Introduction
Although schistosomiasis control programmes have contributed to reducing the prevalence and intensity of infection, the parasitosis still represents a public health problem in many regions of the world (WHO, 2022; Tavakoli Pirzaman et al., Reference Tavakoli Pirzaman, Sepidarkish, Alizadeh, Al-Obidy, Ebrahimi, Kianifard, Sheikhi Nooshabadi, Jafari Tadi, Zolfaghari Dehkharghani, Mousavi, Rezapour, Mohammadnia, Fazlollahpour Naghibi, Bagheri, Asghari, Bayani, Rollinson, Gasser and Rostami2024). In Brazil, the main endemic schistosomiasis mansoni areas are located in the Northeastern and Southeastern States. In other areas of the North, Southeast and South States, the occurrence of cases is low (Katz, Reference Katz2018; Santos and Heller, Reference Santos and Heller2023). As in other countries, a decrease in the number of cases has been observed in Brazil over the years, a result of government programmes implemented since the 1970s. Control measures contributed to reducing morbidity, but not to completely interrupting transmission or reducing prevalence below 5%, thus preventing the occurrence of new transmission foci (Coura and Amaral, Reference Coura and Amaral2004; RS et al., Reference RS, Tauil, Lima and Engels2006; Katz, Reference Katz2018). The State of São Paulo is classified as an area of low transmission, where a small number of autochthonous cases have been continuously reported (Kanamura et al., Reference Kanamura, CS, Glasser, Silva, de Camargo-Neves, Vellosa, Gargioni, Lima, Guercio, Marques and Carvalho2001; Teles, Reference Teles2005; SES-SP/CCD (Secretaria de Estado da Saúde, Coordenadoria de Controle de Doenças). Divisão de Doenças de Transmissão Hídrica e Alimentar, CVE (Centro de Vigilância Epidemiológica ‘Prof. Alexandre Vranjac’), 2009). Data from the National Survey of Schistosomiasis mansoni and Geohelminthiasis (INPEG) indicated a schistosomiasis rate of 0.04% in the state, with 4 cases detected by the Kato–Katz method in the municipalities of Campinas and Guarulhos (Katz, Reference Katz2018).
Cases registered in areas of low transmission are asymptomatic or present non-specific clinical manifestations, due to the low parasitic load, which constitutes a problem for clinical laboratory diagnosis (Pereira Igreja, Reference Pereira Igreja2024). On the other hand, light infections may represent a potential source of transmission (Teles, Reference Teles2005). In areas where the prevalence and intensity of infections are low, parasitological methods have decreased sensitivity, underestimating the number of positive cases (Silva-Moraes et al., Reference Silva-Moraes, Shollenberger, Siqueira, Castro-Borges, Harn, Grenfell, Rabello and Coelho2019). Thus, immunodiagnostic techniques complement epidemiological studies and can be applied to the monitoring of control programmes (Noya et al., Reference Noya, Guevara, Colmenares, Losada and Noya2006; Grenfell et al., Reference Grenfell, Silva-Moraes, Taboada, de Mattos, de Castro and Coelho2012). Investigations in these areas have shown that prevalence rates obtained by serological methods are higher than those of parasitological methods (Kanamura et al., Reference Kanamura, Silva, Chiodelli, Glasser and Dias2002; Gargioni et al., Reference Gargioni, Silva, Thomé, Quadros and Kanamura2008). Serological methodologies, such as enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), circumoval precipitin test (COPT), among others, have been used as tools in the diagnosis of schistosomiasis (Espirito-Santo et al., Reference Espirito-Santo, Sanchez, Sanchez, Alvarado-Mora, Castilho, Gonçalves, Luna and Gryschek2014; Espírito-Santo et al., Reference Espírito-Santo, Alvarado-Mora, Pinto, Sanchez, Dias-Neto, Castilho, Gonçalves, Chieffi, Luna, Pinho, Carrilho and Gryschek2015; Ferrer et al., Reference Ferrer, Villegas, Mughini-Gras, Hernández, Jiménez, Catalano and Incani2020). However, serology does not discriminate between active and treated infections, as circulating antibodies remain after parasitological cure for long periods of time (Silva-Moraes et al., Reference Silva-Moraes, Shollenberger, Siqueira, Castro-Borges, Harn, Grenfell, Rabello and Coelho2019). Thus, in areas of low transmission, the main objective of control actions is to identify and distinguish autochthonous cases from imported ones, preventing the establishment of transmission (Grenfell et al., Reference Grenfell, Silva-Moraes, Taboada, de Mattos, de Castro and Coelho2012).
