as 1. the development of monoclonal antibodies directed to each of two different epitopes, respectively, on a single type of circulating antigenic molecule. One of these monoclonal antibodies would serve as capture antibody. The second would be enzymatically labelled to serve as an indicator reagent.
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2. labeling of this indicator monoclonal antibody would need to be critically controlled to produce an exquisitely sensitive reagent. The use of galactosidase with a higher substrate turnover than peroxidase may result in improved sensitivity (Qian and Wen, 1983). An alternative to the sandwich ELISA may be the use of time-resolved fluorescence (TR-FIA) (Suonpaa et el., 1985) in a sandwich assay with two monoclonal antibodies. The indicator monoclonal antibody is labeled with europium, which results in lanthanide chelates as the fluorescent probe. Because the nonspecific fluorescence associated with serum has a short decay time it is eliminated during the time resolution. Lanthanide chelates have long decay fluorescence and result in a highly sensitive assay. The capability of measurement, at least of alpha-fetoprotein, over a 4-log range is very advantageous. In addition, the biological hazard and disadvantage of the short half-life of radioisotopes are eliminated. The purified target antigen should be produced in large quantities to serve as a standardized reagent for preparing a standard curve in each ELISA. Production of purified antigens will probably necessitate the use of recombinant DNA technology. If appropriate reagents and standardized assays become available, measurement of circulating antigen in patients' sera and urine will be possible.
RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology is widely applied in microbiologic research, and within the past 3.5 yr at least five groups have reported studies dealing with recombinant DNA technology in schistosomiasis. However, target circulation antigen production was not the goal. Interest has been mainly focused on producing a vaccine. Cordingley et al. (1983) first reported the construction of cDNA clone banks from poly(A) +messenger RNA of eggs and adult worms of S. mansoni. Although many translation products were common to eggs and worms, a p40 product was obtained from a mRNA unique to eggs. This p40 molecule appeared to be a potential serodiagnostic reagent. These investigators identified in vitro translation products of 27 and 25 kD, which reacted more frequently with the sera of immune than nonimmune Kenyan children (Butterworth et el., 1985). Recently they have reported the use of mRNA hybrid selection followed by immunoprecipitation of the corresponding translation products (Cordingley et al., 1986). This technique allows screening thousands of clones. Simpson and Knight (1986) screened recombinant cDNA libraries by differential hybridization that had high levels of expression in adult female worms but were not associated with immature female or adult male worms. Thus, cDNA clones were identified that encode a developmentally regulated schistosome gene. In other studies, polypeptide antigens derived by recombinant DNA technology were screened by precipitation with experimental animal and human infection sera (Knight et al. 1986). Some of the products showed potential as diagnostic antigens. Bugra et el. (1983) reported the development of schistosome translation products from mRNA templates. These serve as templates for preparing cDNA libraries and as molecular probes. Saint et al. (1986) prepared a cloned cDNA library from S. japonicum adult worm mRNA. The cDNA was expressed as fusion products with amp3/E, coli ~-galactosidase. Screening with human patient sere identified fewer reactive colonies than screening with sera from immunized rabbits. In summary, investigators have made great strides in recombinant DNA/schistosomiasis research. Conventional approaches have not solved the problems of reagents required for widespread use of standardized serologic assays for schistosomiasis. We now look to this new technology for production of antigens as standards. These are
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required not only for assays that measure antibody levels but also to serve as standards in assays that measure circulating antigens. A standardized assay, the FAST-ELISA, has been discussed. This assay used in c o n j u n c t i o n with microsomal antigen (MAMA) has proved to be suitable for m e a s u r e m e n t of antibody i n both serodiagnosis and seroepidemiology. The high sensitivity of this assay may well permit the measurem e n t of incidence i n an e n d e m i c area. Nonetheless, additional antigens and assays that give greater insight into the disease process pertinent to patient m a n a g e m e n t and control programs should be sought. Measurement of circulating antigen will probably be more appropriate than measurement of antibody. TR-FIA with europiumlabeled conjugates may supply the necessary increased sensitivity. It should be emphasized that essential c o m p o n e n t s for standardization are International Reference Serum pools for each of the three species of schistosomes of major importance to h u m a n s . These reagents are yet to be prepared. The World Health Organization or the International Association of Biological Standards should assume the responsibility for preparing these m u c h - n e e d e d reagents and ascribing International Units to them. REFERENCES Anuar H, Greer GJ, Ow-Yank CK, Sudumaren KD (1984) Detection of Malaysian schistosimiasis in Orang Asli of Peninsular Malaysia using serodiagnostic tests. Southeast Asian l Trap Med Public Health 15:479. Aronstein WS, Strand M (1983) Identification of species-specific and gender-specific proteins and glycoproteins of three human schistosomes. J Parasitol 69:1006. Barakat RM, E1-Gassim EE, Awadalla HN, E1-Molla A, Omer EA (1983) Evaluation of enzymelinked immunosorbant assay (ELISA) as a diagnostic tool for schistosomiasis Trans H Sac Trap Med Hyg 77:109. Boudin C, Chaize J (1982) Depistage de masse de la Bilharziose a Schistosoma mansoni. Erude de la fiabilite de deux techniques: Parasitologique (MIF) et immunologique (ELISA). Cab O.R.S.T.O.M. ser Ent Med Parasitol 20:299, Bruckner DA (1985) Serologic and intradermal tests for parasitic infections. Pediatr Clin N A m 32:1063. Bugra K, Tanaka RD, Boyle WJ, Maclnnis AJ (1983) Isolation of PolyA( + ) RNA from Schistosoma mansoni and immunoprecipitation of its in vitro translation products, l Porasitol 69:486. Butterworth AE, Dalton PR, Dunne DW, Mugambi M, Ouma JH, Richardson BA, Arap Siongok TK, Sturrock RF (1984) Immunity after treatment of human schistosomiasis mansoni. I. Study design, pretreatment observations and the results of treatment. Trans R Sac Trap Med Hyg 78:108. Butterworth AE, Capron M, Cordingley JS, Dalton P'R, Dunne DW, Dariuki HC, Kimani G, Koech D, Mugabi M, Ouma JH, Prentice MA, Richardson BA, Arap Siongok TK, Sturrock RF, Taylor DW (1985) Immunity after treatment of human schistosomiasis mansoni. II. Identification of resistant individuals and analysis of their immune responses. Trans R Sac Trap Med Hyg 79:393. Carlier Y, Nzeyimana H, Bout D, Capron A (1980) Evaluation of circulating antigens by a sandwich radioimmune assay, and of antibodies and immune complexes in Schistosoma mansoni-infectedAfrican parturients and their newborn children. Am l Trap Med Hyg 29:74. Cordingley JS, Taylor DW, Dunne DW, Butterworth AE (1983) Clone banks of cDNA from the parasite Schistosoma mansoni: Isolation of clones containing a potentially immunodiagnostic antigen gene. Gene 26:25. Cordingley JS, Haddow WJ, Nene V, Taylor DW (1986) Identification by message selection of cDNA clones encoding antigens of Schistosoma mansoni. Molec Biochem Parasitol 18:73. Cruise KM, Mitchell GF, Garcia EG, Tiu WU, Hocking RE, Anders RF (1983) Sj23, the target antigen in Schistosoma japonicum adult worms of an immunodiagnostic hybridoma antibody. Parasitol Immunol 5:37. de Cock KM, Hodgen AN, Lillywhite JE, Arap Siongok TK, Lucas SB, Rees PH (1985) Hepatosplenic schistosomiasis in Kenya: An assessment of the enzyme-linked immunosorbent assay (ELISA). Trap Geogr Med 37:285.
