Schistosomiasis mansoni: immunoblot analysis to diagnose and differentiate recent and chronic infection.

One hundred seven patients classified into three different groups (11 with acute schistosomiasis, 58 with chronic schistosomiasis, and 38 children with high IgM-specific antibody titers against schistosome gut-associated antigens living in an endemic schistosomiasis area) were studied by immunoblotting for the presence of IgG, IgM, and IgA antibodies against Schistosoma mansoni soluble adult worm antigen preparation. We used sera from 15 individuals infected with various intestinal parasites, as well as sera from 19 uninfected individuals, as controls. An immunogenic fraction with a molecular weight of 31-32 kD (Sm31/32) was the most frequently recognized by the different antibody isotypes. In the group with acute disease, this fraction was recognized by IgG and IgM antibodies of all patients, and by 10 (90.9%) of 11 samples for IgA antibodies. Approximately 98% of the patients with chronic infections had IgG antibodies against Sm31/32, but only about 10% had IgM and IgA antibodies against this fraction. The IgG immunoblot profiles of the children from the endemic area were similar to those obtained for the group with acute schistosomiasis. This observation suggests recent infection of these children. Our data show that the Sm31/32 protein fraction is highly immunogenic and may be a useful serologic marker for diagnosing and differentiating between acute and chronic schistosomiasis infection.

A comparative epidemiologic study of specific antibodies (IgM and IgA) and parasitological findings in an endemic area of low transmission of schistosoma mansoni.

The diagnostic potential of circulating IgM and IgA antibodies against Schistosoma mansoni gut-associated antigens detected by the immunofluorescence test (IFT) on adult worm paraffin sections was evaluated comparatively to the fecal parasitological method, for epidemiological purposes in low endemic areas for schistosomiasis. Blood samples were collected on filter paper from two groups of schoolchildren living in two different localities of the municipality of Itariri (São Paulo, Brazil) with different histories and prevalences of schistosomiasis. The parasitological and serological data were compared to those obtained for another group of schoolchildren from a non-endemic area for schistosomiasis. The results showed poor sensitivity of the parasitological method in detecting individuals with low worm burden and indicate the potential of the serological method as an important tool to be incorporated into schistosomiasis control and vigilance programs for determining the real situation of schistosomiasis in low endemic areas.

IgM antibodies to Schistosoma mansoni gut-associated antigens for the study of schistosomiasis transmission in Ribeirão Pires, São Paulo.

The potential of an immunofluorescence test for detection of IgM antibodies against Schistosoma mansoni gut-associated antigens (IgM-IFT) was evaluated as a tool for studying aspects related to the schistosomiasis transmission in Ribeirão Pires, in the metropolitan area of the capital of the State of São Paulo, Brazil. Children from a school with about 400 students, 6 to 18 years, were followed-up for two years. In the five surveys, carried out at 6-month intervals, from October 92 to October 94, serological (IgM-IFT) prevalence indices of 5.3%, 5.8%, 6.2%, 2.9% and 3.3% were obtained. These indices were 7 to 10 times higher than the parasitological prevalence indices of 0.5%, 0.5%, 0.7%, 0.4% and 0% determined by the Kato-Katz method. Seroconversion from IFT negative to positive was indicating possible newly acquired S. mansoni infection in three children. But confirmation of infection by fecal examination was possible in only one child. The IgM-IFT can constitute a valuable tool for the improvement of the vigilance program in low endemic areas for schistosomiasis, better characterizing the S. mansoni transmission in such areas.

A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis.

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (São Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 schoolchildren were submitted to ELISA and the data compared to the results of the parasitological method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5%, 43.0% and 56.2% by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Immunofluorescence test on Schistosoma mansoni worm paraffin sections (IgM-IFT) for the study of schistosomiasis transmission in Campinas, São Paulo, Brazil.

The detection of IgM antibodies for Schistosoma mansoni using gut-associated antigens (IgM-IFT) was compared to the parasitological Kato-Katz method for study of the transmission of schistosomiasis in an urban area in Campinas. About 400 schoolchildren whose ages ranged from 6 to 18 years, were observed for a period of two years. Blood samples on filter paper and fecal samples were collected, at intervals of six months. Serological (IgM-IFT) prevalence rates of 1.2%, 4.3%, 3.6%, 2.9% and 3.4% were obtained in five surveys carried out. S. mansoni eggs were detected in only one child out of the 225 children (0.4%) who were submitted to the Kato-Katz method (three slides for each fecal sample) in the 1st survey. Sixty eight children who submitted five blood samples, one for each survey, were found IFT negative throughout the study. No child was found to be IFT positive in all five surveys, and only four children showed IFT positive results in at least four surveys. Seroconversion from IFT negative to positive was observed from the 1st to the 2nd survey in six children, from the 2nd to the 3rd survey in three children, from the 3rd to the 4th survey in four children, and from the 4th to the 5th survey in two cases. However, confirmation of S. mansoni infection using the fecal examination was not possible in any of the cases. Also, in most of them the IFT result oscillated from negative to positive and vice versa. Our data implied that recent transmission of schistosomiasis in the study area was not possible.

