Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity infections in endemic area in Brazil Antigen target in schistosomiasis diagnosis

Background Despite decades of employment of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there need to be better diagnostic tools to detect low intensity infections in low-endemic areas as Brazil. The rationale for development of new diagnostic tools is because in low-endemic settings, the standard Kato-Katz diagnostic method loses its sensitivity and misses low intensity infections. To develop new diagnostic tools, we employed a proteomics approach search for biomarkers associated with schistosome-specific immune responses to develop sensitive and specific new methods for immunodiagnosis. Methods and findings Immunoproteomic analysis was performed on an egg extract of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other helminth parasites was determined using pooled sera from individuals infected with different parasitic helminths. Using this approach we identified 23 spots recognized by schistosome acute and chronic sera samples. To identify immunoreactive spots that are likely glycan epitopes, we compared immunoreactivity of spots treated by sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting they are protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from infection with other helminths and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from schistosome-infected groups in native and deglycosylated conditions and corresponds to a Major Egg Antigen. We expressed the Major egg antigen as a recombinant protein and showed by western blot a similar recognition pattern of that of the native protein. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under ROC curve of 0.95. IgG-ELISA performed better than the conventional K-K (2 slides) identifying 56/64 cases harboring 1-10 egg per gram of feces that were undiagnosed by K-K parasitological technique. Conclusions The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant major egg antigen can be a useful tool to be combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by Kato-Katz method. Further, to overcome the complexity of ELISA in the field, a second-generation of antibody-based rapid diagnostic tests (RDT) can be developed. Author Summary Schistosomiasis remains a serious global public health problem. Detecting parasite eggs in patient stool samples using the Kato-Katz (KK) method is the standard diagnostic recommended by World Health Organization (WHO) for infection by Schistosoma mansoni. As a result of intensive control strategies, many previously high-endemic areas are now considered low-endemic areas and the K-K method does not function well in low-endemic areas and therefore cannot be considered the gold standard. Thus, a new emphasis on strategies to accurately diagnose low-intensity infections was outlined in plan from WHO focusing on elimination of disease as a public health problem. Successful diagnoses and treatment of the majority of infected individuals may result in elimination of a sufficient number of low-burden transmitters and consequently, in the interruption of the disease transmission. In this regard, immunological techniques have proven to be more sensitive and promising for identifying infection in low-intensity of infection positive individuals with negative K-K results. The identification of antigens is the initial step for developing new immunodiagnostic assays. In this study, we used sets of pooled human sera samples from controls to acute and chronic infections to narrow down the number of new candidate antigens via proteomic screening. Using these approaches we initially identified 12 different egg proteins in schistosome-infected individuals (acute and chronic phase). A single antigen identified as Major Egg Antigen was shown to be highly specific as this antigen was not recognized by sera from negative or patients infected with other helminths. The recombinant Major Egg Antigen protein functioned in ELISA as a highly sensitive and specific antigen to detect patient IgG-antibodies. Recombinant Major Egg Antigen performed significantly better to detect low-intensity infections (1 egg per gram of feces) than the K-K method using 2 slides. Therefore, using a proteomic screening approach we were able to identify a potential new candidate antigen for development of far more sensitive diagnostic assays. Further diagnostic assays employing the Major Egg Antigen could be a useful tool on its own or in combination with other methods for diagnosis of schistosome infection in populations living in extreme low-intensity endemic areas of Brazil.