The IFA for the detection of IgM class antibodies (IFA-IgM) has been used, in paraffin sections of adult male worms, for the diagnosis of schistosomiasis in the acute and chronic phases of infection. The technique has also been used in the selection of population groups for parasitological confirmation and in epidemiological surveys (Kanamura et al., Reference Kanamura, CS, Glasser, Silva, de Camargo-Neves, Vellosa, Gargioni, Lima, Guercio, Marques and Carvalho2001, Reference Kanamura, Silva, Chiodelli, Glasser and Dias2002; Gargioni et al., Reference Gargioni, Silva, Thomé, Quadros and Kanamura2008). In a previous study by our group, the applicability of IFA-IgM associated with COPT was demonstrated in an area of low schistosomiasis transmission in the state of Rio de Janeiro. It was shown that IFA-IgM presented greater sensitivity than COPT, and the latter proved to be more specific and could be used as a parameter for identifying active infection, even in the absence of a positive parasitological examination (do Espírito-Santo MC et al., Reference do Espírito-Santo, Pinto, Gargioni, Alvarado-Mora, Pagliusi Castilho, Pinho, de Albuquerque Luna and Borges Gryschek2014). Based on these results, the present study aimed to evaluate COPT in samples positive by IFA-IgM, in order to establish the relationship between the 2 processes in the identification of cases, in serum samples from an immunodiagnostic service for schistosomiasis offered by the Public Health Laboratory of the State of São Paulo.
Additionally, we evaluated the IgA antibody response by IFA-IgA in paraffin sections of worms and related it to the results of reactive COPT, in order to complement the identification of active infections, discriminating recent infections from chronic ones. It is expected, therefore, that the 3 methodologies, when interpreted together, can identify active Schistosoma mansoni infections in their different evolutionary stages. The results can inform the targeting of surveillance and control actions, as well as individual diagnosis. This proposal aligns with the objectives of the World Health Organization (WHO), which aim for the global elimination of schistosomiasis by 2030 (WHO, 2022).
Materials and methods
Samples
Serum samples from the routine diagnostics of the Enteroparasite Center, Instituto Adolfo Lutz de São Paulo, were used. These samples were sent for immunodiagnosis of schistosomiasis, where IgM-IFA was processed from paraffin sections of adult male worms. Routinely, after analysis, the samples were aliquoted and kept at −20 °C. For the study, serum samples showing positive results in the IgM-IFA and containing sufficient aliquots for possible repetitions were selected. Due to the scope of the service offered by the Enteroparasite Center in the diagnosis of schistosomiasis, only samples from patients from municipalities in the State of São Paulo were included. Thus, 685 positive samples were selected. This study meets the requirements of the National Instance for Ethics in Research and was approved by the Research Ethics Committee of the Adolfo Lutz Institute (protocol number 2.420.105).
Maintenance of the experimental biological cycle of S. mansoni
The BH (Belo Horizonte, Minas Gerais) strain of S. mansoni is routinely maintained both at the Centro de Enteroparasitas of the Instituto Adolfo Lutz, São Paulo, Brazil and at the Laboratório de Imunopatologia da Esquistossomose, Faculdade de Medicina da Universidade de São Paulo, Brazil, to obtain adult worms and eggs used as antigen sources. The parasites are maintained through periodic infections in Biomphalaria glabrata Say, 1818 snails (BH strain) and in Mesocrycetus auratus rodents (Golden Hamsters). The protocols used are in accordance with the guidelines of the National Council for the Control of Animal Experimentation (CONCEA, 2018) and were approved by the Animal Experimentation Committee of Faculdade de Medicina da Universidade de São Paulo (protocol number 7499).