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Deelder AM, Kornelis D (1981) Immunodiagnosis of recently acquired Schistosoma mansoni infection. A comparison of various immunological techniques. Trap Geogr Med33:36. Deelder AM, Kornelis D, Van Marck EAE, Eveleigh PL, Van Egmond JG (1980a) Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse, hamster, and human infections. Exp Parasitol 50:16. Deelder AM, Kornelis D, Makbin M, Noordpool HN, Cadfried RM, Rotmans JP, Oostburg BJF (1980b) Applicability of different antigen preparations in the enzyme-linked immunosorbant assay for schistosomiasis mansoni. Am ] Trap IVied Hyg 29:401. de Savigny D, Speiser F (1985) Enzyme immunoassays for tropical parasitic diseases. In Enzymemediated Immunoassay. Eds., Ngo TT, Leahoff HM. New York: Plenum Press, pp. 415-431. Dunne DW, Lucas S, Bickle Q, Pearson S, Madgwick L, Bain J, Doenhoff MJ (1981) Identification and partial purification of an antigen (Wl) from Schistosoma mansoni eggs which is putatively hepatotoxic in T-cell deprived mice. Trans R Sac Trap Med Hyg 75:54. Feldmeier H, Gastl GA, Poggensee U, Kortmann C, Daffalla AA, Peter HH (1985a) Relationship between intensity of infection and immunomodulation in human schistosomiasis. I. Lymphocyte subpopulations and specific antibody responses. Clin Exp Immunol 60:225. Feldmeier H, Gastl GA, Poggensee U, Kortmann C, Daffalla AA, Peter HH (1985b) Relationship between intensity of infection and immunomodulation in human schistosomiasis. II. NK cell activity and in vitro lymphocyte proliferation. Clin Exp Immunol 60:239. Feldmeier H, Noqueira-Queiroz JA, Peixoto-Queiroz MA, Doehring E, Dessaint JP, de Alencar JE, Dafalla AA, Capron A (1986) Detection and quantification of circulating antigen in schistosomiasis by monoclonal antibody. The quantification of circulating antigens in human schistosomiasis mansoni and haematobium: Relationship to intensity of infection and disease status. Clin Exp Immunol 65:232. Hancock K, Tsang VCW (1986) Development and optimization of the FAST-ELISA for detecting antibodies to Schistosoma mansoni. J Immunol Meth 92:167. Hayunga EG, Sumner MP, Stek M, Vannier WE, Chestnut RY (1983) Purification of the major concanavalin A-binding surface glycoprotein from adult Schistosoma mansani. Proc Helminthol 50:219. Hayunga EG, Duncan JF, Stek M, Mollegard I, Sumner MP, Hunter KW (1986) Development of circulating antigen assay for rapid detection of acute schistosomiasis. Lancet ii:716. Higashi GI (1984) Immunodiagnostic tests for protozoan and helminth infections. Diagn Immunol 2:2. Knight M, Simpson AJG, Bickle Q, Hagan P, Moloney A, Wilkins A, Smithers SR (1986) Adult schistosome cDNA libraries as a source of antigens for the study of experimental and human schistosomiasis. Melee Biochem Parasitol 18:235. Lewert RM, Yogore MG, Bias BL (1984) Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. II. Unreliabilityof stool examination in the assessment of incidence. Am J Trap Med Hyg 33:872. Maddison SE (1986) Schistosomiasis. In Immunodiagnosis of Parasitic Diseases, Vol. I. Helminthic Diseases. Eds., Walls KW, Schantz PM. Orlando: Academic Press, Inc., pp. 1-37. Maddison SE, Tsang VCW (1983) Serodiagnosis of schistosomiasis. Clin Immunol Newsl 4:101. Maddison SE, Slemenda SB, Tsang VCW, Pollard RA (1985) Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen (MAMA) in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool. Am J Trap Med Hyg 35:484. Matt KE (1984) Diagnostic tools for Schistosoma japonicum in control programs. Arsheim Forsch/Drug Res 34:1217. Matt KE, Dixon H (1982) Collaborative study on antigens for immunodiagnosis of schistosomiasis. Bull WHO 60:729. Nash TE (1982) Diagnostic serologic responses. In: Schistosame Infection in Humans: Perspectives and Recent Findings. Moderator, Nash TE, Ann Intern Med 97:740. Nash TE, Deelder AM (1985) Comparison of four schistosome excretory-secretory antigens: Phenol sulfuric test active peak cathodic circulating antigen, gut-associated proteoglycan, and circulating anodic antigen. Am ] Trap Med Hyg 34:236. Norden AP, Strand M (1984) Schistosoma mansoni, S. haematobium, and S. japonicum: Identification of genus- and species-specific antigenic glycoproteins. Exp Parasitol 58:333.
