Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. II. Unreliability of stool examination in the measurement of incidence.

It was shown in a previous study that a single quantitative stool examination (by technics currently used in the Philippines) is so sensitive that it underestimated by 50% the prevalence of schistosomiasis japonica in children detected by a single serologic assay (ELISA). This paper proves that measurement of incidence of the infection among these same children by stool examination is unreliable as well. Three important sources of error in the measurement of incidence by stool examination are: (a) the inclusion of already infected children in the group of presumably uninfected children in which conversion (change from negative to positive) is being measured; (b) the insensitivity of stool examination in identifying those who do acquire infection among the initially uninfected; and (c) spontaneous, possibly temporary, stool reversion. An overestimation of incidence results from error (a) because already infected children have 5-7 times the tendency to convert than do uninfected children; error (b) tends to produce an underestimation of incidence since stool examination will detect only about one-half of the children who do become infected; while error (c) also tends to cause an underestimation of incidence and is dependent on the frequency of stool examinations--34% of the conversions observed by multiple examinations may not be detected in a single year-end examination. The equivalent sources of error are insignificant when ELISA is employed to measure incidence. In the 3 years of observation, the annual incidence of schistosomiasis japonica measured by ELISA showed no significant change (13.5%, 16.7%, and 15.6%); on the other hand, the annual incidence measured by stool examination showed a significant reduction from 37.5% down to 16.0% and 15.1%. Significant sources of error invalidate measurements by stool examination; it is therefore concluded that incidence measurements have failed to produce acceptable evidence of reduction in transmission in the area of study in the 3 years of control operations. We believe that insistence on relying almost exclusively on the parasitologic technic can result in an erroneous evaluation of the control program currently being implemented.

Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. III. Selective mass chemotherapy with praziquantel in a control program.

After a pre-control (pre-treatment) prevalence rate of schistosomiasis japonica was established by stool examination and by ELISA in about 400 school children in school year 1979, annual prevalence rates were measured in the following 3 assessment years. From 54-80% of children found infected primarily by stool examination were treated with praziquantel 3-9 months before the assessment examinations. Both stool examination and ELISA showed statistically significant reductions of prevalence from the 1st to the 3rd assessment years (average reduction of 50% measured by stool examination and average reduction of 19% measured by ELISA). These reductions were expected as a result of the biologic activity of the effective schistosomicide; they are therefore not acceptable evidence of reduced transmission levels. By decreasing human sources of snail infection, an effective schistosomicide may have the epidemiologic impact of reducing transmission; assessment prevalence rates can be relevant indices of transmission levels only if the biologic effect of the schistosomicide is dissociated from its epidemiologic impact in a control program in which selective mass chemotherapy plays a dominant role. This dissociation was accomplished in this study by measurements in an essentially untreated group of "new" pupils, i.e., those who entered the school at the beginning of each assessment year. Overall prevalence rates among the "new" pupils did not show a significant reduction until the 3rd assessment year as measured by stool examination (reduction by 36% of pre-control level); as measured by ELISA, a significant reduction (by 15-17%) was detected in the 2nd and 3rd assessment years. The youngest children, and girls more than boys, were the most sensitive indicators of change.(ABSTRACT TRUNCATED AT 250 WORDS)

Seroepidemiology of schistosomiasis japonica by ELISA in the Philippines. I. Underestimation by stool examination of the prevalence of infection in school children.

