Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2196-6
ARTICLE
Evaluation of the sensitivity of IgG and IgM ELISA in detecting Schistosoma mansoni infections in a low endemicity setting M. C. C. Espirito-Santo & M. C. A. Sanchez & A. R. Sanchez & M. V. Alvarado-Mora & V. L. P. Castilho & E. M. N. Gonçalves & E. J. A. Luna & R. C. B. Gryschek
Received: 23 April 2014 / Accepted: 23 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Schistosomiasis is a major public health concern, with 200 million people infected worldwide. In Brazil, this disease has been reported in 19 states, and its prevalence in the city of Barra Mansa in Rio de Janeiro State is 1 %. The parasitological diagnostic methods currently available in these areas lack sensitivity; however, enzyme-linked immunosorbent assays (ELISAs) have been employed successfully for the diagnosis of schistosomiasis by using antibodies against antigens of Schistosoma mansoni adult worms and eggs, and for the detection of circulating antigens. The objective of this study was to determine systematically the prevalence of S. mansoni infection in the peripheral areas of Barra Mansa. A cross-sectional study was conducted from April to December 2011 by using probabilistic sampling that collected 610 fecal samples and 612 serum samples. ELISA-IgG with total extracts and ELISA-IgM with trichloroacetic acid-soluble fractions were employed to detect antibodies against S. mansoni and were compared with the Kato–Katz and Hoffman parasitological techniques. Among the individuals studied, anti-S. mansoni antibodies were detected in 11.16 % (n=71) by ELISA-IgG and in 20.75 % (n=132) by ELISAIgM, while the parasitological techniques showed 0.82 % (n= 5) positivity. The agreement between the two ELISA tests was 85.38 % (n=543), and 8.65 % (n=55) of the serum samples showed positive results in both tests. The higher positivity of the ELISA-IgM test corroborates the results of previous reports and indicates that the test may be a useful tool in epidemiological studies, particularly in areas of low endemicity for S. mansoni. M. C. C. Espirito-Santo (*) : M. C. A. Sanchez : A. R. Sanchez : M. V. Alvarado-Mora : V. L. P. Castilho : E. M. N. Gonçalves : E. J. A. Luna : R. C. B. Gryschek Universidade de São Paulo, São Paulo, Brazil e-mail: [email protected]
Introduction Schistosomiasis is a major public health problem, with 200 million people infected worldwide and 700 million people residing in areas of infection risk [1]. In Brazil, schistosomiasis has been reported in 19 states, and it is estimated that approximately 6 million people are infected and 25 million are at risk of contracting the disease. The national positivity rate is 6.94 %, ranging from 0.04 % in Piauí State to 11.88 % in Pernambuco State [2]. In Rio de Janeiro State, the positivity rate is 1.56 % [2]. Schistosomiasis is currently in the elimination phase of disease control in Brazil; however, despite control measures, this helminthiasis is prevalent in areas with high, medium, and low endemicity, particularly in regions with low-income populations [3]. According to the World Health Organization (WHO), schistosomiasis prevalence should be determined based on stool examinations [4]. The technique developed by Katz et al. [5] made a major contribution to schistosomiasis control by defining the intensity of infection. However, after periodic interventions through schistosomiasis control programs over the past four decades in Brazil and other regions of the world, we are currently faced with a diagnostic challenge, particularly in areas of low endemicity [6]. In these areas, parasitological methods lack the sensitivity required to overcome the low p rev al en ce an d lo w pa ra siti c b urd en (le ss tha n 100 Schistosoma mansoni eggs per gram of feces) [7, 8]. To improve the sensitivity of diagnostic techniques, it is necessary to increase the number of stool samples analyzed. However, obtaining more than one stool sample from the same person requires a considerable logistical effort during fieldwork. Moreover, the collection of samples on different days was found to result in a positivity increase of only 6 %, using the Kato–Katz technique, relative to the results from the first sample alone [9]. In order to address this issue,
Eur J Clin Microbiol Infect Dis
immunodiagnostic techniques have been used in schistosomiasis control programs in certain countries, including Venezuela [10] and China [11], with satisfactory results. The aim of this study was to determine systematically the prevalence of S. mansoni infection in the outlying areas of Barra Mansa, Rio de Janeiro State, using enzyme-linked immunosorbent assays (ELISAs) that detect circulating antibodies. We investigated the presence of IgG antibodies specific for the total extract of S. mansoni (ELISA-IgG) and IgM antibodies specific for the trichloroacetic acid (TCA)-soluble fraction (ELISA-IgM), and compared the results with the parasitological techniques of Kato–Katz (KK) [5] and Hoffman (HH) [12].