Detection of IgM antibodies to Schistosoma mansoni gut-associated antigens for the study of the dynamics of schistosomiasis transmission in an endemic area with low worm burden.

For a period of 2 years, five follow-up measures of prevalence and incidence rates were estimated in a prospective study of S. mansoni infection in a group of schoolchildren who were living in a rural area of the Municipality of Itariri (São Paulo, Brazil), where schistosomiasis is transmitted by Biomphalaria tenagophila. Infection was determined by the examination of three Kato-Katz stool slides, and the parasitological findings were analyzed in comparison to serological data. In the five surveys, carried out at 6-month intervals (March-April and September-October), the prevalences were, respectively, 8.6, 6.8, 9.9, 5.8 and 17.2% by the Kato-Katz, and 56.5, 52.6, 60.8, 53.5 and 70.1% by the immunofluorescence test (IFT). Geometric mean egg counts were low: 57.8, 33.0, 35.6, 47.3 and 40.9 eggs per gram of feces, respectively. Of the total of 299 schoolchildren, who submitted five blood samples at 6-month intervals, one for each survey, 40% were IFT-positive throughout the study, and 22% were IFT-negative in all five surveys. Seroconversion from IFT negative to positive, indicating newly acquired S. mansoni infection, was observed more frequently in surveys carried out during March-April (after Summer holidays), than during September-October. Seasonal trends were not statistically significant for detection of S. mansoni eggs in stool. The results indicate that the use of IgM-IFT is superior to parasitological methods for detection of incidence of S. mansoni infection in areas with low worm burden.

Efficacy of an enzyme-linked immunosorbent assay in the diagnosis of and serologic distinction between acute and chronic Schistosoma mansoni infection.

To determine the efficacy of an ELISA to diagnose and differentiate the clinical phases of schistosomiasis, serum from patients with acute (11) and chronic schistosomiasis (58), from individuals infected with other parasites (53), and from uninfected individuals (40) was analyzed for the presence of anti-schistosomal of IgG, IgM, and IgA antibodies. Immunofluorescence for IgM and IgA antibodies was also performed on serum from all patients and controls. The IgG antibodies against worm antigen (ELISA-w) were detected in all schistosomiasis patients, with only one false-positive result. The IgA antibodies were detected by ELISA-w in 81.8% of patients with acute disease and in only 5.9% of patients with chronic disease. In addition, the mean optical density values for IgM and IgA antibodies was statistically higher in the patients with acute disease than in those with chronic disease. The results of this study show that the use of a crude adult worm extract as an ELISA antigen can provide a serologic method with high sensitivity and specificity for 1) the diagnosis of acute and chronic schistosomiasis and (2) the serologic distinction between the two forms of the disease.

Decay of antibody isotypes against early developmental stages of Schistosoma mansoni after treatment of schistosomiasis patients.

Antibodies to a number of parasite antigens are found in schistosomiasis patients, and antibodies to early developmental stages were demonstrated to be efficient immunologic markers for the diagnosis of schistosomiasis. In the present study, decay patterns of IgM and IgG antibodies against cercariae and schistosomula were investigated, in comparison to antibodies against worms and eggs in schistosomiasis patients after chemotherapy, for an investigation of seroepidemiologic aspects. Data obtained in the study of 359 serum samples from patients with Schistosoma mansoni infection, noninfected individuals, and patients followed-up for a period of 12 to 15 months after treatment provided the basis to postulate a general pattern for the kinetics of antibody decay. Before treatment, the antibody pattern was represented by a unimodal curve, which shifted to a bimodal curve after treatment, and ended with a unimodal curve similar to that for the noninfected group. Different types of antibodies were classified into four categories according to their decay features, and anti-schistosomulum IgM was classified into the moderate-decay category, whereas other antibodies to early parasite stages were classified into the slow-decay category. The present methodology permits the identification of the most suitable antibodies to be detected in field control programs for schistosomiasis or other parasitoses.

Rapid characterization of S. mansoni expression library clones of potential interest.

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing.

Evaluation of the enzyme-linked-immuno-electro-diffusion-assay (ELIEDA) for the diagnosis of Schistosoma mansoni infection with low worm burden.