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Norden AP, Strand M (1985) Identification of antigenic Schistosoma mansoni glycoproteins during the course of infection in mice and humans. Am ] Trop Med Hyg 34:495. Qian ZL, Deelder AM (1983] Schistosoma japonicum: Immunological characterization and detection of circulating polysaccharide antigens from adult worms. Exp Parasitol 55:168. Qian ZL, Wen HC (1983) Schistosome circulating antigens (CSA) as a possible diagnostic parameter for active infections. Acta Leiden 51:37. Ruiz-Tiben E, Hillyer GV, Knight WB, Gomez de Rios I, Woodall JP (1979) Intensity of infection with Schistosoma mansoni: Its relationship to the sensitivity and specificity of serologic tests. Am / Trop Med Hyg 28:230. Saint RB, Beall ]A, Grumont R], Mitchell GF, Garcia EG (1988) Expression of Schistosoma japonicum antigens in Escherichia coil. Molec Biochem Parasito| 18:333. Santoro F, Prata A, Silva AE, Capron A (1981] Correlation between circulating antigens detected by radioimmuno-precipitation-polyethyleneglycol assay (RIPEGA)and CIq-bindingimmune complexes in human schistosomiasis mansoni. Am ] Trop Med Hyg 30:1020. Simpson AJG and Knight M (1986) Cloning of a major developmentallyregulated gene expressed in mature females of Schistosoma mansoni. Molec Biochem Parasitol 18:25. Speiser F (1985) Serodiagnosis of parasitic diseases. Experientia 41:531. Suonpaa MU, Lavi IT, Hemmila IA, Lovgren TN-E (1985] A new sensitive assay for human alpha-fetoprotein using time-resolved flourescence and monoclonal antibodies. Clin Chim Acta 145:341. Taylor DW, Wells PZ (1984) Isolation and antigen analysis of surface tegument membranes from schistosomula of Schistosoma mansoni. Parasitology 89:495. Taylor DW, Cordingley IS, Butterworth AE (19841 Immunoprecipitation of surface antigen precursors from Schistosoma mansoni messenger RNA in vitro translation products. Molec Biochem Parasito] 10:305. Tracy ]W, Domingo EO, Mahmoud AAF (1985) Evaluation of purified Schistosoma japonicum glycoprotein egg antigen for the immunodiagnosis of infection in man. Am ] Trap ivied Hyg 34:92. Tsang VCW, Tsang KR, Hancock K, Kelley MA, Wilson BC, Maddison SE (1983a) Schistosoma mansoni adult microsomal antigens, a serologic reagent. I. Systematic fractionation, quantitation, and characterization of antigenic components. 1 Immuno] 130:1359. Tsang VCW, Tsang KR, Hancock K, Kelley MA, Wilson, BC, Maddison SE {1983bl Schistosoma mansoni adult microsomal antigens, a serologic reagent. II. Specificity of antibody responses to the S. mansoni microsomal antigen (MAMA]. ! Immunol 130:1366. Tsang VCW, Wilson BC, Peralta ]M {1983c) Quantitative, single-tube, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA). Meth Enzymol 92:391. Tsang VCW, Hancock K, Maddison SE, Beatty AL, Moss DM (1984) Demonstration of speciesspecific and cross-reactive components of the adult microsomal antigens from Schistosoma mansoni and S. japonicum (MAMA and lAMA). J ImmunoI 132:2607. Walls KW, Wilson M (1983) Immunoserology in parasitic infections. In Immunodiagnostics. Eds., Aloisi RM, Hyun ]. New York: Alan R. Liss, Inc. pp. 191-214. Wilson M, Sulzer A], Walls KW (1976) Modified antigens in the indirect immunofluorescence test for schistosomiasis. Am I Trop IVied Hyg 23:1072. World Health Organization (WHO) (1985) The control of schistosomiasis. WHO Technical Report Series 728:51. Yogore MG, Lewert RIM,Bias BL (1979) Schistosomiasis japonica in Barrio San Antonio, Basey, Samar in the Philippines. III. The plasma circumoval and precipitin test procedure and use in epidemiologic studies. Southeast Asian / Trop Med Public Health 10:21. Yogore MG, Lewert RM, Bias (1983] Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. I. Underestimation by stool examination of the prevalence of infection in school children. Am J Trop Med Hyg 32:1322. Youden W] {1950) Index for rating diagnostic test. Cancer 3:32.
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REVIEW ARTICLE
The Present Status of Serodiagnosis and Seroepidemiology of Schistosomiasis Shirley E. Maddison
The literature of the past 4-5 yr on serodiagnosis and seroepidemiology of schistosomiasis is reviewed. A variety of assays with different antigens are being used for serodiagnasis. Several purified antigens appear to be sensitive and specific, but have little if any capability of indicating duration of infection, parasite burden, or effect of chemotherapy. The results of long-term posttherapy field studies indicate that serology has a role in monitoring control programs. Standardized serologic assays and the need for International Standard Reference Sera are emphasized. A standardized enzyme-linked immunosorbent assay based an the Falcon Assay Screening Test ®system (FAST-ELISA), and involving a standard reference serum pool, is suitable for both serodiagnosis and field studies. Measurement of circulating antigens as a parameter of active infection is considered to have increased potential, compared with antibody measurement, in management of clinical disease and in control programs. Recombinant DNA technology may be useful for producing standard antigens for use in assays measuring antibody or circulating antigen. Time-resolved immunofluorescence involving europium-labeled conjugates may provide the increased assay sensitivity needed for measurement of circulating antigen. INTRODUCTION In spite of the fact that safe, effective drugs against schistosomiasis in h u m a n s have been available for the past 5 yr, transmission continues in parts of the tropics e n d e m i c for the disease. Identification of schistosome eggs in stool, urine, or tissue constitutes definitive diagnosis. Detecting antibodies to schistosomiasis, however, is valuable in the United States and Europe as an aid to diagnosis in immigrants or in expatriates returning from an e n d e m i c area. In addition, positive serology may be an important lead in diagnosing patients w i t h s y m p t o m s c o m p a t i b l e w i t h ectopic schistosomiasis who are not passing eggs in feces or urine. This review e n c o m p a s s e s the schistosomiasis literature for the past 5 yr. Developments in serodiagnosis, seroepidemiology, n e w serodiagnostic antigens, standardization with emphasis on a rapid enzyme-linked immunosorbent assay (ELISA) known as the FAST-ELISA, detection of antigenemia, and advances in recombinant DNA technology will be discussed. SERODIAGNOSIS
The recent literature i n c l u d e s relatively few reviews of routine serodiagnosis of parasitic infections in general, and schistosomiasis in particular (Nash, 1982; Barakat et al., 1983; Walls and Wilson, 1983; Higashi, 1984; Bruckner, 1985; de Savigny and From the Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Human Services, Atlanta, GA. Address reprint requests to: Shirley E. Maddison, PhD, Division of Parasitic Diseases, Centers for Disease Control, Atlanta, GA 30333. Received January 20, 1987; revised and accepted March 18, 1987.