preserved in 10 % formalin (Indalabor Indaiá Lab Farmacêutico Ltda, Dores do Indaiá, Minas Gerais, Brazil) and two slides were prepared for each participant.
Materials and methods
Host animals and parasites
Study population
Hamsters (Mesocricetus auratus) were injected subcutaneously with approximately 200–300 cercariae of S. mansoni (BH strain) and were analyzed using a perfusion technique [14, 15] after 6–7 weeks of infection. After washing with a physiological solution until complete removal of the blood, the S. mansoni worms were counted and frozen for the preparation of antigens for ELISA.
Schistosomiasis mansoni is endemic in the city of Barra Mansa, Rio de Janeiro State, Brazil, with an estimated prevalence of 1 % [13]. Data for 2001–2008 from the Notifiable Diseases Information System (SINAN 2008) showed that the disease is most prevalent in the neighborhoods of Siderlândia, Santa Clara, São Luís, Nova Esperança, and Cantagalo, which belong to the Barra Mansa River Basin, a tributary of the Paraíba do Sul River. These five neighborhoods, located on the outskirts of the city of Barra Mansa, were selected for the cross-sectional study. Samples of feces and serum were collected randomly from April to December 2011. The sample size was calculated assuming a prevalence of 1 %, and an increase of 30 % was made to compensate for losses. The estimated sample size was 650 individuals residing in the neighborhoods described above. Households were systematically selected (one in six), and individuals were randomly selected by a draw among those who agreed to participate in the study. Subjects who were older than 5 years and had not been treated for schistosomiasis mansoni in the last year were included in the study.
Serum samples for standardization To standardize IgG and IgM ELISA, we used positive and negative control samples and patient sera, which were classified into six groups according to the characteristics described in Table 1. These sera, obtained from patients residing in a schistosomiasis-endemic area in the state of São Paulo, were assessed by an indirect immunofluorescence test (IIFT) and by the KK and HH techniques, and were transferred by Instituto Adolfo Lutz, São Paulo, Brazil.