An immunoprecipitation technique, ELIEDA (enzyme-linked-immuno-electro-diffusion assay), was evaluated for the diagnosis of Schistosoma mansoni infection with low worm burden. One hundred of serum samples from patients excreting less than 600 eggs per gram of feces (epg), with unrelated diseases and clinically healthy subjects were studied. In patients with egg counts higher than 200 epg, the sensitivities of IgM and IgG ELIEDA were 1,000 and 0.923, respectively, not differing from other serologic techniques, such as indirect hemaglutination (IHAT), immunofluorescence (IFT) tests and immuno-electrodiffusion assay (IEDA). However in patients with low egg counts (< 100 epg), the IgG ELIEDA provided better results (0.821) than IgM ELIEDA (0.679), showing sensitivity that did not differ from that of IgG IFT (0.929), but lower than that of IgM IFT (0.964). However, its sensitivity was higher than that found with IHAT (0.607) and IEDA (0.536). The specificity of IgG ELIEDA was comparable to that of other techniques. The data indicate that IgG ELIEDA might be useful for the diagnosis of slight S. mansoni infections, and the cellulose acetate membrane strips can be stored for further retrospective studies.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients.

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM), and lower results with egg antigen, 77.8%(IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r = 0.552 (p = 0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Schistosoma mansoni cercaria and schistosomulum antigens in serodiagnosis of schistosomiasis.

Schistosoma mansoni cercaria and schistosomulum obtained in vitro were used in immunofluorescence (IF) tests and indirect hemagglutination (IHA) tests of 137 study sera, 44 from subjects infected with S. mansoni and 93 from healthy subjects residing outside areas endemic for the disease. The results of these tests were compared with those obtained by testing the same sera using conventional adult worm antigen, and also with the initial clinical and parasitologic diagnoses of the 137 subjects providing the study sera. Regarding sera from acute versus chronic cases, IF testing of the acute sera consistently detected IgA antibodies along with IgM and IgG, the last two being found consistently in chronic sera. Also, the geometric mean of the IgM antibody titers found in the IF tests was higher for acute than for chronic sera. Excluding IF IgA, which was negative for chronic cases, the sensitivity of the other types of tests (IF IgG, IF IgM, and IHA) using cercaria and schistosomulum antigens, both under evaluation, ranged from 0.773 to 0.955, the specificity ranged from 0.957 to 1.000, the efficiency ranged from 0.927 to 0.985, the predictive value of positives ranged from 0.909 to 1.000, and the predictive value of negatives ranged from 0.903 to 0.979. No statistical differences were observed between these results and those obtained with conventional adult worm antigen. This suggests that cercaria and schistosomulum antigens, both of which can be produced more quickly and cheaply than adult worm antigen, could serve as reliable alternatives to adult worm antigen in the serodiagnosis of schistosomiasis mansoni.

[Control of schistosomiasis mansoni in an area of low transmission]. / Controle da esquistossomose mansônica em área de baixa transmissão.

The schistosomiasis is transmitted by Biomphalaria tenagophila in our study area (Pedro de Toledo, São Paulo, Brazil). From 1980 to 1990 epidemiological surveys in a population of 4,000 inhabitants, has shown that: prevalences by Kato-Katz (KKT), immunofluorescence (FT) and intradermal (IDT) techniques were 22.8%, 55.5% and 51.8%, respectively; intensity of infection was low, 58.5 eggs per gram of faeces (epg); there were no symptomatic cases; prevalences were higher in mates, children and rural zone; index of potential contamination was 57.5% in the age group 5 to 20 years; 2/3 of patients were autochthonous; cases were no-randomly aggregated; transmission was focal and only 0.4% of snails were infected; water contacts through recreation showed the most important odds ratio; knowledge, attitudes and practices were satisfactory. From the epidemiological findings a control programme was carried out: yearly faeces exams, chemotherapy, molluscocide, health education and sanitation. Thus, the prevalence decreased sharply to 3.3% and intensity of infection to 30.3 epg; the incidence rates ranged between 0.4% and 2.5% annually; the sanitation became better and the youngsters were the main target in prophylaxis. To improve control, immunodiagnosis has to be conducted and the involvement of the population should be increase. However, we cannot forget that re-infection, therapeutic failure, etc, could play a major role in the maintenance this residual prevalence.

Seroepidemiology of schistosomiasis mansoni.

In population surveys in which the Schistosoma mansoni intensity of infection is low, or in localities where the schistosomiasis control program had success, the parasitologic methods lack in sensitivity. Despite of some limitations, the immunological methods are useful to provide valuable information in such field conditions. Thus, the prevalence of schistosomiasis in untreated population can be determined by the detection of IgG or IgM antibodies, as well as the incidence by the IgA antibodies, employing mainly immunofluorescence (IF) and immunoenzymatic (ELISA), and in some extent hemagglutination (HA) or even skin test. The true prevalence and incidence of schistosomiasis can be estimated using a probabilistic model equation, since knowing before-hand the sensitivity and specificity of employed test. The sensitivity and the specificity of serologic test become higher in low aged group, under 14. The geometric mean IF titers also gives a positive correlation with the intensity of infection. Presently, there are need of serologic tests which are economic and practical in seroepidemiologic inquiries, requiring no specialized personnel to collect population blood or serum samples, and also easily interpret the test results. The reagents for such tests are desired to be stable and reproducible. Moreover, it is expected that the tests can distinguish an active infection.