Indirect IFA for the detection of IgM and IgA antibodies
IFA was processed on serial paraffin sections of adult male worms to focus on the internal components of the digestive tract identified as antigens. The processes for obtaining the worms from the experimental cycle and the preparation of the paraffin histological sections on slides were processed as described by do Espírito-Santo MC et al. (Reference do Espírito-Santo, Pinto, Gargioni, Alvarado-Mora, Pagliusi Castilho, Pinho, de Albuquerque Luna and Borges Gryschek2014).
The detection of IgM and IgA antibodies was in accordance with the protocol described by do Espírito-Santo MC et al. (Reference do Espírito-Santo, Pinto, Gargioni, Alvarado-Mora, Pagliusi Castilho, Pinho, de Albuquerque Luna and Borges Gryschek2014), with some modifications. For this purpose, the serum samples were diluted 1/10 in phosphate-buffered saline solution and deposited on glass slides containing the serial paraffin sections of worms, followed by incubation for 50 min at 37 °C. The dilutions for use of the respective fluorescent conjugates, anti-human immunoglobulin IgM and IgA (Biolab, Rio de Janeiro, Brazil), were determined from block titration using previously characterized positive and negative standard sera. Readings were performed on a fluorescence microscope with an epi-illumination system, at magnifications of 100× and 200×. Samples that showed fluorescence in structures related to the digestive tracts of worms in sections were considered positive, according to criteria described by Kanamura et al. (Reference Kanamura, Silva, Rabello, Rocha and Katz1991) (Figure 1). IgA determination was performed only on samples considered reactive for COPT.
IgA-IFA in a paraffin section of a male S. mansoni worm showing the reading pattern of a positive (A) and negative (B) serum. In (A), the arrow shows the presence of ‘bright apple green’ fluorescence in the worm’s digestive tract. In (B), the arrow shows the total absence of fluorescence in the worm’s digestive tract. Fluorescence microscopy, 400× magnification.
Circumoval precipitin test (COPT)
Obtaining S. mansoni eggs
Viable eggs were obtained from the livers of infected animals used to maintain the evolutionary cycle of S. mansoni. The method used to extract and clean eggs was described by Dresden and Payne (Reference Dresden and Payne1981), with some modifications. Thus, livers from 3 hamsters with 7–8 weeks of infection were cut into small fragments and ground in a blender with ice-cold 1.7% saline solution. Then, the material was screened in metal sieves with meshes of 0.150, 0.75 and 0.038 mm, under negative pressure. The eggs retained on the last sieve were washed in a continuous flow with ice-cold 1.7% saline solution and concentrated by low centrifugation at 1000 rpm for 15 s. The number of eggs in the suspension was determined by microscopic counting and adjusted to contain 300 viable eggs per 10 µL of 1.7% saline. The purified egg suspension was stored in a refrigerator at 4 °C for a maximum of 4 h before processing the reactions.
Preparation for COPT
In an Eppendorf, 25 µL of serum sample and 10 µL of egg suspension (∼300 mature and viable S. mansoni eggs) were added. The mixture was incubated in an oven at 37 °C for 48 h. COPT was performed on all 685 selected samples that were positive for IFA-IgM. To control the reactions, 4 pools (P) of non-glycerinated sera were used, characterized as follows: P1 (5 human sera with positive IgM-IFA and positive COPT); P2 (5 human sera with negative IgM-IFA and negative COPT); P3 (5 sera from rodents with 8 weeks of S. mansoni infection) and P4 (5 sera from uninfected rodents).
Reading and interpreting the COPT
After the incubation period, the entire mixture from each tube (serum + viable egg suspension) was placed between a slide and coverslip (22 × 22 mm) and immediately examined using an optical microscope with 10× and 40× objectives, considering the observation of 100 viable reactive eggs per slide. The results were expressed as the absence or presence of precipitates around the eggs, classified according to their morphological aspect (Figure 2), as described below:
1) Reaction 0 (R0): absence of precipitates surrounding the egg;
2) Reaction 1 (R1): small, rounded reactions with a hyaline appearance;
3) Reaction 2 (R2): slightly elongated, non-septate reactions with a hyaline appearance;
4) Reaction 3 (R3): globular reactions with irregular contours and variable size, showing granular and refringent content under conventional light microscopy;
5) Reaction 4 (R4): reactions consisting of long, septate chains with a granular appearance.