Adult worm total extract Approximately 10,000 S. mansoni adult worms of both sexes were thawed and resuspended at a concentration of approximately 1,000 worms/mL in 10 mM phosphate buffered saline (PBS), pH 7.2. The protease inhibitor phenylmethylsufonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA) was then added at a concentration of 1 mM of final suspension. The suspension was ground in a manual tissue homogenizer in an ice bath for 1 h. After thorough mixing, the suspension was
Table 1 Distribution of sera used in the standardization of IgG and IgM enzyme-linked immunosorbent assay (ELISA) Group Number Characteristics
Collection of serum and stool samples We collected 612 serum samples and 610 stool samples, and 572 of these were paired. The serum samples were aliquoted and stored at −20 °C, transported to São Paulo in coolers containing dry ice, and stored at −20 °C in the lab where they were tested. Stool samples were prepared following the KK (Helm Test®, Bio-Manguinhos, Fiocruz, Rio de Janeiro, Brazil) and HH techniques, and stored at 4 °C until shipment. In the KK technique, two slides were sewn up and stored in boxes fixed with a rigid polypropylene cover lined with cork (Prolab, São Paulo, Brazil). In the HH technique, the samples were
1 2 3 4
10 29 30 3
IIFT-Sm and SPT-Sm positive IIFT-Sm and SPT-Sm negative IIFT-Sm positive and SPT-Sm negative IIFT-Sm, SPT-Sm, and SPT-other helminths positive
5
11
6
13
IIFT-Sm positive, SPT-Sm negative, and SPT-other helminths positive IIFT-Sm and SPT-Sm negative and SPT-other helminths positive
IIFT-Sm: indirect immunofluorescence test for S. mansoni SPT-Sm: stool parasitologic technique of Kato–Katz for S. mansoni SPT-other helminths: stool parasitologic technique of Kato–Katz and Hoffman for helminths
Eur J Clin Microbiol Infect Dis
subjected to two centrifugations at 4 °C for 45 min at 10,000× g. The supernatant was then removed and the protein content was measured using the DC Protein Assay reagent (detergentcompatible colorimetric assay kit; Bio-Rad, Hercules, CA, USA) by employing a modified Lowry method [16, 17]. The extract was aliquoted and stored in a freezer at −80 °C until further use. This total extract was used for the sensitization of microplates to IgG (ELISA-IgG) antibodies. TCA-soluble fraction A TCA-soluble fraction was prepared from the crude extract of S. mansoni adult worms, according to a previously described method [18], with modifications. An equal volume of 10 % TCA was added to the total adult worm extract. After mixing vigorously for three cycles of 1 min using a vortexer (Vortex-Genie 2, Scientific Industries Inc., Bohemia, NY, USA), the suspension was subjected to centrifugation at 4 °C for 45 min at 10,000×g. The supernatant (containing the TCA-soluble fraction) was removed and then dialyzed against PBS on a cellulose membrane that retains substances with a molecular weight of 12,000 or greater (Sigma-Aldrich), with continuous stirring overnight at 4 °C. We subsequently determined the protein content of the antigen solution (TCAsoluble fraction), which was then aliquoted and stored in a freezer at−80 °C until further use. The TCA-soluble fraction was used for the sensitization of microplates to IgM (ELISAIgM) antibodies. Enzyme-linked immunosorbent assays (ELISAs) Initially, block titrations were performed to determine the optimal concentration of antigens, dilutions of samples and conjugates, and reaction time at each stage of the test and were compared to known serum samples. ELISA-IgM Polystyrene plates (Costar High Binding 3590, Corning, NY, USA.) were sensitized with 50 μL/well of the TCA-soluble fraction of the S. mansoni total extract and 20 μg/mL of 0.1 M carbonate/bicarbonate buffer (pH 9.6) and incubated for 2 h at 37 °C, followed by 18 h at 4 °C in a humid chamber. After washing three times with PBS containing 0.05 % Tween-20 (PBS-T), the plates were blocked for nonspecific sites with 200 μL of 5 % skim milk in PBS-T (PBS-TM 5 %) and incubated for 2 h at 37 °C in a humid chamber. The plates were then washed again with PBS-T. Serum samples were diluted (1:100) in PBS-T with 2 % skim milk (PBS-TM 2 %) and tested in duplicate (50 μL/well). After incubation for 30 min at 37 °C in a humid chamber, the plates were washed three times with PBS-T and 50 μL of a μchain-specific anti-human IgM peroxidase conjugate (SigmaAldrich) diluted 1:5,000 in PBS-TM 2 % was added. After 30 min of incubation in a humid chamber, the plates were