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Speiser, 1985; Maddison, 1986). Bruckner, in a discussion of the immunodiagnosis of parasitic infections commonly found in U.S. children did not include schistosomiasis, because transmission does not occur in the continental United States but is limited to the island of Puerto Rico. Bruckner pointed out that because of the complex life cycles of many parasitic organisms, few serologic tests for parasitic infections could be accurately used for their predictive values. A variety of assays are used for the serodiagnosis of schistosomiasis, but most laboratories report test results as positive or negative; few have the capability of furnishing the physician with information relative to the activity status of the infection. Speiser (1985) in Switzerland used a rapid multiantigen ELISA for antibody screening and relied on the differential level of response to the battery of crude antigens to presumptively diagnose the causative organisms. He advocated, however, the development and use of purified specific antigens. At the Centers for Disease Control (CDC), where reference diagnosis has been performed for years, serum specimens were tested by indirect immunofluorescence (IIF) (Wilson et al., 1974). The assay, which used frozen sections of adult worms of Schistosoma mansoni and anti-IgG heavy and light chain conjugate, was positive in 85% of parasitologically proven cases (Walls and Wilson, 1983). No correlation was found, however, between titer and onset of disease or severity of symptoms. Nash (1982) identified specific fluorescence of the gut epithelial cells of adult schistosome worms (GASP antigen). U.S. expatriates infected within the previous 4 yr all had increased IgM and IgG reactivity to GASP compared with the serum reactivity of chronically infected patients from Puerto Rico and Brazil. Even in very light, recent infections, anti-GASP response was detectable. In contrast, when Nash (1982) fractionated the proteoglycan antigens of adult worms by diethylaminoethyl (DEAE) ionexchange chromatography, he found that antibody response in radioimmunoassay to a phenol sulfuric test active peak (PSAP) was dependent on intensity of infection as determined by fecal egg counts. In addition, acute versus chronic infections in the PSAP assay were differentiated by the predominant IgM antibody response in acute infections. Norden and Strand (1985), in an experimental study, used radioimmunoprecipitation of adult male worm glycoproteins to differentiate acute and chronic infections. Sera from acute infections precipitated glycoproteins of 55, 52, and 35 kD: sara from chronically infected patients precipitated all major glycoproteins ranging from 400 to 12 kD. Infections of Dutch and U.S. tourists in Ethiopia have allowed serologic studies of early infections within 8 wk-1 yr of exposure to S. mansoni (Deelder and Kornelis, 1981; Maddison and Tsang, 1983). In the study of the Dutch tourists, all individuals who had been in contact with water harboring cercariae had demonstrable antibody, whether or not they were passing schistosome eggs or had clinical symptoms. Highest antibody titers were observed with gut-associated polysaccharide antigens, both in IIF and ELISA. Maddison and Tsang (1983) observed moderate levels of reactivity with a standardized plate ELISA (Maddison et al., 1985) in the sera of the U.S. tourists. All the tourists had low fecal egg counts. The sera were obtained within 6 wk-2 mo of exposure. S. mansani adult microsomal antigen (MAMA)(Tsang et al., 1983a) was used in this study. SEROEPIDEMIOLOGY Seroepidemiology of schistosomiasis has not been an integral component of control programs in endemic areas, with the possible exception of the People's Republic of China. This may reflect the insensitivity and nonspecificity of crude schistosomal
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antigens and the insensitivity and cumbersomeness of assays available. Thus, at the present time the information provided by seroepidemiologic studies is essentially limited to determining the prevalence of past and present infection, but not disease, in an endemic area. Mott (1984) concluded from the evaluation initiated by the World Health Organization (WHO) of S. japonicum egg and adult worm antigens that immunodiagnosis may play an important role in the monitoring and surveillance phases of control programs for schistosomiasis japonica. The WHO Expert Committee on schistosomiasis (Technical Report Series 728, 1985) suggested that serologic tests for S. jQponicum infections be developed further because of their potential usefulness. Nontheless, recent field studies have made significant contributions to the epidemiology of schistosomiasis in endemic areas in both Southeast Asia and Africa. A retrospective study using ELISA and the circumoval precipitin test (COPT) to examine sera collected between 1974 and 1975 suggested that S. japonicum-like infections may be more prevalent in some areas of Malaysia than was previously thought (Anuar et al., 1984). Yogore et al. (1983) reported that ELISA using a crude egg antigen of S. japonicum was considerably more sensitive than a single stool examination using a modified merthiolate-formaldehyde procedure in a study in the Philippines. Serology was performed on plasma samples obtained from whole blood collected in heparinized capillary tubes. This approach to collecting blood was a marked improvement over the drying on and elution of whole blood from filter paper (Yogore et al., 1979). The ELISA had a sensitivity of 98% and specificity of 95%. The ELISA was also more reliable than stool examinations for determining incidence of schistosomiasis in the same population (Lewert et al., 1984). Lewert et al. strongly advocated the use of ELISA with crude egg antigen for monitoring schistosomiasis programs in the Philippines. In Upper Volta, Boudin and Chaize (1982) compared ELISA using a crude adult S. mansoni worm antigen to stool examinations for eggs of S. mansoni by the merthiolate-iodine-formalin method. Both serologic and coprologic examinations were insensitive and the ELISA also showed nonspecificity. In a study of 124 adult Kenyans with chronic splenomegaly, 100% of the 29 parasitologically proved infections with S. mansoni were positive serologically (de Cock et al., 1985). However, the crude egg antigen used in the ELISA probably resulted in a number of false positives in patients with splenomegaly in whose stools schistosome eggs were not found. Feldmeier et al. (1985a, 1985b) are conducting their immunologically oriented field study in Sudan. The sensitivity of their egg counts was increased by performing repeated modified Kato thick smear examinations for S. mansoni and repeated urine filtration for S. haematobium. Crude extracts of adult worms of S. mansoni and S. haematobium were used in ELISA for IgG class antibody responses and in a radioallergosorbent test (RAST) for IgE antibody responses, respectively. In the latter assay, the conjugate was labeled with B-galactosidase, thus, avoiding the use of radioisotopes. The 44 school children studied were infected with both S. mansoni and S. haematobium. T helper/T suppressor cell ratio and NK cell function decreased with increasing worm burden. T suppressor cells and IgE and IgG responses to crude S. mansoni and S. haematobium adult worm antigens increased. Posttherapy studies on this group of children will be of particular interest. The field study in the Machakos District of Kenya being undertaken by Butterworth et al. (1984, 1985) is also yielding valuable information. Selection of the 129 children in the study was based on their having a widely ranging intensity of infection. At one end of the spectrum, children had high intensity of infection with low water contact and were predicted to be nonimmnne. At the other end of the spectrum, children had low intensity of infection with high water contact and were predicted to be immune. Pretreatment evaluations were made with ELISA using crude adult
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worm antigen and soluble egg antigen (SEA) and the cation exchange purified CEF6 egg antigen of Dunne et al. (1981). All children in this study were positive when tested with each of the three antigens. The children were treated with oxamniquine. A rise in the levels of reactivity against the crude adult worm antigen was observed 5 wk after treatment. No significant changes were observed with the egg antigens at this time. Followup studies at 3-mo intervals over one year evaluated the level of intensity of infection and immunologic response. Water contact was monitored throughout the study. The results clearly indicated, as predicted, that the high intensity infection-low water contact children were nonimmune and, thus, susceptible to reinfection. This was manifest by return of high fecal egg counts within I yr of treatment. In contrast, the children who were predicted to be immune showed low intensity infections throughout the followup, in spite of frequent water contact with sites harboring infected snails. The investigators associated the acquisition of resistance to a function of the children's age and not to previous egg-induced pathology. The mean age of the resistant group was 2 yr higher than that of the susceptible group. Butterworth et al. (1985) did not rule out the possibility that duration of infection, rather than absolute age, had a strong bearing on the acquisition of resistance. No clear-cut correlation was observed between antibody responses and resistance in this study. Antibodies mediating eosinophil-dependent schistosomulum killing rose markedly in both groups, suggesting that both groups were being exposed to cercariae. After the sharp rise of antibody levels to adult worm antigens in this study as a result of treatment, these levels fell to pretreatment values within 6 mo in both groups, and thereafter remained constant. In contrast, responsiveness to egg antigen remained constant in the nonimmune children throughout the period of observation; the resistant children showed a progressive decrease. The ability of the resistant and nonimmune children to recognize different antigenic components was studied by immunoprecipitation of mRNA translation products (Taylor et al., 1984). A marked heterogeneity in responses to the various peptides was observed, but immune children showed a higher frequency of response to mRNA translation products of 27 and 24 kD than did the susceptible children. Continuation of this study by Butterworth et al. should yield greater insight into immune mechanisms and environmental factors that influence the course of schistosomiasis in a highly endemic area. The findings, thus, will enable more effective control strategies to be devised.