In a longitudinal seroepidemiological study in the Schistosoma japonicum endemic area of Leyte, the ELISA technique to determine prevalence and incidence rates in elementary school children was compared with similar determinations made by a modified quantitative stool examination (MIFC). In the area of this study, Barrio Salvador, Tanauan, Leyte, the ongoing schistosomiasis japonica control program in the Philippines is dependent on stool examination by MIFC and/or the quantitative thick smear (Kato-Katz) for measurement of prevalence and incidence. Over a 3-year period with multiple periodic examinations, infection rates were measured and the serologic technique was compared to stool examination in 598 untreated children (mostly 7-10 years of age) of Salvador Elementary School. A group of 150 school children from a non-endemic area, Milagros, Masbate, provided sera as a reference negative control. ELISA results are expressed as ELISA activity (EAc) in reference to a positive control serum pooled from parasitologically confirmed cases, dilutions of which were always included in each assay. A convenient positive-negative discrimination level was chosen based on the EAc values obtained from 170 stool-positive Salvador pupils and the 150 pupils of the non-endemic area. Using the chosen discrimination level, ELISA in this study had a sensitivity of 98% and a specificity of 96%. ELISA was significantly more sensitive than stool examination in detecting infections; only 28% of the children were stool positive on a single examination in contrast with 56% positive by ELISA. A single stool examination underestimated serologic positives by 50% while two stool examinations 4 months apart reduced the underestimate to 29%. The underestimation varied by age and sex, and showed no consistent pattern in this regard. Stool-positive children had a wide variation of egg counts with a geometric mean of 6.4 eggs/g of stool, with 52% of the stool positives excreting only 1-5 eggs/g. A high percentage of infected children have a misdiagnosis of infection by stool examination. This has, in the past, resulted in many being misclassified as noninfected. This erroneous classification has serious consequences on the measurement of prevalence and incidence, on studies of clinical manifestations of the disease, and on the evaluation of serologic techniques for diagnosis. Stool examination does not give an accurate measurement of prevalence, and therefore it cannot be relied upon for the evaluation of the current control program. It is recommended that the capability to undertake serodiagnostic tests for schistosomiasis japonica be encouraged and adopted in the Philippines for field

Immunoglobulin M-Immunoglobulin G mixed cryoglobulinemia in Schistosoma japonicum-infected rabbits.

Cryoprecipitation was observed in defibrinated serum from Schistosoma japonicum-infected rabbits. Ouchterlony immunodiffusion, sodium dodecyl sulfate-polyacrylamide gels, and anti-cryoprecipitate antiserum demonstrated the presence of immunoglobulin G (IgG), IgM, C3, fibrinogen, and an alpha-macroglobulin. Parasite antigen was not detected. IgG and IgM from pooled cryoprecipitates failed to react with each other in Ouchterlony immunodiffusion gels after separation by column chromatography. The data suggest that the IgG and IgM are not specifically complexed with each other but may simply be aggregates of altered proteins.

Efficacy of purified schistosoma japonicum egg antigens for ELISA serodiagnosis of human Schistosomiasis japonica: specificity and sensitivity.

At present, there is no consensus that purified schistosome egg antigens offer any advantage in the diagnosis of schistosomiasis by enzyme linked immunosorbent assay (ELISA). Previously, we demonstrated by multiple techniques that the major serologic antigens in Schistosoma japonicum soluble egg antigen (SEA) are glycoproteins, and that the glycoproteins with highest specificity and sensitivity are hydrophobes. We therefore tested these materials for their specificity, sensitivity and cost effectiveness in the ELISA. In this study we used five SEA fractions that varied in their purity and antigenicity. The order of immunologic specific activity in the ELISA, measured by titration of a standard sera pool, was: hydrophobic glycoproteins (highest), crude SEA glycoproteins, hydrophilic glycoproteins, crude SEA, and SEA proteins (lowest). Complexity (purity) of these materials were (in rank order), hydrophilic glycoproteins (purest), hydrophobic glycoproteins, crude glycoproteins, SEA proteins, and crude SEA (most complex). Epidemiologic sensitivity in the ELISA was tested on limited but well characterized populations. At high antigen coating concentration (0.5 microgram/well), the only antigen fraction with poor sensitivity was SEA proteins. There was little difference in epidemiologic sensitivity between the purer fractions with highest immunologic sensitivity (hydrophobic glycoproteins and crude SEA glycoproteins) and the crude SEA which possesses intermediate immunologic sensitivity. Differences in epidemiologic sensitivity were most pronounced when wells were coated at an antigen concentration (0.1 microgram/well) where crude SEA began to fail. Specificity for all preparations, assessed by reactivity with sera from patients with other trematode infections and with cestode and nematode infections, was excellent. The clinical sensitivity of the ELISA employing crude S. japonicum SEA is so high, and the specificity so good, that the increased immunologic sensitivity of partially purified antigens had little effect on epidemiologic sensitivity. This is not true for the S. mansoni ELISA where crude antigens had inferior sensitivity and specificity.

Immune complex glomerulonephritis and amyloidosis in Schistosoma japonicum infected rabbits.