washed with PBS-T and 100 μL of a chromogenic mixture consisting of tetramethylbenzidine (TMB) and H2O2 was added. After 10 min of incubation in the dark, the reaction was quenched with 25 μL of 2 N H2SO4. The absorbance was then measured at 450 nm by using an ELISA plate reader (Multiskan® MCC/340, Titertek, Huntsville, AL, USA). ELISA-IgG The reaction conditions described above for ELISA-IgM were also used for ELISA-IgG, except that polystyrene plates (Nunc™ PolySorp, Roskilde, Denmark), a 10 μg/mL total antigen extract of S. mansoni, and an Fcspecific anti-human IgG peroxidase conjugate (SigmaAldrich) diluted 1:20,000 were used instead in this analysis. To determine the threshold of reactivity (cut-off) of ELISAIgG and ELISA-IgM, receiver operating characteristic (ROC) curves were constructed. We used 13 and 29 samples from patients who tested positive and negative, respectively, for S. mansoni in the parasitological examination and IIFT, and 13 samples from patients who tested positive for other helminth parasites in the parasitological examination and negative for S. mansoni in IIFT. The reactivity index (RI) of the samples was calculated by using the equation RI=sample absorbance/cut-off. Serum samples with RI≥1.00 were considered reagents. Statistical analysis Statistical analyses were performed using SPSS 15.0, GraphPad Prism 6.02, SigmaStat 3.5, and Microsoft Excel 2010. The levels of significance were set accepting a type 1 error of 5 % (α=0.05). The ROC curves were used to determine the sensitivity and specificity of the tests, the 95 % confidence intervals (CIs), and the likelihood ratios. Population characteristics were determined using absolute frequencies; the relative frequency, mean, and standard deviation of the ages were also calculated. We evaluated the proportion of positive results for each test (prevalence), according to each of the techniques used. The techniques were compared and a marginal association was detected using McNemar’s test [19]. The agreement between the results of the techniques was assessed using Cohen’s kappa (K) index and their range with 95 % confidence. We evaluated the possible association of the positivity of each technique to age, gender, neighborhood, and the use of river water using the Chi-square test (χ2). Fisher’s exact test or the likelihood ratio test [19] were used when the sampling was insufficient for applying the χ2 test. Ethical issues Informed consent was obtained individually from all the participants before the collection of blood samples (Resolution
Eur J Clin Microbiol Infect Dis 25 21.41 20 PosiƟvity (%)
No. 196, October 10, 1996, National Board of Health, revoked by Resolution No. 466, December 12, 2012, National Board of Health). This study was approved by Ethics Committees of the Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da Universidade de São Paulo and the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (0405/09). This study received funding from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), approved on 02/18/2011 under number 2010/52615-0.
15
12.42
11.60 8.99
10 5
2.61
0 IgM
Ethical aspects of experiments with animals The experimental research projects complied with Laws 6.638/79 and 9605/98, Decree 24.645/34, the Ethical Principles in Animal Experimentation (COBEA), the Principles for Research Involving Animals (Geneva, 1985), and other instructions governing animal research. The work was started after approval of research project number CEP-IMT 2011/096 was granted by the Animal Research Ethics Committee of the Instituto de Medicina Tropical de São Paulo da Universidade de São Paulo (IMTSP-USP), São Paulo, Brazil.
Results The total extract obtained from 10,000 adult worms showed a concentration of 8.850 mg/mL and a TCA-soluble fraction of 0.495 mg/mL. The diagnostic performance of ELISA-IgM and ELISA-IgG calculated by the ROC curves [20] (Fig. 1) indicated a sensitivity (95 % probability confidence interval; CI) of 100.0 % (75.29 to 100.0 %), a specificity of 97.62 % (87.43 to 99.94 %), and a likelihood ratio of 42.00, with cutoffs of 0.210 and 0.320, respectively.