ANTIGENS FOR DETECTION OF ANTIBODIES
It is obvious from this discussion of serodiagnosis and seroepidemiology that serologic antigens and assays were individual for each study described. Sensitive, specific antigens available in costeffective quantities and standardized assays are required for accurate serodiagnosis and comparability between various field studies and use in seroepidemiology. WHO initiated antigen evaluation studies with both S. mansoni (Mott and Dixon, 1982) and S. japonicum (Mott, 1984} reagents. Numerous reports on schistosome antigen purification and characterization have appeared during the past 3 yr. Strand et al. have concentrated on the glycoproteins of both adult worms and eggs (Aronstein and Strand, 1983; Norden and Strand, 1984}. Genus-, species-, stage-, and sex-specific glycoproteins were identified. As mentioned above, acute and chronic infections could be differentiated on the basis of reactions in immunoprecipitation with antigen bands from adult worms. An immunogenic protein of 23 kD was isolated from the surface of adult worms of S. japonicum and identified by a monoclonal antibody (Cruise et al., 1983). The
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antigen was recognized by immunoprecipitation with the serum of an infected Philippine patient. This low molecular-weight component was considered to be a potentially important diagnostic antigen. Hayunge et al. (1983) also purified surface and other glycoproteins from adult worms of S. mansoni. Their antigens reacted with sera of infected humans. In studies directed to identification of surface antigens as potential vaccines, Taylor and Wells (1984) isolated the tegument membranes of schistosomula of S. mansoni. Immunoblot indicated that human sera showed considerable variation in antibody response to the 35 peptides demonstrable by twodimensional gel electrophoresis. Tracy et al. (1985) purified a glycoprotein antigen from eggs of S. japonicum by absorption to concanavalin A-sepharose 4B followed by repetitive gel filtration chromatography. Conventional plate ELISA, in an initial evaluation of this antigenic component has indicated that it is sensitive. This test detected homologous infection in patients with egg counts of <1000 eggs/g of feces. A marked degree of species specificity was evidenced by antigenic reactivity with the sera of only three of 24 patients with S. haematobium eggs ~2000/10 ml urine, and none of 20 patients infected with S. mansoni. Deelder et al. (1980a) in the Netherlands, and Nash (1982) in the U.S., each independently, isolated two very similar glycoproteins. Deelder determined that both his components could also be identified as circulating antigens; only one was found to be a circulating antigen by Nash. A useful collaborative report has been published by Nash and Deelder (1985), showing that the gut-associated proteoglycan (GASP) and circulating anodic antigen (CAA) are identical. This particular antigenic component is now to be known as GASCAP (gut-associated circulating anodic proteoglycan). In contrast, a phenol sulfuric test active peak (PSAP) from the DEAE fractionation of the proteoglycan antigens by Nash was found to be different than the cathodic circulating antigen (CCA) of Deelder. Use of CAA in an ELISA has resulted in a sensitive assay for infection with S. mQnsoni (Deelder et al., 1980b). The limited yield of antigen, however, precludes its wide-scale usage (Deelder and Kornelis, 1981). Another antigen of diagnostic promise is the CEF6 antigen with the active W1 component from eggs of S. mansoni (Dunne et al., 1981). CEF6 is isolated by carboxymethyl cellulose and shows a high degree of species specificity. As already mentioned, Butterworth et al. (1984, 1985) have included its use in their field study; however, their results to date do not show that the purified CEF6 antigen is markedly superior to the crude SEA. Microsomal antigens from adult worms were developed by Tsang et al. (1983a, 1984). The microsomal antigen from S. mansoni is known as MAMA, and the corresponding antigen of S. japonicum as JAMA. These have proved to be specific, sensitive, stable and costeffective in production. A high degree of species specificity is evident in ELISA (Tsang et al., 1983b) and immunoblot (Tsang et al., 1984). The production of the comparable JAMA from S. haematobium is in progress.
STANDARDIZED ELISA
In addition to sensitive, specific, costeffective antigens, quantitative, standardized serologic assays are required for accurate serodiagnosis and for comparison of results between laboratories. A standardized assay for antibody measurement involves the use of a standard serum pool from which a standard curve is prepared in each run of the assay. The reactivity of the unknowns are determined from the standard curve. We evaluated MAMA in a standardized microtest plate ELISA in which enzyme
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reactivity was chemically stopped after 30 min (Maddison et al., 1985). A reference serum pool, arbitrarily designated as having 100 activity units per microliter was used to prepare a standard curve in each plate. The levels of activity of the unknown sera were determined from the standard curve on a continuously variable response scale and recorded as activity units per microliter. Although this assay proved to be sensitive and specific, it had limitations because the enzyme reaction was being measured when it was no longer kinetic. In addition, because the reactions in our standardized assay were allowed to proceed for 0.5 hr, the moderate and highly reactive sera required further dilution in order for the activity levels to lie within the range of the single-log standard curve. The disadvantages of ELISA in which the reactions are chemically stopped are as follows: 1. The activity-stopping agent may not "freeze" color development 2. End-point color intensity (absorbance) is seldom linear with respect to antibody concentration 3. Enzyme reaction is linear with respect to activity only during brief initial phase. Most reaction times chosen grossly exceed the linear phase, and the assay is not truly quantitative. In contrast, single-well ELISA based on enzyme rate kinetics yields quantitative linear measurements for antigen or antibody. The rate analyzer used in the kinetic ELISA (Tsang et al., 1983c) for monitoring of the isolation and characterization of the microsamal antigens allowed such quantitative serologic analysis. However, the rate analyzer is expensive and would not be available in regional centers in developing countries. Such centers are usually familiar with the principles of ELISA. Hancock and Tsang (1986) recently developed a standardized kinetic-dependent FIGURE 1. Lid of microtitration plate holding eight strips of polystyrene sticks with attached beads.
Serology of Schistosomiasis
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FIGURE 2. (A} Standard curve for 100-1000 U of the standard serum pool. Note that two equations, one linear and the other exponential, are necessary to accurately describe the data. (B) Standard curve for 10-100 U of the standard serum pool is best described by a linear equation, y = mx + b. The coefficient of determination (r2} = 0.976. (C) Standard curve for 100-1000 U of the standard serum pool is best described by an exponential equation, y = ae bx, r2 = 0.989. For each curve, each unit value was assayed in triplicate. After Hancock and Tsang, 1986; reproduced by kind permission of the authors and the editors of Journal of Immunological Methods. plate FAST-ELISA, based on the Falcon assay screening test (FAST®)1 system using MAMA. The FAST system consists of strips of 12 polystyrene beads on short sticks. The strips snap into the lid of a microtest plate (Fig. 1). The lid with one or more strips of beads is inverted into troughs or wells of a microtitration plate. A standard reference serum pool was an integral part of the assay. To prepare the standard curve for each plate, increasing a m o u n t s of the standard reference serum pool were reacted with both MAMA and conjugate i n excess. A 2-log range of activities from 10 to 1000 u n i t s of the standard reference serum pool was measurable. To describe the data, two c u r v e s are needed. The activity of the standard serum pool from 10 to 100 u n i t s is best described by a linear equation and from 100 to 1000 by an e x p o n e n t i a l curve (Fig. 2). 1Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Dept. of Health and Human Services.
