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

Abstract

Background Despite decades of use 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 needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. Methods and findings Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S . mansoni -infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1–10 eggs per gram of feces that were undiagnosed by KK 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 MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

Evaluation of schistosomula crude antigen (SCA) as a diagnostic tool for Schistosoma mansoni in low endemic human population

“…The identified 10–12 kDa immunoreactive protein in our study was much smaller than 62 kDa protein that stimulated CD4 + Th cells and 37 kDa diagnostic protein known as the major egg antigen (MEA) from SEA 16 , 17 . Although other immunoreactive proteins of higher molecular weight (25–37 kDa) were identified in the Western blot assay, the intensity of the reaction was weaker and these proteins showed no consistency in the subsequent dot-blot immunoassay performed.…”

“…So, the reference standard was a positive KK or SG or both. Sera samples from healthy individuals living in a schistosomiasis non-endemic area in Belo Horizonte, Minas Gerais, who had no history of previous Schistosoma infection, were used as the negative control (Age 21–70 years old) 16 . Fecal samples from these individuals were also screened for S. mansoni and other helminths by KK and SG.…”

“…The tails were removed and the process was repeated until all tails were removed. The suspension containing schistosomula was sonicated using six 10 s pulses on full power with 1 min on ice between each sonication 16 . The suspension was centrifuged at 20,000× g for 10 min.…”

“…The membranes were thereafter incubated under shaking at room temperature for 1 h 30 min with anti-human IgG peroxidase-conjugated antibody, diluted 1:2000 in the immunoblotting buffer. After three washes at 10 min/wash, the immunoreactive proteins were developed using ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, Illinois, USA) and images were captured using chemiluminescence detection in ImageQuant LAS 4000 (GE Healthcare, Chicago, Illinois, USA) 16 . The Western blot analyses were performed in triplicate.…”

Estudo Culicidiano No Centro De Ciências Agrárias (Cca) Universidade Federal Do Piauí (Ufpi): Vigilância Entomológica

“…Grande parte desses parasitos podem infectar humanos e provocar doenças como paragonimose(KARKI et al, 2021), clonorquiose(LUN et al, 2005) e equinostomose(GRACZYK; FRIED, 1998). Porém, a principal enfermidade causada por esses indivíduos é a esquistossomose, parasitose que acomete mais de 200 milhões de pessoas em todo o mundo(ROCHA et al, 2016;SILVA-MORAES et al, 2019). Desde a descrição do S. mansoni no Brasil, diversas investigações em malacologia vem sendo realizadas por todo o território brasileiro, e devido a esses estudos houve a revelação de uma diversidade de trematódeos transmitidas por moluscos, tanto gastrópodes como bivalves (PINTO;.…”