Fig. 1 Receiver operating characteristic (ROC) curve obtained using samples from patients positive for parasitological (Schistosoma mansoni) and immunofluorescence tests (n=13), patients negative for parasitological and immunofluorescence tests (n=29), and patients negative for
IgG
IgG + IgM
only IgM
only IgG
Fig. 2 Prevalence of IgG and IgM anti-S. mansoni antibodies in 612 sera from Barra Mansa, Rio de Janeiro State, Brazil. p
ARTICLE
Evaluation of the sensitivity of IgG and IgM ELISA in detecting Schistosoma mansoni infections in a low endemicity setting M. C. C. Espirito-Santo & M. C. A. Sanchez & A. R. Sanchez & M. V. Alvarado-Mora & V. L. P. Castilho & E. M. N. Gonçalves & E. J. A. Luna & R. C. B. Gryschek
Received: 23 April 2014 / Accepted: 23 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Schistosomiasis is a major public health concern, with 200 million people infected worldwide. In Brazil, this disease has been reported in 19 states, and its prevalence in the city of Barra Mansa in Rio de Janeiro State is 1 %. The parasitological diagnostic methods currently available in these areas lack sensitivity; however, enzyme-linked immunosorbent assays (ELISAs) have been employed successfully for the diagnosis of schistosomiasis by using antibodies against antigens of Schistosoma mansoni adult worms and eggs, and for the detection of circulating antigens. The objective of this study was to determine systematically the prevalence of S. mansoni infection in the peripheral areas of Barra Mansa. A cross-sectional study was conducted from April to December 2011 by using probabilistic sampling that collected 610 fecal samples and 612 serum samples. ELISA-IgG with total extracts and ELISA-IgM with trichloroacetic acid-soluble fractions were employed to detect antibodies against S. mansoni and were compared with the Kato–Katz and Hoffman parasitological techniques. Among the individuals studied, anti-S. mansoni antibodies were detected in 11.16 % (n=71) by ELISA-IgG and in 20.75 % (n=132) by ELISAIgM, while the parasitological techniques showed 0.82 % (n= 5) positivity. The agreement between the two ELISA tests was 85.38 % (n=543), and 8.65 % (n=55) of the serum samples showed positive results in both tests. The higher positivity of the ELISA-IgM test corroborates the results of previous reports and indicates that the test may be a useful tool in epidemiological studies, particularly in areas of low endemicity for S. mansoni. M. C. C. Espirito-Santo (*) : M. C. A. Sanchez : A. R. Sanchez : M. V. Alvarado-Mora : V. L. P. Castilho : E. M. N. Gonçalves : E. J. A. Luna : R. C. B. Gryschek Universidade de São Paulo, São Paulo, Brazil e-mail: [email protected]
Introduction Schistosomiasis is a major public health problem, with 200 million people infected worldwide and 700 million people residing in areas of infection risk [1]. In Brazil, schistosomiasis has been reported in 19 states, and it is estimated that approximately 6 million people are infected and 25 million are at risk of contracting the disease. The national positivity rate is 6.94 %, ranging from 0.04 % in Piauí State to 11.88 % in Pernambuco State [2]. In Rio de Janeiro State, the positivity rate is 1.56 % [2]. Schistosomiasis is currently in the elimination phase of disease control in Brazil; however, despite control measures, this helminthiasis is prevalent in areas with high, medium, and low endemicity, particularly in regions with low-income populations [3]. According to the World Health Organization (WHO), schistosomiasis prevalence should be determined based on stool examinations [4]. The technique developed by Katz et al. [5] made a major contribution to schistosomiasis control by defining the intensity of infection. However, after periodic interventions through schistosomiasis control programs over the past four decades in Brazil and other regions of the world, we are currently faced with a diagnostic challenge, particularly in areas of low endemicity [6]. In these areas, parasitological methods lack the sensitivity required to overcome the low p rev al en ce an d lo w pa ra siti c b urd en (le ss tha n 100 Schistosoma mansoni eggs per gram of feces) [7, 8]. To improve the sensitivity of diagnostic techniques, it is necessary to increase the number of stool samples analyzed. However, obtaining more than one stool sample from the same person requires a considerable logistical effort during fieldwork. Moreover, the collection of samples on different days was found to result in a positivity increase of only 6 %, using the Kato–Katz technique, relative to the results from the first sample alone [9]. In order to address this issue,
Eur J Clin Microbiol Infect Dis
immunodiagnostic techniques have been used in schistosomiasis control programs in certain countries, including Venezuela [10] and China [11], with satisfactory results. The aim of this study was to determine systematically the prevalence of S. mansoni infection in the outlying areas of Barra Mansa, Rio de Janeiro State, using enzyme-linked immunosorbent assays (ELISAs) that detect circulating antibodies. We investigated the presence of IgG antibodies specific for the total extract of S. mansoni (ELISA-IgG) and IgM antibodies specific for the trichloroacetic acid (TCA)-soluble fraction (ELISA-IgM), and compared the results with the parasitological techniques of Kato–Katz (KK) [5] and Hoffman (HH) [12].