100
S.E. Maddison
The run-to-run variation of the reactivity of a serum was evaluated by repeatedly testing a serum in triplicate over a 1-mo period. Although the CV of the mean daily absorbance was 20.3%, the CV of the activity units per microliter as determined from the standard curve was only 13.6%; thus, the use of a standard curve reduces the run-to-run variation significantly. Figure 3 s h o w s the results of testing 234 sera. The previously recognized high degree of species-specificity of MAMA (Tsang et al., 1984) is reflected in the l o w level of reactivity of sera from S. japonicum-infected patients compared with that of sera from S. mansoni patients. The diagnostically significant level of reactivity was determined statistically by using the J index (Youden, 1950; Ruiz-Tiben et al., 1979). The data from the h o m o l o g o u s S. mansoni infections were used for the true positives: the normal CDC controls and the patients with other helminthic or liver infection were considered to be true negatives. Both the sensitivity and specificity of the assay are 99%. The FAST-ELISA with MAMA for infection with S. mansoni has been incorporated into our reference diagnostic laboratory at CDC. This assay does not give insight into duration of infection or intensity of infection. Immunoblots with extracts of the respective adults w o r m s are used for suspect S. haematobium and S. japonicum infections. When suitable antigens are available, w e will apply FAST-ELISA to other FIGURE 3. Results from assay of sera from 234 individuals. The groups of sera are identified on the x-axis. The numbers immediately above the x-axis are the number of individuals in the corresponding group that is immediately below the x-axis. On the y-axis is the FAST-ELISA result, expressed in U/p.l. Note that the scale in not continuous. All results on and above the line at 10 U/p.1 are positive, all results below are negative. After Hancock and Tsang, 1986; reproduced by kind permission of the authors and the editors of Journal of Immunological
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Serology of Schistosomiasis
101
helminthic systems. Because each strip of 12 beads is independent, more than one type of antigen strip can be placed in a single lid. A preliminary field study has been made with the FAST-ELISA for schistosomiasis in Cameroon. The only equipment used was two Eppendorf pipettes and a garden spray for washing the beads. The assay appears to be surprisingly rugged, but minor difficulties were encountered with the stability of the hydrogen peroxide. This problem has been resolved by use of a very stable TMB Microwell Peroxidase Substrate System (Kirkegaard and Perry Laboratories, Inc. Gaithersburg, MD). In this study blood collected into phosphate buffered saline (PBS) containing 0.2% sodium azide as a preservative (A. Sulzer, personal communication) was suitable for transport and subsequent testing. A volume-marked capillary tube was used to collect 0.1 ml of whole blood from a finger-stick into 1.9 ml of PBS. When dilution was taken into account no significant difference was observed between the levels of reactivity of serum and the blood in PBS. This method of collecting and processing blood from finger-sticks is vastly superior to filter paper. DETECTION OF ANTIGENEMIA Antibody detection for routine serodiagnosis of schistosomiasis currently provides little, if any, information about the course or intensity of infection, or the effect of chemotherapy because antibodies persist in chronic infections and are slow to indicate significant change after chemotherapy. Detection and quantitation of worm products--surface glycoproteins and proteins that have been sloughed off, as well as secretory/excretory products--in serum or urine would identify active infection and give insight into the effect of chemotherapy. In contrast to some mycotic, viral, and bacterial diseases, however, antigen detection in parasitic disease in general, and schistosomiasis in particular, has not yet become a diagnostic procedure. French investigators with workers in Burundi (Carlier et al., 1980) and Brazil (Santoro et al., 1981) identified circulating antigen and circulating immune complexes in patients' sera. The amounts of the schistosome products were small, however, and necessitated concentration of serum and/or use of radioisotopic assays. Decider et al. (1980a) demonstrated circulating S. mansoni polysaccharide antigens (CAA and CCA) in heavily infected experimental animals. Qian and Decider (1983) quantitated CAA by ELISA in the serum of rabbits experimentally infected with S. japonicum. Circulating antigens in mice harboring 100 worms were detectable as early as I wk after exposure (Hayunga et al., 1986). This was achieved in a competitive-inhibition ELISA using a purified cercarial antigen of apparent molecular weight of 41 kD. CAA and CCA recently have been studied in mixed infection S. mansoni and S. haematobium patients (Feldmeier et al., 1986). A radioimmunometric assay could detect 150 ng/ml of CCA. The monoclonal antibody used appeared to be speciesspecific, because a correlation was observed between the amount of CCA and number of S. mansoni eggs in the stool, but not the number of S. haematobium eggs in urine. The circulating antigen assay was not as sensitive as the detection of circulating antibody by ELISA. Because of the short half-life of radioisotopes and the hazards associated with their use, alternates to isotopic assays are being sought. The development of a sensitive ELISA for measuring circulating antigens and specific immune complexes in schistosomiasis, however, will probably necessitate demanding steps, such as 1. the development of monoclonal antibodies directed to each of two different epitopes, respectively, on a single type of circulating antigenic molecule. One of these monoclonal antibodies would serve as capture antibody. The second would be enzymatically labelled to serve as an indicator reagent.
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2. labeling of this indicator monoclonal antibody would need to be critically controlled to produce an exquisitely sensitive reagent. The use of galactosidase with a higher substrate turnover than peroxidase may result in improved sensitivity (Qian and Wen, 1983). An alternative to the sandwich ELISA may be the use of time-resolved fluorescence (TR-FIA) (Suonpaa et el., 1985) in a sandwich assay with two monoclonal antibodies. The indicator monoclonal antibody is labeled with europium, which results in lanthanide chelates as the fluorescent probe. Because the nonspecific fluorescence associated with serum has a short decay time it is eliminated during the time resolution. Lanthanide chelates have long decay fluorescence and result in a highly sensitive assay. The capability of measurement, at least of alpha-fetoprotein, over a 4-log range is very advantageous. In addition, the biological hazard and disadvantage of the short half-life of radioisotopes are eliminated. The purified target antigen should be produced in large quantities to serve as a standardized reagent for preparing a standard curve in each ELISA. Production of purified antigens will probably necessitate the use of recombinant DNA technology. If appropriate reagents and standardized assays become available, measurement of circulating antigen in patients' sera and urine will be possible.
RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology is widely applied in microbiologic research, and within the past 3.5 yr at least five groups have reported studies dealing with recombinant DNA technology in schistosomiasis. However, target circulation antigen production was not the goal. Interest has been mainly focused on producing a vaccine. Cordingley et al. (1983) first reported the construction of cDNA clone banks from poly(A) +messenger RNA of eggs and adult worms of S. mansoni. Although many translation products were common to eggs and worms, a p40 product was obtained from a mRNA unique to eggs. This p40 molecule appeared to be a potential serodiagnostic reagent. These investigators identified in vitro translation products of 27 and 25 kD, which reacted more frequently with the sera of immune than nonimmune Kenyan children (Butterworth et el., 1985). Recently they have reported the use of mRNA hybrid selection followed by immunoprecipitation of the corresponding translation products (Cordingley et al., 1986). This technique allows screening thousands of clones. Simpson and Knight (1986) screened recombinant cDNA libraries by differential hybridization that had high levels of expression in adult female worms but were not associated with immature female or adult male worms. Thus, cDNA clones were identified that encode a developmentally regulated schistosome gene. In other studies, polypeptide antigens derived by recombinant DNA technology were screened by precipitation with experimental animal and human infection sera (Knight et al. 1986). Some of the products showed potential as diagnostic antigens. Bugra et el. (1983) reported the development of schistosome translation products from mRNA templates. These serve as templates for preparing cDNA libraries and as molecular probes. Saint et al. (1986) prepared a cloned cDNA library from S. japonicum adult worm mRNA. The cDNA was expressed as fusion products with amp3/E, coli ~-galactosidase. Screening with human patient sere identified fewer reactive colonies than screening with sera from immunized rabbits. In summary, investigators have made great strides in recombinant DNA/schistosomiasis research. Conventional approaches have not solved the problems of reagents required for widespread use of standardized serologic assays for schistosomiasis. We now look to this new technology for production of antigens as standards. These are
Serology of Schistosomiasis
103
required not only for assays that measure antibody levels but also to serve as standards in assays that measure circulating antigens. A standardized assay, the FAST-ELISA, has been discussed. This assay used in c o n j u n c t i o n with microsomal antigen (MAMA) has proved to be suitable for m e a s u r e m e n t of antibody i n both serodiagnosis and seroepidemiology. The high sensitivity of this assay may well permit the measurem e n t of incidence i n an e n d e m i c area. Nonetheless, additional antigens and assays that give greater insight into the disease process pertinent to patient m a n a g e m e n t and control programs should be sought. Measurement of circulating antigen will probably be more appropriate than measurement of antibody. TR-FIA with europiumlabeled conjugates may supply the necessary increased sensitivity. It should be emphasized that essential c o m p o n e n t s for standardization are International Reference Serum pools for each of the three species of schistosomes of major importance to h u m a n s . These reagents are yet to be prepared. The World Health Organization or the International Association of Biological Standards should assume the responsibility for preparing these m u c h - n e e d e d reagents and ascribing International Units to them. REFERENCES Anuar H, Greer GJ, Ow-Yank CK, Sudumaren KD (1984) Detection of Malaysian schistosimiasis in Orang Asli of Peninsular Malaysia using serodiagnostic tests. Southeast Asian l Trap Med Public Health 15:479. Aronstein WS, Strand M (1983) Identification of species-specific and gender-specific proteins and glycoproteins of three human schistosomes. J Parasitol 69:1006. Barakat RM, E1-Gassim EE, Awadalla HN, E1-Molla A, Omer EA (1983) Evaluation of enzymelinked immunosorbant assay (ELISA) as a diagnostic tool for schistosomiasis Trans H Sac Trap Med Hyg 77:109. Boudin C, Chaize J (1982) Depistage de masse de la Bilharziose a Schistosoma mansoni. Erude de la fiabilite de deux techniques: Parasitologique (MIF) et immunologique (ELISA). Cab O.R.S.T.O.M. ser Ent Med Parasitol 20:299, Bruckner DA (1985) Serologic and intradermal tests for parasitic infections. Pediatr Clin N A m 32:1063. Bugra K, Tanaka RD, Boyle WJ, Maclnnis AJ (1983) Isolation of PolyA( + ) RNA from Schistosoma mansoni and immunoprecipitation of its in vitro translation products, l Porasitol 69:486. Butterworth AE, Dalton PR, Dunne DW, Mugambi M, Ouma JH, Richardson BA, Arap Siongok TK, Sturrock RF (1984) Immunity after treatment of human schistosomiasis mansoni. I. Study design, pretreatment observations and the results of treatment. Trans R Sac Trap Med Hyg 78:108. Butterworth AE, Capron M, Cordingley JS, Dalton P'R, Dunne DW, Dariuki HC, Kimani G, Koech D, Mugabi M, Ouma JH, Prentice MA, Richardson BA, Arap Siongok TK, Sturrock RF, Taylor DW (1985) Immunity after treatment of human schistosomiasis mansoni. II. Identification of resistant individuals and analysis of their immune responses. Trans R Sac Trap Med Hyg 79:393. Carlier Y, Nzeyimana H, Bout D, Capron A (1980) Evaluation of circulating antigens by a sandwich radioimmune assay, and of antibodies and immune complexes in Schistosoma mansoni-infectedAfrican parturients and their newborn children. Am l Trap Med Hyg 29:74. Cordingley JS, Taylor DW, Dunne DW, Butterworth AE (1983) Clone banks of cDNA from the parasite Schistosoma mansoni: Isolation of clones containing a potentially immunodiagnostic antigen gene. Gene 26:25. Cordingley JS, Haddow WJ, Nene V, Taylor DW (1986) Identification by message selection of cDNA clones encoding antigens of Schistosoma mansoni. Molec Biochem Parasitol 18:73. Cruise KM, Mitchell GF, Garcia EG, Tiu WU, Hocking RE, Anders RF (1983) Sj23, the target antigen in Schistosoma japonicum adult worms of an immunodiagnostic hybridoma antibody. Parasitol Immunol 5:37. de Cock KM, Hodgen AN, Lillywhite JE, Arap Siongok TK, Lucas SB, Rees PH (1985) Hepatosplenic schistosomiasis in Kenya: An assessment of the enzyme-linked immunosorbent assay (ELISA). Trap Geogr Med 37:285.