Precipitate patterns observed in serum samples subjected to COPT. The arrows demonstrate reaction patterns R1 (A), R2 (B), R3 (C) and R4 (D) by optical microscope, 400× magnification.
Specific reactions were considered to be those described as R3, associated with recent infections, and R4 with chronic infections, according to the patterns also observed by Long et al. (Reference Long, Pelley, Blas, Yogore and Lewert1980) in human infections by S. japonicum. R1 and R2 were considered non-specific, as described by Pellegrino and de Mello (Reference Pellegrino and de Mello1975).
Data analysis
The Chi-square statistical test with P ≤ 0.05 was used to verify the association between the reactive patterns for COPT and IFA-IgA.
Results
Of the 685 sera selected and positive in the IFA-IgM, all were submitted to COPT, which defined 2 groups of samples according to the periovular precipitate patterns found: group (G1) with 548 (80.0%) samples considered non-reactive for COPT, since they presented eggs with an absence of reactions or reactions of patterns R1 and/or R2. In this group, R0 reactions were found in 15 (2.7%) of the sera, R1 in 24 (4.4%), R2 in 174 (31.8%) and R1/R2 in 335 (61.1%). Group (G2) was composed of 137 (20.0%) samples considered reactive because they presented specific patterns of precipitates R3, R4 or mixed R3/R4. In this second group, R3 reactions were found in 57 (41.6%) of the sera, R4 in 45 (32.8%) and R3/R4 in 35 (25.5%). Some sera from group G2 also simultaneously presented non-specific R1 and R2 reactions, data not shown.
Based on these results and considering the 2 morphological types of periovular precipitates that defined the positivity of the sera in group G2, IFA-IgA was performed on paraffin sections in the 137 samples with the association of IFA-IgM (+)/COPT (+) results. Of these samples, 64 (46.7%) were positive for immunoglobulin A. The results are detailed in Table 1. Emphasis is placed on the exclusively R3 reactions (57), where IFA-IgA was positive in 47 (82.5%) sera (P ≤ 0.001). Mixed R3/R4 reactions were associated with 12 (34.3%) samples with IFA-IgA and R4 in only 5 (11.1%).
Distribution of the number of positive and negative sera for IFA-IgA according to the R3 and R4 precipitate patterns in COPT, in 137 sera from group G2
Table 1 Long description
Counts and percentages of IFA-IgA negative and IFA-IgA positive sera are listed by COPT patterns R3, R3/R4, and R4, with totals. For R3, IFA-IgA negative was 10 at 17.5 percent and IFA-IgA positive was 47 at 82.5 percent, marked with an asterisk, total 57. For R3/R4, IFA-IgA negative was 23 at 65.7 percent and IFA-IgA positive was 12 at 34.3 percent, total 35. For R4, IFA-IgA negative was 40 at 53.3 percent and IFA-IgA positive was 5 at 11.1 percent, total 45. Overall totals were 73 IFA-IgA negative at 53.3 percent and 64 IFA-IgA positive at 46.7 percent, across 137 samples.
* Chi-square test with P ≤ 0.001.
Discussion
The State of São Paulo is considered an area of low transmission, with foci identified in regions such as Baixada Santista, Vale do Ribeira, Vale do Paraíba, the Metropolitan Region of Campinas, and some municipalities in Greater São Paulo, including the capital. With systematic surveillance actions in recent decades, there has been a significant reduction in the transmission and notification of autochthonous cases in the state (SES-SP/CCD (Secretaria de Estado da Saúde, Coordenadoria de Controle de Doenças). Divisão de Doenças de Transmissão Hídrica e Alimentar, CVE (Centro de Vigilância Epidemiológica ‘Prof. Alexandre Vranjac’), 2009), as well as a decrease in reported imported cases (RS et al., Reference RS, Tauil, Lima and Engels2006).