preserved in 10 % formalin (Indalabor Indaiá Lab Farmacêutico Ltda, Dores do Indaiá, Minas Gerais, Brazil) and two slides were prepared for each participant.
Materials and methods
Host animals and parasites
Study population
Hamsters (Mesocricetus auratus) were injected subcutaneously with approximately 200–300 cercariae of S. mansoni (BH strain) and were analyzed using a perfusion technique [14, 15] after 6–7 weeks of infection. After washing with a physiological solution until complete removal of the blood, the S. mansoni worms were counted and frozen for the preparation of antigens for ELISA.
Schistosomiasis mansoni is endemic in the city of Barra Mansa, Rio de Janeiro State, Brazil, with an estimated prevalence of 1 % [13]. Data for 2001–2008 from the Notifiable Diseases Information System (SINAN 2008) showed that the disease is most prevalent in the neighborhoods of Siderlândia, Santa Clara, São Luís, Nova Esperança, and Cantagalo, which belong to the Barra Mansa River Basin, a tributary of the Paraíba do Sul River. These five neighborhoods, located on the outskirts of the city of Barra Mansa, were selected for the cross-sectional study. Samples of feces and serum were collected randomly from April to December 2011. The sample size was calculated assuming a prevalence of 1 %, and an increase of 30 % was made to compensate for losses. The estimated sample size was 650 individuals residing in the neighborhoods described above. Households were systematically selected (one in six), and individuals were randomly selected by a draw among those who agreed to participate in the study. Subjects who were older than 5 years and had not been treated for schistosomiasis mansoni in the last year were included in the study.
Serum samples for standardization To standardize IgG and IgM ELISA, we used positive and negative control samples and patient sera, which were classified into six groups according to the characteristics described in Table 1. These sera, obtained from patients residing in a schistosomiasis-endemic area in the state of São Paulo, were assessed by an indirect immunofluorescence test (IIFT) and by the KK and HH techniques, and were transferred by Instituto Adolfo Lutz, São Paulo, Brazil.