Epidemiologically, the incidence of renal pathology in patients with chronic parasitic infections is higher than expected. In particular, schistosomiasis may have an association with renal failure. 24 New Zealand White rabbits were, therefore infected with 250 or 500 Schistosoma japonicum cercariae of the Philippine-Leyte strain and studied for eight months to determine if rabbits with long-term infections were suitable hosts for the study of schistosomal nephropathy. Clinical evidence for renal disease consisted of proteinuria, haematuria, and casts. Of the 18 surviving infected animals, six had trace amounts of protein in their urine, three had significant proteinuria ranging from 100 to 300 mg%, four exhibited haematuria and 14 were positive for the presence of proteinaceous cast formation. The clinical findings correlated with the histological data. Periodic open renal biopsies on a subgroup of the animals revealed no changes until about the sixth month. At eight months after infection, five (28%) of the 18 rabbits had amyloid deposits and 15 (83%) had some degree of renal change which included mild focal, diffuse intracapillary, and crescentic glomerulonephritis with mesangial and subendothelial complex trapping. Periodic-acid Schiff staining graphically demonstrated wire loops and tubular casts. Immunofluorescence showed that 15 (83%) of the infected animals exhibited diffuse mesangial and peripheral capillary wall deposition of IgG while 14 contained IgM (78%). The third component of complement was found in only five (28%) of the infected rabbits. Parasite antigen could not be detected in the glomeruli of any of the animals. Kidneys from age-matched controls were within normal limits. Electron microscopy of glomeruli from several animals demonstrated the presence of subendothelial and mesangial immune complex deposition similar to that seen in systemic lupus erythematosus. These findings show that schistosomiasis japonica in the rabbit offers an excellent model system for studying not only the renal pathology associated with human schistosomiasis but also the pathogenesis of amyloidosis which is a frequent sequela observed in a variety of chronic inflammatory infections.

Fractionation of Schistosoma japonicum soluble egg antigen glycoproteins by hydrophobic interaction chromatography.

We are currently studying the soluble egg antigens of Schistosoma japonicum in an attempt to determine which antigens are potent immunogens. Previously, we demonstrated by Ouchterlony immunodiffusion and inhibition of the circumoval precipitin test that the glycoprotein fraction of soluble egg antigens contains the antigens which are most immunogenic in natural infections. The soluble egg antigen glycoproteins have now been further fractionated via hydrophobic interaction chromatography on phenyl Sepharose. We found that there were at least two antigens involved in the circumoval precipitin reaction. Both the hydrophilic antigen which we call japonicum antigen glycoprotein II (JAG II) and a mixture of hydrophobic antigens (JAG III and the JAG IV complex) were capable of causing a 50% inhibition of the COP reaction around S. japonicum eggs. JAG II was not a major serological antigen of S. japonicum since it gave only a weak precipitin line upon Ouchterlony immunodiffusion analysis with pooled sera from Filipino patients with chronic S. japonicum infections. Hydrophobic interaction chromatography yielded preparations which were sufficiently pure for use in radioimmunoassays. By radioimmunoassay, the best antigens among the glycoproteins were moderately hydrophobic JAG III and the JAG IV complex. They had large amounts of antibody directed toward them in patients with schistosomiasis japonica and exhibited little reactivity with S. mansoni. The hydrophilic glycoproteins JAG I and II were poor immunogens and extensively cross-reacted with S. mansoni. This cross-reactivity means that diagnostic tests with crude soluble egg antigens would run the risk of potential false-negative results in patients with other trematode infections.

Characterization of the glycoprotein antigens which mediate the Schistosoma japonicum circumoval precipitin reaction.