Thus, following the global trend in the epidemiology of schistosomiasis, the low prevalence associated with the reduction of the parasitic load of infected individuals constitutes a new challenge for surveillance and control, requiring the incorporation of new strategies, such as early recognition of cases and treatment of symptomatic and asymptomatic individuals, aiming at the elimination of autochthonous transmission (WHO, 2022). Among the recommended strategies, the incorporation of more sensitive diagnostic methodologies in relation to parasitological examinations stands out (Silva-Moraes et al., Reference Silva-Moraes, Shollenberger, Siqueira, Castro-Borges, Harn, Grenfell, Rabello and Coelho2019; Ramos et al., Reference Ramos, da Silva-Pereira, Oliveira, Fonseca and Graeff-Teixeira2023).
The IFA-IgM has been used as a serological test for schistosomiasis, both for seroepidemiological surveys in areas of low transmission (Kanamura et al., Reference Kanamura, Dias, Silva, Glasser, Patucci, Vellosa and Antunes1998; Gargioni et al., Reference Gargioni, Silva, Thomé, Quadros and Kanamura2008; Carvalho do Espirito-Santo et al., Reference Espirito-Santo, Sanchez, Sanchez, Alvarado-Mora, Castilho, Gonçalves, Luna and Gryschek2014) and for complementary diagnosis to parasitological stool examinations (SES-SP/CCD (Secretaria de Estado da Saúde, Coordenadoria de Controle de Doenças). Divisão de Doenças de Transmissão Hídrica e Alimentar, CVE (Centro de Vigilância Epidemiológica ‘Prof. Alexandre Vranjac’), 2009). In a recent review of performance indicators for serological tests in Brazil, Ramos et al. (Reference Ramos, da Silva-Pereira, Oliveira, Fonseca and Graeff-Teixeira2023) reported sensitivity rates ranging from 60 to 97% and specificity from 84 to 100% based on 5 studies that used IFA-IgM for epidemiological purposes. The authors highlighted as negative points for the use of the test the complexity of obtaining paraffin sections and the impossibility of automation, characteristics that would restrict large-scale population screening. However, the widespread use of IFA-IgM in previous studies, especially in samples from the State of São Paulo, and the high stability of paraffin sections at room temperature have shown its practicality in the storage and distribution of sections, which allows the technique to be performed by other laboratories, facilitating network diagnosis (Kanamura et al., Reference Kanamura, Dias, Silva, Glasser, Patucci, Vellosa and Antunes1998, Reference Kanamura, Silva, Chiodelli, Glasser and Dias2002).
In fact, experience has shown that a single serological diagnosis, based solely on antibody detection, is not sufficient for identifying active and treated cases and also restricts its application in post-treatment monitoring. These limitations have led to the development of new methodologies or the improvement of existing ones for antibody detection (Silva-Moraes et al., Reference Silva-Moraes, Shollenberger, Siqueira, Castro-Borges, Harn, Grenfell, Rabello and Coelho2019; Chala, Reference Chala2023). In other countries, immunodiagnosis of schistosomiasis has been used to define cases in areas of low transmission, showing good results. In Venezuela, Noya et al. (Reference Noya, Guevara, Colmenares, Losada and Noya2006) proposed new criteria for case definition, in addition to a positive stool parasitological examination. The authors used COPT associated with other serological methods as markers of active infection for S. mansoni and in the selection of individuals for treatment. Similarly, in recent decades in China, methodologies such as COPT and ELISA have been improved and applied in the screening of patients with S. japonicum infection for specific chemotherapy (Zhu, Reference Zhu2005).