Adult worm total extract Approximately 10,000 S. mansoni adult worms of both sexes were thawed and resuspended at a concentration of approximately 1,000 worms/mL in 10 mM phosphate buffered saline (PBS), pH 7.2. The protease inhibitor phenylmethylsufonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA) was then added at a concentration of 1 mM of final suspension. The suspension was ground in a manual tissue homogenizer in an ice bath for 1 h. After thorough mixing, the suspension was
Table 1 Distribution of sera used in the standardization of IgG and IgM enzyme-linked immunosorbent assay (ELISA) Group Number Characteristics
Collection of serum and stool samples We collected 612 serum samples and 610 stool samples, and 572 of these were paired. The serum samples were aliquoted and stored at −20 °C, transported to São Paulo in coolers containing dry ice, and stored at −20 °C in the lab where they were tested. Stool samples were prepared following the KK (Helm Test®, Bio-Manguinhos, Fiocruz, Rio de Janeiro, Brazil) and HH techniques, and stored at 4 °C until shipment. In the KK technique, two slides were sewn up and stored in boxes fixed with a rigid polypropylene cover lined with cork (Prolab, São Paulo, Brazil). In the HH technique, the samples were
1 2 3 4
10 29 30 3
IIFT-Sm and SPT-Sm positive IIFT-Sm and SPT-Sm negative IIFT-Sm positive and SPT-Sm negative IIFT-Sm, SPT-Sm, and SPT-other helminths positive
5
11
6
13
IIFT-Sm positive, SPT-Sm negative, and SPT-other helminths positive IIFT-Sm and SPT-Sm negative and SPT-other helminths positive
IIFT-Sm: indirect immunofluorescence test for S. mansoni SPT-Sm: stool parasitologic technique of Kato–Katz for S. mansoni SPT-other helminths: stool parasitologic technique of Kato–Katz and Hoffman for helminths
Eur J Clin Microbiol Infect Dis
subjected to two centrifugations at 4 °C for 45 min at 10,000× g. The supernatant was then removed and the protein content was measured using the DC Protein Assay reagent (detergentcompatible colorimetric assay kit; Bio-Rad, Hercules, CA, USA) by employing a modified Lowry method [16, 17]. The extract was aliquoted and stored in a freezer at −80 °C until further use. This total extract was used for the sensitization of microplates to IgG (ELISA-IgG) antibodies. TCA-soluble fraction A TCA-soluble fraction was prepared from the crude extract of S. mansoni adult worms, according to a previously described method [18], with modifications. An equal volume of 10 % TCA was added to the total adult worm extract. After mixing vigorously for three cycles of 1 min using a vortexer (Vortex-Genie 2, Scientific Industries Inc., Bohemia, NY, USA), the suspension was subjected to centrifugation at 4 °C for 45 min at 10,000×g. The supernatant (containing the TCA-soluble fraction) was removed and then dialyzed against PBS on a cellulose membrane that retains substances with a molecular weight of 12,000 or greater (Sigma-Aldrich), with continuous stirring overnight at 4 °C. We subsequently determined the protein content of the antigen solution (TCAsoluble fraction), which was then aliquoted and stored in a freezer at−80 °C until further use. The TCA-soluble fraction was used for the sensitization of microplates to IgM (ELISAIgM) antibodies. Enzyme-linked immunosorbent assays (ELISAs) Initially, block titrations were performed to determine the optimal concentration of antigens, dilutions of samples and conjugates, and reaction time at each stage of the test and were compared to known serum samples. ELISA-IgM Polystyrene plates (Costar High Binding 3590, Corning, NY, USA.) were sensitized with 50 μL/well of the TCA-soluble fraction of the S. mansoni total extract and 20 μg/mL of 0.1 M carbonate/bicarbonate buffer (pH 9.6) and incubated for 2 h at 37 °C, followed by 18 h at 4 °C in a humid chamber. After washing three times with PBS containing 0.05 % Tween-20 (PBS-T), the plates were blocked for nonspecific sites with 200 μL of 5 % skim milk in PBS-T (PBS-TM 5 %) and incubated for 2 h at 37 °C in a humid chamber. The plates were then washed again with PBS-T. Serum samples were diluted (1:100) in PBS-T with 2 % skim milk (PBS-TM 2 %) and tested in duplicate (50 μL/well). After incubation for 30 min at 37 °C in a humid chamber, the plates were washed three times with PBS-T and 50 μL of a μchain-specific anti-human IgM peroxidase conjugate (SigmaAldrich) diluted 1:5,000 in PBS-TM 2 % was added. After 30 min of incubation in a humid chamber, the plates were