Seroepidemioplogy of schistosomiasis mansoni

In population surveys in wich the Schistosoma mansoni intensity of infection is low, or in localities where the schistosomiasis control program had success the parasitologic methods lack in sensitivity. Despite of some limitations the immunological methods are useful to provide valuable information in such field conditions. Thus, the prevalaence of schistosomiasis in untreated population can be determined by the detection of IgG or IgM antibodies, as well as the incidence by the IgA antibodies , employing mainly immunofluorescence (IF) and immunoenzymatic (ELISA), and in some extent hemagglutination (HA) or even skin test. The true prevalence and incidence of schistosomiasis can be estimated using a probabilistic model equation, since knowing before-hand the sensitivity and specificity of emploved test. The sensitivity and the specificity of serologic test become higher in low aged group, under 14. The geometric mean IF titers also gives a positive correlation with the intensity of infection. Presently there are need of serologic tests wich are economic and pratical in soroepidemiologic inquires, requiring no specialized personnel to collect population blood or serum and also easily interpret the test results. The reagents for such tests are desired to be stable and reproducible. Moreover, it is expected that the tests can distinguish an ative infection

Schistosomiasis mansoni in the municipality of Pedro de Toledo (Säo Paulo, Brazil) where the Biomphalaria tenagophila is the snail host. I-Prevalence in human population

Devido à escassez de dados epidemiológicos sobre esquistossomose mansônica onde Biomphalaria tenagophila é vetor foi desenvolvido em 1980 o presente trabalho, no município paulista de Pedro de Toledo. Foram examinadas fezes de 4741 pesssoas (Método de Kato-Katz) com prevalência de 22,8%; entre essas, 583 foram tratadas para a endemia anteriormente e 4158 näo medicadas; as prevalências nos dois grupos foram, respectivamente, 31,7% e 21,6%. Por investigaçäo epidemiológica constatou-se que 83,6% dos casos foram autóctones da área estudada. Prevalência dos autóctones näo tratados foram maiores no homem do que na mulher, a intensidade no último grupo foi baixa: 58,5 ovos g de fezes (média geométrica). De acordo com grupos etários, se correlacionaram bem (rs=0,745) as intensidades de infecçäo e as prevaleência. A infecçäo ocorreu, na zona rural, principalmente, durante lazer e trabalho doméstico. Somente 0,4% de 1137 moluscos foram positivos para Schistosoma mansoni. Esse índice foi, aparentemente, o mesmo em estudo de 1978 quando a prevalência humana era de 12,0%. A área estudada apresentou difernças e semelhanças epidemiológicas em relaçäo às áreas onde B. glabrata é o principal hospedeiro intermediário

Schistosomiasis mansoni in the municipality of Pedro de Toledo (São Paulo, Brazil) where the Biomphalaria tenagophila is the snail host. I--Prevalence in human population.

Due to the scarce information about the epidemiological features of schistosomiasis in which the vector is Biomphalaria tenagophila, an investigation was carried in Pedro de Toledo in 1980 where such peculiarity is observed. Stool examinations (Kato-Katz method) were performed in 4,741 individuals (22.8% positive to Schistosoma mansoni eggs) of this 583 had previously received chemotherapy and 4,158 remainders, untreated. The schistosomiasis prevalence in those two groups where respectively 31.7% and 21.6%. Epidemiological investigation showed that 83.6% were autochthonous cases from the studied area: the autochthonous prevalence rate, and the intensity of infection in the untreated autochthonous cases were higher in males than in females; the intensity in the latter untreated group was low, 58.5 eggs/g feces (geometric mean). Moreover, according to the age groups the intensity of infections correlated well (rs = 0.745) with the prevalence rates. Schistosomiasis was verified to occur mostly during the leisure time and by the use of water streams for housework in rural zone. Only 0.4% out of 1,137 snails was positive for S. mansoni cercariae, apparently unchanged from the 1978 study when the human prevalence was 12.0%. The studied area presented differences and similarities in relation to the other Brazilian areas were the main intermediate host is B. glabrata.

Schistosomiasis mansoni: immunodiagnosis aspects and search for an immunological marker related to therapeutic efficacy.

The recent findings on immunodiagnosis of schistosomiasis mansoni have shown that purified Schistosoma mansoni antigens do not provide maximum positivity. Therefore, the authors suggest the use of semi-purified antigens for diagnostic purposes. So far, no serological marker for cured patients as shown by negative stool examination was found. However, a tendency of IgG antibody titre decrease was observed, when egg antigen was used.