Regarding the samples analysed in this study, 685 positive sera for IFA-IgM were selected, originating from spontaneous demand for presumptive diagnosis of schistosomiasis and from sero-epidemiological surveys related to outbreaks, exclusively for the state of São Paulo. From the panel of sera showing a positive result in the IFA-IgM, COPT was performed, a serological technique that, according to the literature, shows greater concordance with the parasitological examination of feces (Zhu, Reference Zhu2005; Noya et al., Reference Noya, Guevara, Colmenares, Losada and Noya2006) and which constitutes a tool for detecting active cases in areas of low transmission (Spencer et al., Reference Spencer, Noya, Noya and Masroua1991). Based on this premise, in the present study, all selected samples positive in the IFA-IgM test were analysed by COPT. Given the variability of the reactions found in each serum, a qualitative positivity criterion was adopted, considering as positive reactions those in which there was the presence of precipitates described here as R3 and R4, which characterize recent and chronic infections, respectively. However, other criteria are pointed out, such as the intensity of the precipitates and the percentage of reactive eggs. Spencer et al. (Reference Spencer, Noya, Noya and Masroua1991) established quantitative criteria for COPT, considering as positive only those samples that presented percentages of reactive eggs equal to or above 15%. However, the authors did not make clear the reason for this criterion and whether they considered reactions of non-specific patterns in this percentage. Although some studies have also adopted quantitative parameters in the interpretation of COPT results (Vendrame et al., Reference Vendrame, Carvalho, Yamamoto, Nakhle, Carvalho and Chieffi2001; Noya et al., Reference Noya, Guevara, Colmenares, Losada and Noya2006), the occurrence of other morphological patterns of precipitates in the reaction, both in samples from patients with schistosomiasis and negative individuals, makes the interpretation of the results difficult (Pellegrino and de Mello, Reference Pellegrino and de Mello1975).
Given the need to select samples from patients with active infection and the absence of fecal parasitology results, the present study combined findings from positive IgM-IFA and COPT. Consequently, 137 samples (20%) were selected, based on the morphological patterns of precipitates surrounding the eggs. Thus, in the combination of positive IFA-IgM and COPT results, 137 (20%) samples were selected that, based on the morphological patterns of the precipitates around the eggs, reinforced the hypothesis that the reactions described here as R3 may be related to recent infections, the mixed R3/R4 as a transitional stage between acute and chronic and the R4 as chronic infections caused by S. mansoni. These data fully agree with those inferred by Lewert et al. (Reference Lewert, Yogore, Martin and Blas1980) for S. japonicum eggs in human infections and in experimental models.
Based on the results of the COPT, the IFA-IgA was incorporated into the study, since there is consensus among some authors that immunoglobulins of this class are present in greater quantity in sera of patients with recent infection (Kanamura et al., Reference Kanamura, Hoshino-Shimizu, Camargo and da Silva1978, Reference Kanamura, Silva, Rabello, Rocha and Katz1991, Reference Kanamura, Dias, Silva, Glasser, Patucci, Vellosa and Antunes1998; Silva-Moraes et al., Reference Silva-Moraes, Shollenberger, Siqueira, Castro-Borges, Harn, Grenfell, Rabello and Coelho2019). It has been shown that the IgA response in schistosomiasis mansoni is directed against antigenic components that coat the inner part of the digestive tract of adult worms and against soluble egg antigens (Kanamura et al., Reference Kanamura, Silva, Rabello, Rocha and Katz1991; Rabello et al., Reference Rabello, Garcia, Silva, Rocha, Chaves and Katz1995). Due to the nature of the antigen used, this immunoglobulin can be detected by IFA in frozen sections of adult worms and granulomas, and in paraffin sections of adult worms (Kanamura et al., Reference Kanamura, Hoshino-Shimizu, Camargo and da Silva1978, Reference Kanamura, Silva, Rabello, Rocha and Katz1991) or by the ELISA method when soluble egg antigens are used (Rabello et al., Reference Rabello, Garcia, Silva, Rocha, Chaves and Katz1995). Although this marker is more frequent in patients in the acute phase, it may also be present in some individuals in the chronic phase of S. mansoni infection (Kanamura et al., Reference Kanamura, Silva, Rabello, Rocha and Katz1991; Rabello et al., Reference Rabello, Garcia, Silva, Rocha, Chaves and Katz1995).