The circumoval precipitin test is a serological test used for diagnosis of schistosomiasis japonica. Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans. Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum. Crude egg antigens were separated into protein and glycoprotein fractions by lectin chromatography on concanavalin A Sepharose. The glycoprotein fraction produced two intense precipitin lines upon immunodiffusion analysis with human chronic infection sera. The protein fraction produced two faint precipitin lines which did not cross-react with those of the glycoprotein fraction. The glycoprotein fraction contained 90% of the circumoval precipitin inhibitory activity. Isoelectric focusing of (125)I-labeled concanavalin A Sepharose fractions revealed at least four groups of potential S. japonicum antigens, termed JAG I, II, and III, and a JAG IV complex. These had isoelectric points ranging from 3.2 to 6.7. In these respects, the S. japonicum egg antigen glycoproteins are similar to those of Schistosoma mansoni. The glycoproteins were further separated by diethylaminoethyl ion-exchange chromatography. On immunodiffusion analysis it was found that one of the strong Ouchterlony precipitin lines was associated with glycoproteins that did not adsorb to diethyl-aminoethyl columns, whereas the second Ouchterlony precipitin was heterodisperse, being present in the first, second, and third of the four peaks eluted from the diethylaminoethyl column. Immunoelectrophoresis of the diethylaminoethyl fractions demonstrated that the antigen present in highest concentration in soluble egg antigen glycoproteins, JAG II, was extremely heterodisperse in its behavior on diethylaminoethyl columns. This is unlike the S. mansoni antigens which can be easily separated by diethylaminoethyl ion-exchange chromatography.

Histological changes elicited in Schistosoma japonicum infected rabbits following curative chemotherapy with 4-isothiocyano-4'-nitro-diphenylamine (C9333-Go/CGP 4540).

New Zealand white rabbits were infected with 250 or 500 cercariae of the Philippine-Leyte strain of Schistosoma japonicum. 26 weeks later the experimental group were given one dose of 25 mg/kg formulated CGP 4540. Six weeks after dosing the treated rabbits and untreated controls were killed and examined. Parasitological cure was complete in all those treated. The liver and intestinal granulomata were greatly diminished in size and there was a dramatic decrease in the number of inflammatory cells in the hepatic tissue.

Analysis of the immunoglobulins responsible for the circumoval precipitation reaction.

Two morphologically distinct types of circumoval precipitates (COP) have been observed in human Schistosoma japonicum infections. An elongated segmented COP occurs in chronic human infections. An unsegmented "reaction of recent infection" (RRI) occurs in serum from humans with recently acquired infections and is morphologically similar to the reaction observed in the sera of mice infected with S. japonicum. Sera from infected mice and humans were separated by G-200 chromatography to determine whether the unsegmented "RRI" was due to IgM antibody and the segmented COP reaction due to IgG. There was an elevation of the 19S fraction of sera of mice with 10 and 16 week infections. In addition, the murine 7S fraction was elevated in the 16 week infections. The COP activity was confined to the 7S fraction in the murine sera. Sera from Philippine patients which produced reactions of recent infection (acute sera), segmented COP reactions (chronic sera), and mixed reactions (believed to be from a transition stage between acute and chronic schistosomiasis) were tested. All human sera had elevation of both the 19S and 7S fractions of the acute serum. However, COP-reactive antibodies were confined to the 7S fraction of sera from the transition stage and acute stage infections. The results suggest that although IgM antibodies do in certain cases participate in the COP and produce reactions of recent infection, antibody class is not responsible for the different morphology of this reaction.

Schistosomiasis japonica in Barrio San Antonio, Basey, Samar in the Philippines. IV. "Atypical" precipitates in the circumoval precipitin test--a reaction of recent infection.

Morphologically distinct circumoval precipitin reactions, not having the usual "segmented" appearance, are characteristic of recently acquired infection of man with Schistosoma japonicum. In contrast to the precipitins seen in plasma of those with chronic infections, reactions of recent infection (RRI) are commonly, large, vacuolated blebs or, if elongate, are smooth and vacuolated in structure. Recognition of these RRI provides a practical, simple method to study incidence of infection and the techniques used are readily accepted by children in primary school grades.

Schistosomiasis japonica in Barrio San Antonio, Basey, Samar , The Philippines. I. Epidemiology and morbidity.