control and eliminate the disease is diagnosis and treatment of active cases at the 174 primary care level [4,8]. Currently diagnosis continues to be detection of schistosome 8 175 eggs in stools by microscopic examination using the Kato-Katz technique (K-K). K-K is 176 the World Health Organization (WHO) reference for diagnosis [9]. The K-K method is 177 low-cost and suitable for detection of high-intensity infections. However, the K-K 178 technique has poor sensitivity for detection of low-intensity infections that are seen in 179 residents living in low-endemic areas (<10% prevalence, <100 epg). The low-sensitivity 180 of the K-K method results in misdiagnosis (schistosome negative) of infected 181 individuals, who due to lack of diagnosis, continue to contribute to disease transmission 182 and skew actual disease prevalence. Previous studies in Brazil demonstrated that 183 prevalence of disease has been underestimated by a factor of 2-4, due to the inability of 184 the K-K method to detect low-intensity infections [10][11][12][13][14]. 185 As a result of intensive control strategies employing praziquantel (PZQ), many 186 previously high-endemic areas are now considered low-endemic areas where due to lack 187 of sensitivity, K-K cannot be used as the gold standard for diagnosis [15]. Therefore, 188 new diagnostic methods need to be developed to detect low-intensity infections. The 189 ability to accurately diagnose low-intensity infections was outlined in plans focusing on 190 elimination of disease as a public health problem [7,16,17]. Methods that can 191 accurately diagnosis the majority of individuals will contribute to the goal of 192 elimination of low-burden transmitters and consequently, in the interruption of the 193 disease transmission. In this regard, molecular and immunological techniques have 194 proven to be more sensitive and promising for identifying infection in infected 195 individuals that are negative by K-K coproscopy results [10,13,14,[18][19][20]. 196 Significant progress has been seen in the development of antigen-based rapid 197 diagnostic tests (RDT), as its assembly is user-friendly in the field. The 198 immunochromatographic point-of-care (POC) test that detects circulating cathodic 199 antigen (CCA) in urine has been commercially available since 2008 [21,22]. Although 200 POC-CCA has been suggested to be a suitable substitute for K-K in S. mansoni 201 prevalence mapping [22][23][24][25], its performance is still debatable in low-endemic areas 202 [26,27]. The majority of studies validating POC-CCA were conducted in Africa, 203 whereas few (10) studies were conducted in Brazil, which has a significantly different 204 prevalence and morbidity profile. In contrast to Africa, most of the low-intensity 205 infections in Brazil are denoted as < 25 epg [11,12,20,[27][28][29][30][31][32][33]. 206 Antibody-based methods have high sensitivity in detecting low-intensity 207 infections and are capable of identifying loads of 1 epg [14,19,[34][35][36][37][38][39]. Their use as 208 screening tools combined with parasitological evaluations has decreased the false-209 negative cases seen when the unique analysis by 2 K-K slides is applied in endemic 210 settings. Furthermore, another useful application of these tests is the ability to detect 211 acute infections in individuals from non-endemic areas recently exposed to 212 schistosomiasis-endemic settings. Since antibodies to the parasite develop during the 213 first weeks after infection, they can be detected before eggs in the feces to yield higher 214 sensitivities. In clinical practice, positive serology in K-K negative people from non-215 endemic countries is usually sufficient to prescribe treatment with PZQ [40][41][42].

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In regards to developing an antibody-based test, the choice of antigen is the most 217 challenging. Crude antigens, such as soluble eggs antigens (SEA) and worm antigens 218 (SWAP), are frequently used, but they can exhibit low-sensitivity and cross-reactivity 219 with other helminthes [43,44]. A combination of proteomic and serological analyses 220 have served as promising experimental approaches for screening new biomarkers in the 221 diagnostic field [45][46][47]. However, there is a limited number of serological-proteomic 222 studies involving Schistosoma spp. and most of them are related to searching for 223 vaccine candidates using animal models [47][48][49][50][51][52]. Only one immunoproteomic analysis related to S. mansoni and human samples has been performed to date, but it also focuses 225 on the search for vaccines candidates [52]. 226 Since the lack of effective diagnostic assays in low-endemic areas is one of the 227 factors that contributes to transmission and there is a need for more specific biomarkers 228 in immunodiagnostic development, in this present work, we adopted immunoproteomic 229 analysis to identify a new antigen candidate for schistosomiasis diagnosis. We used 230 different sets of pooled sera from acute and chronically infected patients, as well as 231 from helminth positive, but schistosome-negative individuals, to select highly specific 232 schistosome antigens. Antibodies against schistosome eggs have been considered useful 233 antigens for the diagnosis of schistosomiasis [35,42,53]. Therefore, we screened 234 soluble egg extracts (SEE) by two-dimensional western blotting (2D-WB). While many 235 studies have focused on the serologic-proteomics of adult worms, we focused on egg 236 antigens. Antigens from eggs are highly immunogenic, but less specific, due to cross 237 reactions provided by glycan epitopes [44,54]. To achieve higher specificity from a 238 recombinant antigen, we compared native SEE extracts to those oxidized by Sodium 239 Metaperiodate (SMP), to identify those antigens whose antigens were glycan-based. 240 We identified 23 immunoreactive spots, which resolved to 12 differently 241 characterized proteins. One protein was uniquely recognized by schistosome-infected 242 patients and not recognized by sera from patients infected with other helminths or 243 negative control sera. We produced and purified a recombinant protein for this antigen 244 developing a conventional Enzyme-linked Immunosorbent Assay to detect antigen-245 specific IgG (IgG-ELISA). The recombinant egg antigen showed high sensitivity for 246 detection of low-intensity infections that were misdiagnosed by the standard K-K (2          The 2D-PAGE provided good resolution of spots in pH range with minimal 566 streaking. In order to identify the antigens recognized by antibodies in pooled sera, a 567 corresponding 2D-PAGE was performed in parallel so that WB (native and SMP-568 oxidized) could be performed to exclude any variation that might arise from the use of 569 different antigen preparations (Fig 1).

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In native 2D-WB, 23 immunoreactive spots were recognized by the pooled identification of spots is presented in Table 2.

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The 23 spots were resolved into 12 proteins. LC/MS analysis revealed instances 580 in which different spots were derived from the same protein: for example, spots 11, 17, immunoreactive spots that were barely visible in stained gels (e.g., spot 1). Spots 10, 19, 586 20 and 21 were not identified due to low abundance. The most identified proteins were related to housekeeping proteins. These include structural/muscle proteins, enzymes 588 (mostly components of the glycolytic pathway) and chaperone proteins.