The use of COPT and IFA-IgA, presented here, is being proposed for the first time for confirmation of cases and respective stages of infection. Although the results point to the relationship of IgA with R3 reactions in recent infections, no data were found in the literature that correlated the concomitant presence of IgA antibodies to globular precipitates in sera of patients with acute or recent infections. The statistically significant association between R3 pattern COPT and IFA-IgA, found in this study, reinforces the hypothesis that this result is related to recent infections. The occurrence of mixed reactions (R3/R4) with IFA-IgA in 34.3% of the samples may, in fact, be related to the transition phase of the infection, as shown by Lewert et al. (Reference Lewert, Yogore, Martin and Blas1980) for S. japonicum. As previously mentioned, IgA antibodies decrease in the serum of patients throughout the infection by S. mansoni, a fact that may explain the presence of this marker in only 11.1% of the R4 pattern samples by COPT.
The limitations of laboratory diagnosis of schistosomiasis in areas of low endemicity are well known, where a single methodology (parasitological or serological), when used individually, is not sufficient to detect cases (Silva-Moraes et al., Reference Silva-Moraes, Shollenberger, Siqueira, Castro-Borges, Harn, Grenfell, Rabello and Coelho2019; Chala, Reference Chala2023). In this study, an association of serological tests using different markers was proposed, with the aim of improving case prospecting and relating them to the infection phase and autochthonous status in the absence of stool parasitology test results. The installed capacity of the Public Health Laboratory served as the basis for these procedures; the laboratory has utilized the IFA-IgM test on paraffin sections of adult worms as a tool for the serological diagnosis of schistosomiasis (Ramos et al., Reference Ramos, da Silva-Pereira, Oliveira, Fonseca and Graeff-Teixeira2023).
As limitations of the study, it should be noted that both IFA and COPT are operator-dependent, very laborious techniques that rely on maintaining the parasite’s life cycle, making them unfeasible for many laboratories. Additionally, the need for viable eggs in the COPT limits its application to a restricted number of samples and epidemiological surveys (Carvalho do Espirito-Santo et al., Reference Espirito-Santo, Sanchez, Sanchez, Alvarado-Mora, Castilho, Gonçalves, Luna and Gryschek2014). However, a positive result is significant, as it may indicate the presence of eggs in host tissues, regardless of the parasitological examination result (Noya et al., Reference Noya, Guevara, Colmenares, Losada and Noya2006).
As final considerations, the IgA-IFA test was incorporated into our procedures after observing its relationship with the R3 precipitation pattern of COPT, both markers of the recent stage of the disease. In this way, the association of the 3 proposed serological techniques will contribute specifically to increasing the detection of cases and speeding up the laboratory response, especially in situations of low parasite load, where the parasitological examination remains negative in several samples. Thus, the search for markers for recent infections is recommended, which, once confirmed, can trigger Epidemiological Surveillance actions to assess the autochthonous nature of cases. This approach aligns with the goals established by the WHO for areas of low endemicity (WHO,2022).
Future perspectives should focus on studies to characterize and purify S. mansoni egg excretion-secretion antigens, as well as epitopes located on the inner wall of the worms’ digestive tract, aiming at the production of recombinant proteins, either isolated or as part of chimeric proteins, suitable for use in enzyme immunoassay platforms, thereby enabling large-scale application and the decentralization of diagnosis.
Author contributions
Inception and design of the study: P.L.S.P.; organization of samples for the study and standardization of diagnostic techniques: F.C.; data analysis and statistical testing: F.C., P.L.S.P., M.C.C.E.S. and F.M.P.; first draft of the paper: P.L.S.P., F.M.P., M.C.C.E.S. and R.C.B.G.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors declare that there are no conflicts of interest.
Ethical standards
This study meets the requirements of the National Instance for Ethics in Research and was approved by the Research Ethics Committee of the Adolfo Lutz Institute (protocol number 2.420.105). The protocols used to maintain the experimental biological cycle of Schistosoma mansoni comply with the guidelines of the National Council for the Control of Animal Experimentation and were approved by the Ethics Committee on Animal Use of the University of São Paulo Medical School (protocol number 7499).
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