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Deelder AM, Kornelis D (1981) Immunodiagnosis of recently acquired Schistosoma mansoni infection. A comparison of various immunological techniques. Trap Geogr Med33:36. Deelder AM, Kornelis D, Van Marck EAE, Eveleigh PL, Van Egmond JG (1980a) Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse, hamster, and human infections. Exp Parasitol 50:16. Deelder AM, Kornelis D, Makbin M, Noordpool HN, Cadfried RM, Rotmans JP, Oostburg BJF (1980b) Applicability of different antigen preparations in the enzyme-linked immunosorbant assay for schistosomiasis mansoni. Am ] Trap IVied Hyg 29:401. de Savigny D, Speiser F (1985) Enzyme immunoassays for tropical parasitic diseases. In Enzymemediated Immunoassay. Eds., Ngo TT, Leahoff HM. New York: Plenum Press, pp. 415-431. Dunne DW, Lucas S, Bickle Q, Pearson S, Madgwick L, Bain J, Doenhoff MJ (1981) Identification and partial purification of an antigen (Wl) from Schistosoma mansoni eggs which is putatively hepatotoxic in T-cell deprived mice. Trans R Sac Trap Med Hyg 75:54. Feldmeier H, Gastl GA, Poggensee U, Kortmann C, Daffalla AA, Peter HH (1985a) Relationship between intensity of infection and immunomodulation in human schistosomiasis. I. Lymphocyte subpopulations and specific antibody responses. Clin Exp Immunol 60:225. Feldmeier H, Gastl GA, Poggensee U, Kortmann C, Daffalla AA, Peter HH (1985b) Relationship between intensity of infection and immunomodulation in human schistosomiasis. II. NK cell activity and in vitro lymphocyte proliferation. Clin Exp Immunol 60:239. Feldmeier H, Noqueira-Queiroz JA, Peixoto-Queiroz MA, Doehring E, Dessaint JP, de Alencar JE, Dafalla AA, Capron A (1986) Detection and quantification of circulating antigen in schistosomiasis by monoclonal antibody. The quantification of circulating antigens in human schistosomiasis mansoni and haematobium: Relationship to intensity of infection and disease status. Clin Exp Immunol 65:232. Hancock K, Tsang VCW (1986) Development and optimization of the FAST-ELISA for detecting antibodies to Schistosoma mansoni. J Immunol Meth 92:167. Hayunga EG, Sumner MP, Stek M, Vannier WE, Chestnut RY (1983) Purification of the major concanavalin A-binding surface glycoprotein from adult Schistosoma mansani. Proc Helminthol 50:219. Hayunga EG, Duncan JF, Stek M, Mollegard I, Sumner MP, Hunter KW (1986) Development of circulating antigen assay for rapid detection of acute schistosomiasis. Lancet ii:716. Higashi GI (1984) Immunodiagnostic tests for protozoan and helminth infections. Diagn Immunol 2:2. Knight M, Simpson AJG, Bickle Q, Hagan P, Moloney A, Wilkins A, Smithers SR (1986) Adult schistosome cDNA libraries as a source of antigens for the study of experimental and human schistosomiasis. Melee Biochem Parasitol 18:235. Lewert RM, Yogore MG, Bias BL (1984) Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. II. Unreliabilityof stool examination in the assessment of incidence. Am J Trap Med Hyg 33:872. Maddison SE (1986) Schistosomiasis. In Immunodiagnosis of Parasitic Diseases, Vol. I. Helminthic Diseases. Eds., Walls KW, Schantz PM. Orlando: Academic Press, Inc., pp. 1-37. Maddison SE, Tsang VCW (1983) Serodiagnosis of schistosomiasis. Clin Immunol Newsl 4:101. Maddison SE, Slemenda SB, Tsang VCW, Pollard RA (1985) Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen (MAMA) in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool. Am J Trap Med Hyg 35:484. Matt KE (1984) Diagnostic tools for Schistosoma japonicum in control programs. Arsheim Forsch/Drug Res 34:1217. Matt KE, Dixon H (1982) Collaborative study on antigens for immunodiagnosis of schistosomiasis. Bull WHO 60:729. Nash TE (1982) Diagnostic serologic responses. In: Schistosame Infection in Humans: Perspectives and Recent Findings. Moderator, Nash TE, Ann Intern Med 97:740. Nash TE, Deelder AM (1985) Comparison of four schistosome excretory-secretory antigens: Phenol sulfuric test active peak cathodic circulating antigen, gut-associated proteoglycan, and circulating anodic antigen. Am ] Trap Med Hyg 34:236. Norden AP, Strand M (1984) Schistosoma mansoni, S. haematobium, and S. japonicum: Identification of genus- and species-specific antigenic glycoproteins. Exp Parasitol 58:333.
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Norden AP, Strand M (1985) Identification of antigenic Schistosoma mansoni glycoproteins during the course of infection in mice and humans. Am ] Trop Med Hyg 34:495. Qian ZL, Deelder AM (1983] Schistosoma japonicum: Immunological characterization and detection of circulating polysaccharide antigens from adult worms. Exp Parasitol 55:168. Qian ZL, Wen HC (1983) Schistosome circulating antigens (CSA) as a possible diagnostic parameter for active infections. Acta Leiden 51:37. Ruiz-Tiben E, Hillyer GV, Knight WB, Gomez de Rios I, Woodall JP (1979) Intensity of infection with Schistosoma mansoni: Its relationship to the sensitivity and specificity of serologic tests. Am / Trop Med Hyg 28:230. Saint RB, Beall ]A, Grumont R], Mitchell GF, Garcia EG (1988) Expression of Schistosoma japonicum antigens in Escherichia coil. Molec Biochem Parasito| 18:333. Santoro F, Prata A, Silva AE, Capron A (1981] Correlation between circulating antigens detected by radioimmuno-precipitation-polyethyleneglycol assay (RIPEGA)and CIq-bindingimmune complexes in human schistosomiasis mansoni. Am ] Trop Med Hyg 30:1020. Simpson AJG and Knight M (1986) Cloning of a major developmentallyregulated gene expressed in mature females of Schistosoma mansoni. Molec Biochem Parasitol 18:25. Speiser F (1985) Serodiagnosis of parasitic diseases. Experientia 41:531. Suonpaa MU, Lavi IT, Hemmila IA, Lovgren TN-E (1985] A new sensitive assay for human alpha-fetoprotein using time-resolved flourescence and monoclonal antibodies. Clin Chim Acta 145:341. Taylor DW, Wells PZ (1984) Isolation and antigen analysis of surface tegument membranes from schistosomula of Schistosoma mansoni. Parasitology 89:495. Taylor DW, Cordingley IS, Butterworth AE (19841 Immunoprecipitation of surface antigen precursors from Schistosoma mansoni messenger RNA in vitro translation products. Molec Biochem Parasito] 10:305. Tracy ]W, Domingo EO, Mahmoud AAF (1985) Evaluation of purified Schistosoma japonicum glycoprotein egg antigen for the immunodiagnosis of infection in man. Am ] Trap ivied Hyg 34:92. Tsang VCW, Tsang KR, Hancock K, Kelley MA, Wilson BC, Maddison SE (1983a) Schistosoma mansoni adult microsomal antigens, a serologic reagent. I. Systematic fractionation, quantitation, and characterization of antigenic components. 1 Immuno] 130:1359. Tsang VCW, Tsang KR, Hancock K, Kelley MA, Wilson, BC, Maddison SE {1983bl Schistosoma mansoni adult microsomal antigens, a serologic reagent. II. Specificity of antibody responses to the S. mansoni microsomal antigen (MAMA]. ! Immunol 130:1366. Tsang VCW, Wilson BC, Peralta ]M {1983c) Quantitative, single-tube, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA). Meth Enzymol 92:391. Tsang VCW, Hancock K, Maddison SE, Beatty AL, Moss DM (1984) Demonstration of speciesspecific and cross-reactive components of the adult microsomal antigens from Schistosoma mansoni and S. japonicum (MAMA and lAMA). J ImmunoI 132:2607. Walls KW, Wilson M (1983) Immunoserology in parasitic infections. In Immunodiagnostics. Eds., Aloisi RM, Hyun ]. New York: Alan R. Liss, Inc. pp. 191-214. Wilson M, Sulzer A], Walls KW (1976) Modified antigens in the indirect immunofluorescence test for schistosomiasis. Am I Trop IVied Hyg 23:1072. World Health Organization (WHO) (1985) The control of schistosomiasis. WHO Technical Report Series 728:51. Yogore MG, Lewert RIM,Bias BL (1979) Schistosomiasis japonica in Barrio San Antonio, Basey, Samar in the Philippines. III. The plasma circumoval and precipitin test procedure and use in epidemiologic studies. Southeast Asian / Trop Med Public Health 10:21. Yogore MG, Lewert RM, Bias (1983] Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. I. Underestimation by stool examination of the prevalence of infection in school children. Am J Trop Med Hyg 32:1322. Youden W] {1950) Index for rating diagnostic test. Cancer 3:32.