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To evaluate the presence of glycosylated epitopes on the 23 immunoreactive 590 spots, 2D-WB were performed using SMP-treated membranes and then compared to the 591 native one (Table 3). After oxidation, only 12/23 spots maintained immunoreactivity, 592 indicating they potentially have protein epitopes. From these 12 spots, 11 spots cross-           The agreement between rMEA-IgG-ELISA and the reference method 691 determined by 24 slides of K-K and 2 procedures of SG showed substantial 692 concordance (κ = 0.75). When the current adopted K-K (2 slides) was compared, it 693 demonstrated a fair concordance (κ = 0.32) underdiagnosing 57 true cases. The 694 positivity rate from rMEA-IgG-ELISA (58.8%) and the reference method (62.8%) 695 showed no significant difference (χ 2 , p = 0.24) ( Table 5).     Antibody-based immunodiagnostics have greater sensitivity than parasitological 725 methods in low-endemic areas [14,19,[34][35][36][37][38][39]. Serum immunoreactivity to schistosome 726 antigens allows detection of infections with loads as low as 1 epg. Even though the 727 antibody-detection methods are currently not the first choice for endemic areas, due to 728 their inability to discriminate current infection from previous exposure, as well as the 729 inability to monitor treatment effectiveness, they have been accepted as a complementary tool during epidemiological survey [13,19,34,62]. One of the 731 difficulties in developing antibody-detection methods is the choice of antigen. Crude 732 antigens, such as SEA and SWAP, are not ideal in terms of sensitivity and specificity.

733
To overcome this, the search for new antigens and subsequent production by 734 recombinant strategies has been proposed as an alternative to improve antibody 735 detection. In this study, we aimed to identify parasite markers for development of highly 736 sensitive and specific new immunological tests. By using serum from individuals from a 737 low-endemic area in Brazil in 2D-WB, we were able to screen antibody targets directed 738 to egg extracts and identify an antigen, which shows potential for diagnosis of low-739 intensity schistosomiasis infections. which could be resolved by means of a differential diagnosis using an antigen specific 776 for that phase. Mutapi et al. (2005), using a similar approach to this work, but with S. . MEA has been described as highly antigenic in infected humans [71]. 806 The profile of cytokines obtained from peripheral blood mononuclear cells (PBMCs) 807 from S. mansoni infected patients and stimulated with purified MEA was associated 808 with reduced granuloma formation and an anti-pathological vaccine [72].

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MEA was previously suggested as a diagnostic by Nene et al. (1986), since it  Although antibody detection cannot differentiate between active and past 818 infection, its main advantage is in the detection of individuals recently exposed (pre-819 patent phase) and those with low parasitic loads that are not detected using K-K (2 820 slides) [13,14,19,74]. Among serological tests, ELISA is widely used for the diagnosis 821 of schistosomiasis, due to its low relative cost and ability to run a lot of samples at the 822 same time. In an outbreak in Southeast in Brazil, the IgG-ELISA was used to diagnose 823 tourists from a non-endemic area recently exposed to an infected river considered an  The rMEA-IgG-ELISA performed significantly better than the currently adopted 852 K-K (2 slides) for detection of low-intensity infections. The K-K exhibited a sensitivity 853 of 38.71% with 57 false negative cases, ranging from 1-10 epg. In this case, undiagnosed individuals would not receive treatment, possibly develop serious disease, 855 and contribute to maintenance of transmission. On the other hand, rMEA-IgG-ELISA 856 showed 87.5% (56/64) of sensitivity in the group of ≤ 10 epg, which is indicative of the 857 majority of cases of schistosome infection in Brazil [10-12, 20, 27, 28]. Additional 858 studies utilizing other S.mansoni recombinant proteins also showed better results than

869
In the work presented here, we demonstrated that the immunoproteomic analysis 870 was successful in selecting a good candidate for use in the diagnosis of schistosomiasis.

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The rMEA showed performance in ELISA superior to the current gold standard K-K 872 since high levels of IgG were identified in individuals harboring 1-10 epg missed in 873 primary parasitological analysis (K-K 2 slides). We believe the IgG-ELISA can be a 874 useful tool to be combined with other techniques in low-endemic areas to determine the 875 true prevalence of schistosome infection. The rMEA-IgG-ELISA results suggest that 876 this assay can be valuable when used as a screening tool during epidemiological surveys 877 followed by more specific assays as a robust parasitological evaluation. To overcome 878 the complexity of ELISA in the field, a second-generation of antibody-based RDTs has already been proposed, as well as the detection of antigen together in a multiplex strip 880 on a reader [60]. In this study, we have demonstrated this initial step successfully.

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We would like to thank the people from the rural communities of Montes Claros, 884 Minas Gerais for their collaboration and the warm reception during the field activities. 885 We are grateful for the excellent cooperation with the technical team of the Zoonosis