Barrio San Antonio is a coastal settlement on the San Juanico straits in the municipality of Basey on the island of Samar, the Philippines. It has a population of approximately 1,900 in 320 households. Initially, 851 residents (45%) of 240 households participated in this survey to determine the prevalence and intensity of Schistosoma japonicum infection, and morbidity as indicated by associated hepatomegaly and splenomegaly. As a result of an initial single stool examination of 1 ml by a modified formalin concentration technique, 40% of this population was found to be infected. On subsequent examinations, with the addition of serologic techniques and recording the history of therapy, approximately 70% of this population was found to be infected with S. japonicum after the age of 10 years. If judged by the number of eggs produced per milliliter of feces, infection intensity in this population might be considered to be low. However, 25% (73 of 391 positives on single examination) exhibited schistosomal hepatomegaly or hepato-splenomegaly and had a mean egg count of 10.9. Those infected but without hepatomegaly had a slightly lower mean egg count of 8.4. Of the infected males with hepatomegaly, 75% were 19 years of age or less and 45% of these were producing less than 10 eggs/ml of stool. Of infected females with hepatomegaly, only 28% were 19 years of age or less, and 68% of these were producing less than 10 eggs/ml. Abdominal pain, distress, diarrhea, and dysentery were significantly more frequent in the infected than uninfected persons, and this frequency was related to egg output. The heights and weights of these infected individuals were less than those of the uninfected members of this population and significantly less than the Filipino norm. The small percentage of the infected population (6.6%) that were producing 51% of the eggs had a mean egg count of approximately 260 with a mean age of 33.7. In this group, 7 of the 22 individuals were in the age group 10--14 years and 15 were above 20 years of age. Although some individuals of Barrio San Antonio have sought therapy, this population and area have been largely unstudied and have not up to the present been involved in control or mass chemotherapy programs. The findings of this survey give an opportunity to determine the impact of such programs when they are instituted in this area.

Schistosomiasis japonica in Barrio San Antonia, Basey Samar, in the Philippines: III. The plasma circumoval precipitin test-procedure and use in epidemiologic studies.

A plasma circumoval precipitin test is proposed as the basic diagnostic tool for epidemiologic studies on schistosomiasis japonica in the Philippines. The collection of plasma, the preparation of the COP test slide, and the reading and interpretation of results are described in detail with pertinent comments. The cost, advantages and usefulness of the test are also discussed.

Lymphocyte responsiveness to phytohemagglutinin and to worm and egg antigens in human schistosomiasis japonica.

Using a whole blood culture technique, lymphocyte responsiveness was assayed as 3H thymidine incorporation in 41 individuals infected with Schistosoma japonicum. In comparison with controls, response to phytohemagglutinin (PHA) was suppressed but was unrelated to egg excretion levels or duration of infection. Reactivity to whole worm antigens was significantly greater in individuals with a higher level of egg excretion and presumably greater infection intensity. The response to egg antigens significantly decreased with age and duration of infection. Some individuals with a depressed responsiveness to PHA maintain a high reactivity to parasite antigens, suggesting that different subpopulations of lymphocytes may be involved in these measurements.

Schistosomiasis japonica in Barrio San Antonio, Basey, Samar in the Philippines: II. Quantitative fecal examination and circumoval precipitin tests.

An improved quantitative stool examination technique (MFCT) and two variations in the performance of the circumoval precipitin test, one using whole serum (SCOPT) and the other employing the eluate from finger prick blood dried on filter paper (FPCOPT), were evaluated in a field study to detect infection with S. japonicum among residents of an endemic barrio in Samar. The sensitivity of SCOPT was above 90%, that of MFCT was between 80 to 90%, while that of FPCOPT was only about 50%. Intensity of infection as judged by fecal egg excretion and duration of infection as indicated by age were shown to be determinants of serum reactivity (and hence sensitivity of the two serodiagnostic tests). The relative insensitivity of FPCOPT observed in this work, also evident in some published data of previous workers, puts to doubt the wisdom of using this technique as the procedure of choice in epidemiologic surveys in the Philippines as currently favored in that country. Atypical positive reactions in circumoval precipitin tests using whole serum were detected which may make the differentiation of relatively recent from old infections possible.

Splenic suppressor cells and cell-mediated cytotoxicity in murine schistosomiasis.

An impairment of the capacity to generate alloantigen-specific cytotoxic T lymphocytes (CTL) was observed in mixed lymphocyte cultures (MLC) established with spleen cells from mice infected with Schistosoma mansoni. This impairment, which was observed as early as the eighth week of infection, could be abrogated by the fractionation of spleen cell suspensions by the carbonyl iron/magnet method prior to the establishment of MLC. Cocultivation of normal spleen cells with increasing numbers of splenocytes from S. mansoni-infected syngeneic mice resulted in a dosage-dependent suppression of CTL generation. This "infectious suppression" was not sensitive to antiserum against mouse thymic lymphocyte antigen (MTLA). The present studies suggest the role of a macrophage rather than a T cell as the suppressor cell in this model of cell-mediated immunity in schistosome-infected mice.