washed with PBS-T and 100 μL of a chromogenic mixture consisting of tetramethylbenzidine (TMB) and H2O2 was added. After 10 min of incubation in the dark, the reaction was quenched with 25 μL of 2 N H2SO4. The absorbance was then measured at 450 nm by using an ELISA plate reader (Multiskan® MCC/340, Titertek, Huntsville, AL, USA). ELISA-IgG The reaction conditions described above for ELISA-IgM were also used for ELISA-IgG, except that polystyrene plates (Nunc™ PolySorp, Roskilde, Denmark), a 10 μg/mL total antigen extract of S. mansoni, and an Fcspecific anti-human IgG peroxidase conjugate (SigmaAldrich) diluted 1:20,000 were used instead in this analysis. To determine the threshold of reactivity (cut-off) of ELISAIgG and ELISA-IgM, receiver operating characteristic (ROC) curves were constructed. We used 13 and 29 samples from patients who tested positive and negative, respectively, for S. mansoni in the parasitological examination and IIFT, and 13 samples from patients who tested positive for other helminth parasites in the parasitological examination and negative for S. mansoni in IIFT. The reactivity index (RI) of the samples was calculated by using the equation RI=sample absorbance/cut-off. Serum samples with RI≥1.00 were considered reagents. Statistical analysis Statistical analyses were performed using SPSS 15.0, GraphPad Prism 6.02, SigmaStat 3.5, and Microsoft Excel 2010. The levels of significance were set accepting a type 1 error of 5 % (α=0.05). The ROC curves were used to determine the sensitivity and specificity of the tests, the 95 % confidence intervals (CIs), and the likelihood ratios. Population characteristics were determined using absolute frequencies; the relative frequency, mean, and standard deviation of the ages were also calculated. We evaluated the proportion of positive results for each test (prevalence), according to each of the techniques used. The techniques were compared and a marginal association was detected using McNemar’s test [19]. The agreement between the results of the techniques was assessed using Cohen’s kappa (K) index and their range with 95 % confidence. We evaluated the possible association of the positivity of each technique to age, gender, neighborhood, and the use of river water using the Chi-square test (χ2). Fisher’s exact test or the likelihood ratio test [19] were used when the sampling was insufficient for applying the χ2 test. Ethical issues Informed consent was obtained individually from all the participants before the collection of blood samples (Resolution
Eur J Clin Microbiol Infect Dis 25 21.41 20 PosiƟvity (%)
No. 196, October 10, 1996, National Board of Health, revoked by Resolution No. 466, December 12, 2012, National Board of Health). This study was approved by Ethics Committees of the Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da Universidade de São Paulo and the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (0405/09). This study received funding from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), approved on 02/18/2011 under number 2010/52615-0.
15
12.42
11.60 8.99
10 5
2.61
0 IgM
Ethical aspects of experiments with animals The experimental research projects complied with Laws 6.638/79 and 9605/98, Decree 24.645/34, the Ethical Principles in Animal Experimentation (COBEA), the Principles for Research Involving Animals (Geneva, 1985), and other instructions governing animal research. The work was started after approval of research project number CEP-IMT 2011/096 was granted by the Animal Research Ethics Committee of the Instituto de Medicina Tropical de São Paulo da Universidade de São Paulo (IMTSP-USP), São Paulo, Brazil.
Results The total extract obtained from 10,000 adult worms showed a concentration of 8.850 mg/mL and a TCA-soluble fraction of 0.495 mg/mL. The diagnostic performance of ELISA-IgM and ELISA-IgG calculated by the ROC curves [20] (Fig. 1) indicated a sensitivity (95 % probability confidence interval; CI) of 100.0 % (75.29 to 100.0 %), a specificity of 97.62 % (87.43 to 99.94 %), and a likelihood ratio of 42.00, with cutoffs of 0.210 and 0.320, respectively.
Fig. 1 Receiver operating characteristic (ROC) curve obtained using samples from patients positive for parasitological (Schistosoma mansoni) and immunofluorescence tests (n=13), patients negative for parasitological and immunofluorescence tests (n=29), and patients negative for
IgG
IgG + IgM
only IgM
only IgG
Fig. 2 Prevalence of IgG and IgM anti-S. mansoni antibodies in 612 sera from Barra Mansa, Rio de Janeiro State, Brazil. p
